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Protein Electrophoresis
BIT 230
Electrophoresis
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Separate proteins based on
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Size (Molecular Weight - MW)
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Isoelectric Point
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SDS PAGE
Isoelectric focusing
Allows us to
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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 polyacrylamide
smaller pores than ???
Is DNA smaller or bigger than protein?
SDS-PAGE
Sodium 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 MW
no interference from 3D structure or charge
Smaller proteins move faster and further
Smaller the proteins being separated --Higher % of acrylamide
Reducing Agents
DDT dithiothreitol
BME B-mercaptoethanol
Break disulfide bonds
Completely disrupt the 3D structure of proteins
Miscellaneous Terms
Stacking Gel - minimizes effect of different volumes
Separating gel
Anode (-)
Cathode (+)
Combs
Plates
Spaces
Polymerization
Precast gels
Dye front 5mm from bottom
Resolution
Linear vs Gradient Gels
range of % polyacrylamide
used for samples with large range of MW
Two-Dimensional Gel Electrophoresis
Stage 1 Isoelectric Focusing
separate based on pI (isoelectric point)
pH where a protein is electrically neutral
sum 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)
Graph
Measure distance unknown protein travelled
Compare 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