Protein Isolation and Protein Isolation and QuantificationQuantification

ABE Workshop 2007ABE Workshop 2007

DNA

RNA

Protein

How to isolate total protein How to isolate total protein

Lyse the cell, Lyse the cell, Solubilize the proteins:Solubilize the proteins: To Solubilize To Solubilize membrane membrane

proteinprotein, we have to use , we have to use detergentsdetergents in the protein in the protein extraction bufferextraction buffer

The often used detergents in the protein The often used detergents in the protein extraction bufferextraction buffer

Nonionic detergentsNonionic detergents (milder)(milder) Triton X-100Triton X-100: break lipid-lipid interaction and : break lipid-lipid interaction and lipid-protein interactionlipid-protein interaction

Anionic detergents Anionic detergents (more denaturing)(more denaturing) SDSSDS: protein-protein interaction: protein-protein interaction Sodium DeoxycholateSodium Deoxycholate: protein-protein interaction: protein-protein interaction

Proteases inhibitorsProteases inhibitors Upon lysis of the cell, Upon lysis of the cell, proteasesproteases are released are released

into the lysateinto the lysate What are proteases?What are proteases? Where are the proteases from when isolating Where are the proteases from when isolating

the protein?the protein?

What are proteases?What are proteases?

Protease: Protease: (proteinases(proteinases, , peptidasespeptidases or or proteolytic enzymesproteolytic enzymes) are enzymes that ) are enzymes that breakbreak peptide bondspeptide bonds between amino acids of proteins between amino acids of proteins

Where are the proteases from when Where are the proteases from when isolating the protein?isolating the protein?

Animal cells: Lysosomes, contain a large variety of hydrolytic enzymes that degrade proteins and other substances

Plant cells: Vacuole, many hydrolytic enzymes found in vacuole resemble those present in Lysosomes of animal cells

other organelles also have proteases

How to prevent the proteins from How to prevent the proteins from degradation by protease?degradation by protease?

the protein isolation is carried out at the protein isolation is carried out at low low temperaturetemperature to minimize the activities of to minimize the activities of these proteasesthese proteases

To further optimize the results, we use the To further optimize the results, we use the proteases inhibitorsproteases inhibitors

Often used chemical protease inhibitors Often used chemical protease inhibitors in protein isolationin protein isolation

EDTA (or EGTA)EDTA (or EGTA): chelating the : chelating the Ca2+,Ca2+,

PMSFPMSF: a general serine protease : a general serine protease inhibitor. It is the most common inhibitor. It is the most common inhibitor used in protein inhibitor used in protein purification. Soluble in purification. Soluble in isopropanol. isopropanol.

The The protease inhibitorsprotease inhibitors cocktail cocktail: : a a mixturemixture of several protease of several protease inhibitors with inhibitors with broad specificitybroad specificity

The protein quantificationThe protein quantification

UV 280 absorption UV 280 absorption ::

Colorimetric methodsColorimetric methods::

BiuretBiuret

Lowry Lowry

BradfordBradford

UV absorption methodUV absorption method

The amino acids tryptophan, tyrosine and The amino acids tryptophan, tyrosine and phenylalanine absorb light in the phenylalanine absorb light in the UV wavelengthUV wavelength

Since the absorption is proportional to Since the absorption is proportional to concentration, this is a useful way to quantitates concentration, this is a useful way to quantitates protein concentration (for proteins containing Trp) protein concentration (for proteins containing Trp)

Disadvantages of UV absorption Disadvantages of UV absorption methodmethod

If some proteins do not contain these amino acids, If some proteins do not contain these amino acids, it will not absorb UV light, it will not absorb UV light,

Nucleic acids (DNA, RNA) contaminant will also Nucleic acids (DNA, RNA) contaminant will also absorb UV light, absorb UV light,

Colorimetric methodsColorimetric methods we can we can modify modify the protein sample with appropriate the protein sample with appropriate

reagentsreagents so as to produce a so as to produce a color reactioncolor reaction and and measure protein concentration using a measure protein concentration using a spectrophotometer. spectrophotometer.

Advantages of Colorimetric methodsAdvantages of Colorimetric methods

1. Cheap cuvette! (cheap glass or plastic versus 1. Cheap cuvette! (cheap glass or plastic versus quartz quartz) quartz quartz)

2. Not contaminating absorbance from nucleic 2. Not contaminating absorbance from nucleic acids! acids!

Colorimetric methods I: Bradford MethodColorimetric methods I: Bradford Method

A dye known as A dye known as Coomassie Brilliant BlueCoomassie Brilliant Blue was was developed by the textile industry. It was developed by the textile industry. It was noticed to stain skin as well as the textiles. noticed to stain skin as well as the textiles.

This dye (which normally absorbs at This dye (which normally absorbs at 465nm465nm) ) binds to proteins and to absorb strongly at binds to proteins and to absorb strongly at 595nm595nm. .

The assay is sensitive, but somewhat The assay is sensitive, but somewhat non-non-linear linear

A widely-used method of measuring protein concentration

A colorimetric assayAmount of blue color

proportional to amount of protein

Absorbance read using 500-750nm light

Lowry et al, 1951

Lowry MethodLowry Method

Lowry MethodLowry Method

Two reactions make the blue color develop:

Reaction 1

Cu2+ + peptide bonds → Cu1+-peptide bond complex, produces purple-blue color

Reaction 2

Folin reagent + Cu1+-complex → reduced Folin reagent, produces blue-green

Making a standard curve Making a standard curve

with BSA (bovine serum albumin)with BSA (bovine serum albumin)

A graph that correlates Absorbance with protein concentrationStandard Curve generated by doing a Lowry Assay on protein solutions of known concentrationStandard Curve must be done each time unknowns are being tested

The SDS-PAGEThe SDS-PAGE

PAGEPAGE

Gels are cast by polymerizing a solution of Gels are cast by polymerizing a solution of acrylamide acrylamide monomersmonomers into into polyacrylamide chainspolyacrylamide chains

Gel pore sizeGel pore size can be varied by adjusting the concentrations can be varied by adjusting the concentrations of polyacrylamide of polyacrylamide

Smaller proteins migrate faster than larger proteins through Smaller proteins migrate faster than larger proteins through the gelthe gel

Native proteinsNative proteins

SDS (sodium dodecyl sulfate) binds to and SDS (sodium dodecyl sulfate) binds to and coat the proteincoat the protein

SDSSDS

1. SDS disrupts some of the noncovalent 1. SDS disrupts some of the noncovalent interactions that stabilize protein interactions that stabilize protein quaternary and tertiary structures, quaternary and tertiary structures, facilitates denaturation.  facilitates denaturation.  2. SDS also has a negative electrical 2. SDS also has a negative electrical charge and binds to proteins in a constant charge and binds to proteins in a constant mass ratiomass ratio of 1.4 : 1, so that the total of 1.4 : 1, so that the total amount of detergent bound is directly amount of detergent bound is directly proportional to the molecular weight of proportional to the molecular weight of the protein.  the protein.  3. The ‘coating’ of negatively charged 3. The ‘coating’ of negatively charged SDS overwhelms the inherent charges of SDS overwhelms the inherent charges of protein molecules and gives them a protein molecules and gives them a uniform charge to mass ratio.  uniform charge to mass ratio.  4. This allows proteins to be separated on 4. This allows proteins to be separated on the basis of their relative sizes, the basis of their relative sizes,

SDSSDS

all polypeptide chains are then forced into all polypeptide chains are then forced into extended extended conformations conformations

SDS treatment SDS treatment eliminates the effect of differences in eliminates the effect of differences in shapeshape

individual polypeptide chains migrate as a negatively individual polypeptide chains migrate as a negatively charged charged SDS-protein complexSDS-protein complex through the porous through the porous polyacrylamide gel polyacrylamide gel

speed of migration is proportional to the size of the proteins speed of migration is proportional to the size of the proteins smaller polypeptides running faster than larger polypeptidessmaller polypeptides running faster than larger polypeptides

How about covalent link?How about covalent link?

SH

HS

S-S

DTT/Me

NoncovalentNoncovalent

covalentcovalent

Heating the sampleHeating the sample

Heating your samples at 99ºC completed Heating your samples at 99ºC completed denaturationdenaturation of of the protein molecules, ensuring that they were in completely the protein molecules, ensuring that they were in completely linear form. linear form.  This allowed SDS to bind all regions of each protein This allowed SDS to bind all regions of each protein equally.equally.

Protein loading bufferProtein loading buffer

Protein gel Protein gel loadingloading bufferbuffer contains Tris contains Tris bufferbuffer to to maintain constant pHmaintain constant pH

glycerol to increase sample density, glycerol to increase sample density, the strong ionic detergent the strong ionic detergent SDSSDS (sodium (sodium

dodecylsulfate), dodecylsulfate), β-mercaptoethanolβ-mercaptoethanol, a reducing agent. .  Beta-, a reducing agent. .  Beta-

mercaptoethanol eliminates mercaptoethanol eliminates disulfide bondsdisulfide bonds in in proteins by proteins by reducingreducing them (adding hydrogen them (adding hydrogen atoms).   atoms).   

HeatingHeating

Running the gelRunning the gel

Stacking gelStacking gel

To obtain optimal resolution of proteins, a “stacking” gel is To obtain optimal resolution of proteins, a “stacking” gel is poured over the top of the “resolving” gel. poured over the top of the “resolving” gel.

The stacking gel The stacking gel

lower concentration of acrylamide (larger pore size), lower concentration of acrylamide (larger pore size),

lower pH lower pH

different ionic contentdifferent ionic content This allows the proteins in a lane to be concentrated into a tight This allows the proteins in a lane to be concentrated into a tight

band before entering the band before entering the running or resolving gelrunning or resolving gel produces a gel with tighter or better separated protein bandsproduces a gel with tighter or better separated protein bands

Gel stainingGel staining Once proteins have been fractionated by electrophoresis, to Once proteins have been fractionated by electrophoresis, to

make them visible, make them visible, stainingstaining with a material that will bind with a material that will bind to to proteinsproteins but but not polyacrylamide.  not polyacrylamide. 

the most common one: staining with the most common one: staining with Coomassie BlueCoomassie Blue.  .  This is a dye that binds most proteins uniformly based on This is a dye that binds most proteins uniformly based on

interactions with the carbon-nitrogen backbone.  interactions with the carbon-nitrogen backbone.  The dye is dissolved in a solution that contains both The dye is dissolved in a solution that contains both

methanolmethanol and and acetic acidacetic acid

gel-drying framesgel-drying framesfor drying of SDS-PAGE gelsfor drying of SDS-PAGE gels

Gel dryingGel drying SDS-PAGE gels between two SDS-PAGE gels between two moistenedmoistened sheets of sheets of

Gel Drying Film (from Promega) on the bench. Gel Drying Film (from Promega) on the bench. Clamp the Gel Drying FrameClamp the Gel Drying Frame Dry over nightDry over night It is important to remove all the air bubbles from It is important to remove all the air bubbles from

between the two sheets of gel drying films. Air between the two sheets of gel drying films. Air bubbles may cause the gel to crack during dryingbubbles may cause the gel to crack during drying

ReferencesReferences http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.htmlhttp://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. J. Biol.

Chem.Chem.193, 265–275193, 265–275 www.bio-itworld.comwww.bio-itworld.com/ archive/091103/russell.html/ archive/091103/russell.html http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmohttp://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmo

cz/gfp.htmcz/gfp.htm

TransferTransfer

In this procedure, a sandwich of In this procedure, a sandwich of gelgel and solid support and solid support membranemembrane (Nitrocellulose or PVDF) is (Nitrocellulose or PVDF) is compressed in a cassette and compressed in a cassette and immersed in buffer between two immersed in buffer between two parallel electrodes. parallel electrodes.

A current is passed at right A current is passed at right angles to the gel, which causes angles to the gel, which causes the separated proteins to the separated proteins to electrophorese out of the gel and electrophorese out of the gel and onto the solid support membraneonto the solid support membrane

Transfer the protein from the gel to the Transfer the protein from the gel to the membranemembrane

Transfer of the proteins fractionated by Transfer of the proteins fractionated by SDS-PAGE to a solid support membrane SDS-PAGE to a solid support membrane (Western blotting) can be accomplished (Western blotting) can be accomplished by by electroblottingelectroblotting