Protein Electrophoresis

BIT 230

Electrophoresis Separate proteins based on

Size (Molecular Weight - MW) SDS PAGE

Isoelectric Point Isoelectric focusing

Allows us to characterize (degradation, MW) quantify determine purity of sample compare proteins from different sources step in Western blot

How does it work?

Proteins are Charged. HOW?

Add protein sample to negative side of polyacrylamide

Add electric field

Proteins migrate through pores of polyacrylamidesmaller pores than ???Is DNA smaller or bigger than protein?

SDS-PAGESodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis

developed by Laemmli (1970)Denatured Gel

SDS is a negatively charge detergent which DENATURES the protein (what does this mean?)and gives all proteins a uniform charge SHOW FIGURE 6

This gel separates based on MWno interference from 3D structure or charge

Smaller proteins move faster and further

Smaller the proteins being separated --Higher % of acrylamide

Reducing AgentsDDT dithiothreitol

BME B-mercaptoethanol

Break disulfide bondsCompletely disrupt the 3D structure of proteins

Miscellaneous TermsStacking Gel - minimizes effect of different volumesSeparating gelAnode (-)Cathode (+)CombsPlatesSpacesPolymerizationPrecast gelsDye front 5mm from bottomResolution

Linear vs Gradient Gelsrange of % polyacrylamideused for samples with large range of MW

Two-Dimensional Gel ElectrophoresisStage 1 Isoelectric Focusing

separate based on pI (isoelectric point)

pH where a protein is electrically neutralsum of + and - are equal

Stage 2 SDS-PAGE

How to Detect Proteins?Coomassie Blue (0.1 ug)

Silver Staining (2 ng)

How to Quantify Proteins

•Desitometry

Molecular Weight Determination

Method 1:Amino Acids approx 110 daltons# residues x 110 dalton/residue = MW

Method 2:Run SDS PAGE with known standards (MW markers)GraphMeasure distance unknown protein travelledCompare on standard curve

Non-denatured Gels

Also called native

Based on size and charge

Greater the charge the greater the mobility

typical pH 8.8 (most proteins negative at this pH)

Immunoblots (Westerns)

•Run proteins on denatured gel (SDS-PAGE)

•Transfer (blot) proteins onto membrane (nylon , nitrocellulose)

•Probe the membrane with 1o antibody (recognizes your protein)

•Add 2o antibody (this antibody is linked to an enzyme)

•Substrate is added

•Color change occurs