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Some Biophysical Techniques

Gels, Columns, and Centrifuges

Outline1. Overview: chromatography,

electrophoresis, and centrifugation2. Observing charge

1. Isoelectric focusing2. Ion-exchange chromatography

3. Observing size1. Denatured

1. SDS-PAGE2. Native (or denatured)

1. Gel filtration or size exclusion chromatography2. Centrifugation

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Chromatography- General

(a) Chromatography column separation

(b) Resulting chromatogram

• Horton 1996, p. 67.

Electrophoresis- General

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Isoelectric Focusing

IEF needs

• Stable pH gradient• Carrier ampholytes• Sample• Detergents and other agents to solubilize

and stabilize sample• Staining of the gel to visualize it (to be

discussed under SDS-PAGE)

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IEF biophysics

• Equilibrium technique• The protein stops migrating when pH =

pKa of the protein• Requires time to reach equilibrium• Temperature dependent migration

IEF summary

• IEF separates proteins according to their isoelectric points on a pH gradient gel

• Migration in the electric field stops when the pH = pKa

• Staining the gel is necessary.

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LIQUID CHROMATOGRAPHY

ION-EXCHANGE: by Charge (least attracted to resin comes out first)

++++++++++

++++++++++

load -+

-+

++++++++++

++++++++++

-

--

--

-

Elute w/NaCl

+ -

++++++++++

++++++++++

--

-

--

--

Increase Conc. NaCl

++++++++++

++++++++++

--- -- -

-

Ion-exchange chromatography

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Common ion-exchange media

Chromatogram for ion exchange

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Measuring size: general considerations

• The size of a protein varies depending on its conformation

• For measurements interested in peptide length, the protein must be denatured first

• Two main techniques for approximating peptide length:– SDS-PAGE– Gel filtration chromatography under

denaturing conditions

Measuring size

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SDS-PAGE

• Components– Acrylamide gel– SDS– Buffers

• Electrodes, power supply and chamber• Denatured protein sample• Staining procedure

The components

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Acrylamide gel• Polymerization of acrylamide and methylene bis-

acrylamide catalyzed by ammonium persulfate and accelerated by N,N,N’,N’-tetramethylenediamine

Controlling Pore Size

• Typically, we use Laemmli gels which are 2.6%C.

%100)()(

)(%

%100100

)()(%

_

xgidebisacrylamgacrylamide

gidebisacrylamC

linkcross

xmL

gidebisacrylamgacrylamideT

ionconcentratacrylamide

+=

=−

+=

=

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Controlling Pore Size

Concentrates bandStacking gel: 3-4%

6,000-250,0005-20 gradient

6,000-50,00015

10,000-100,00012

15,000-150,00010

20,000-175,0008

30,000-200,0006

Protein MW range%T of resolving gel

Changing parameters:Effect of %T (most common)

• G. S. Makowskiand M. L. Ramsby in Creighton, Protein Structure: A Practical Approach 2/E, p. 15.

• Zig-zag shows position of albumin band.

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Changing parameters:Effect of %C (cross-linker)

• G. S. Makowskiand M. L. Ramsby in Creighton, Protein Structure: A Practical Approach 2/E, p. 18.

• Zig-zag shows position of albumin band.

Calibration curves

• Plot of MW vs. migration distance is hyperbolic, but plotting log MW vs. distance yields a linear working range.

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Laemmli discontinuous gel

• Stacking gel of large pore size and low pH (~6.8)• Resolving gel of desired pore size and higher pH

(8.8)• Kohlrausch boundary enables a high voltage

gradient between rapidly migrating chloride ions and slowly migrating glycine, focusing the sample band

• pKa of glycine = 2.4 and 9.8. At 8.8, it is more (-) as the amine becomes more deprotonated

Laemmli discontinuous gel:time course

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Sample considerations

• SDS– Denatures the proteins– Provides uniform charge density so that

migration is not due to intrinsic charge of protein but only due to size

– Approximately one SDS molecule to two residues, or 1.4 g SDS per g protein

• Proteins are heated to 100°C in excess SDS (usually 10% SDS buffer, 10% 2-mercaptoethanol or dithiothreitol

Buffer components

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Staining of gels with Coomassie Brilliant Blue

• Coomassie R250 is used for staining proteins in gels

• It forms noncovalentcomplexes that render the protein insoluble

• The dye binds more strongly to proteins than to the acrylamide gel

• Detection of down to 0.5 µg/cm2 of protein in a band

• Peak at 549 nm

Staining and destaining

• Methanol/acetic acid solution with the Coomassie R250 (0.1 % w/v) is used to stain in solvent that is 40% v/v methanol/ 10% v/v acetic acid in water solution. The same solvent without the dye is used to destain.

• Destaining reduces the background.• Prolonged destaining leads to removal of stain

from the protein bands.• The solution is reusable.

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Staining with silver• Staining with silver can detect as little as below 1

ng of protein per band• Silver nitrate in acidic solution is added• Preferential binding to proteins and nucleic acids

vs. the gel seems to occur• Reduction of silver ion to metallic silver by the

protein, and then by formaldehyde in alkaline conditions leads to deposition of silver.

• Prolonged reaction leads to darkening of the stain so timing is essential.

• Process seems to be similar to photography

Variation: 2-D electrophoresis• 1st dimension: Isoelectric focusing• 2nd dimension: SDS-PAGE

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Comparison: Coomassie vs. silver

• Wu et al. Malaria Journal 2006 5:67 doi:10.1186/1475-2875-5-67.http://www.malariajournal.com/content/5/1/67/figure/F1?highres=y 2D electrophoresis profile of Coomassie Blue (upper) and silver (lower) stained iRBC ghosts from 3D7 (left) and A4 (right). The first dimension was run on pH4-7 IEF strips followed by 12.5% SDS-PAGE. The two major changes in the protein profiles are ringed (see text for details). High quality tif files showing the original 2D gels are available for 3D7 iRBC ghosts stained with Coomassie Blue (Additional file 1) and Silver (Additional file 2), and for A4 iRBC ghosts stained with Coomassie Blue (Additional file 3) and silver (Additional file 4).

2-D PAGE at Ateneo

• Video clip

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Variation on SDS-PAGE:Western blotting

Summary for SDS-PAGE

• Separation of denatured proteins according to size

• Long molecules are trapped in the gel and travel a shorter distance

• SDS denatures and maintains charge:sizeratio

• A log MW vs. distance curve of size standards allows determination of approximate size of the sample

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Outline1. Overview: chromatography,

electrophoresis, and centrifugation2. Observing charge

1. Isoelectric focusing2. Ion-exchange chromatography

3. Observing size1. Denatured

1. SDS-PAGE2. Native (or denatured)

1. Gel filtration or size exclusion chromatography2. Centrifugation

Size exclusion or gel filtration chromatography

• This technique can be used for denatured or native samples, depending on the buffer conditions used.

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Size exclusion/ gel filtration chrom

• http://www.science.fau.edu/chemistry/Mari/biochemlab/manual.html, accessed 2007 Aug. 27.

Closeup of the bead

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Size exclusion• Hydrodynamic method: soft: depends on

hydration, dynamics• The separation medium should be inert, and

conditions should maintain inertness. Usually, this means some ionic strength that would prevent interactions.

)(log))((

10 practicalMWBAVtheoryVVKVV

elution

voidtotalavvoidelution

−=−+=

Where A and B are constants based on properties of the column, within the fractionation range of the column.

Denaturing conditions• 8M Urea or 6M guanidine in the buffer system, plus disulfide

bonds broken by DTT or beta-mercaptoethanol• Elution volume related to effective hydrodynamic volume and

viscosity

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Outline1. Overview: chromatography,

electrophoresis, and centrifugation2. Observing charge

1. Isoelectric focusing2. Ion-exchange chromatography

3. Observing size1. Denatured

1. SDS-PAGE2. Native (or denatured)

1. Gel filtration or size exclusion chromatography2. Centrifugation

Measuring size

Folded, native proteins

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Folded globular proteins

Experimental considerations

• Packing of the support• Band broadening, which limits the amount

of sample that can be loaded onto the column to get good resolution

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What if the protein is not monomeric?

• Denatured and SDS-PAGE information can give monomeric state

• Native state size exclusion chromatography gives native information, which can lead to a conclusion that the protein is a dimer, trimer, tetramer, etc.

Application of SEC:desalting column

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Centrifugation- General

Density gradient centrifugation

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Centrifugation basics

• Relative centrifugal force (RCF) can be calculated from the centrifugal radius (r) in cm and the rotational speed (n) in rpm.

RCF = 1.118 x 10-5 *r *n2

• The rotational speed is determined asrpm = square root of [RCF/(1.118 x 10-5) *

r (cm)]

Sedimentation Velocity:The Svedberg Equation

• Mr is relative molecular mass (dimensionless)• S is the sedimentation coefficient• Subscripts 20,w refer to conditions of 20°C, in water, and

ρ (rho) is the density of the solvent• V is the partial specific volume (reciprocal of anhydrous

macromolecular density), typically ~0.73 mL/g for proteins, carbohydrate ~0.6 mL/g. Can be obtained based on amino acid data also or determined from density measurements.

)1

)((,20,20

0,20

0

ww

wr v

RTDsM

ρ−=

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Equilibrium sedimentation

• Requires long run times• Not a transport method, unlike

electrophoresis, sedimentation velocity, or gel filtration

Equilibrium sedimentation• Data analyzed as plot of Absorbance vs.

radius:• S.E. Harding in Creighton 2/E 1997, p. 237.

22 )1(2ln

ωρvRT

drAdM r −

×=

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Summary of centrifugation

• Useful as a preparative method for fractionating organelles, for example

• Analytical centrifugation requires hybrid centrifuge + optical detection systems

• Measurements can be based on velocity or on equilibrium position