Biophysics IIBiophysics II

ByByAProf Xiang Yang LiuAProf Xiang Yang LiuBiophysics LabBiophysics LabDepartment of Physics Department of Physics NUSNUS

Announcement

Term Test ndash Mar 2 2007 Fri 1048700 Venue S13-0507 1048700 Time 830-930am (1 hr) Closed book One A4 ldquocheat sheetrdquo is allowed

Outline

ELECTROPHORESIS Discussion you are supposed to prepare

your questions to be discussed in the lecture

Example

Example

Example

If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi

mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Announcement

Term Test ndash Mar 2 2007 Fri 1048700 Venue S13-0507 1048700 Time 830-930am (1 hr) Closed book One A4 ldquocheat sheetrdquo is allowed

Outline

ELECTROPHORESIS Discussion you are supposed to prepare

your questions to be discussed in the lecture

Example

Example

Example

If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi

mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Outline

ELECTROPHORESIS Discussion you are supposed to prepare

your questions to be discussed in the lecture

Example

Example

Example

If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi

mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Example

Example

Example

If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi

mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Example

Example

If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi

mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Example

If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi

mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

ELECTROPHORESIS

Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

ELECTROPHORESIS

The great majority of the polymers of biological interest are electrically charged

Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups

They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

ELECTROPHORESIS

electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis General Principles Electrophoresis The transport of particles by

an electrical field The charged molecule is not alone but in the

presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Diffusion through a solvent

The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein

D = kTf

f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent

Stokesrsquo Law for a sphere of radius a

fo = 6a

the viscosity of the solvent

Do = kT6a

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis General Principles An idealized simplified situation an isolated charged

particle in a nonconducting medium

The force experienced by a particle in an electrical field is given by Coulombrsquos law

F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -

fv (f the frictional factor) The viscous resistance of the medium just balances

the driving force fv = F = ZeE

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis General Principles

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = vE = Zef If the particle happens to be spherical Stokesrsquos law

applies U = Ze6a

The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix

The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis General Principles The kind of supporting matrix used depends on the type of

molecules to be separated and on the desired basis for separation charge molecular weight or both

Almost all electrophoresis of biological macromolecules is at

present carried out on either polyacrylamide or agarose gels

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis

Each macromolecular solution is applied in a thin layer in one well

If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation

Usually a dye of high mobility is added its migration serves to mark the progress of the experiment

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis

The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by

Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Ferguson plots

A very simple relationship between relative mobility and gel concentration

logUri = logUrio - kiC

where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Electrophoresis

Ferguson plots for a number of commonly encountered solutions

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Example

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the

molecular weights of polypeptide chains the protein to be studied is first heated in a dilute

solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also

added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain

weight and is nearly independent of the charge on the polypeptide

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to

many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight

the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA

there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility

logUri = logUrio - kiC

DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)

References

Section 54 in Principles of Physical Biochemistry

References

Section 54 in Principles of Physical Biochemistry