Protein Electrophoresis BIT 230. Electrophoresis Separate proteins based on Size (Molecular Weight - MW) SDS PAGE Isoelectric Point Isoelectric focusing.

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Presentation transcript:

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 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 2 o antibody (this antibody is linked to an enzyme) Substrate is added Color change occurs