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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