SDS-PAGE + Western Blot Laboratory What is Electrophoresis? • Electrophoresis (to carry with electricity) is the migration of charged molecules in an electric field toward the electrode with the opposite charge • SDS-PAGE (Sodium dodecyl sulfate – polyacrylamide gel electrophoresis) is a form of electrophoresis that treats samples with SDS to denature proteins Why is SDS-PAGE used? • How many proteins are in my sample? • What are the molecular weights of the proteins? • What differences are there in proteins from different sources? • How pure is my protein of interest? Why are proteins separated on SDSPAGE? • Small size of proteins • Gel matrix of polyacrylamide is tighter than agarose – resolve smaller molecules • Polyacrylamide – small DNA • Agarose – large protein - Agarose gels will never resolve as tightly as polyacrylamide gels Two phases of polyacrylamide. • Upper stacking gel = 4% acrylamide • Lower resolving gel = 15% • Discontinuous system – results in all proteins separating or resolving at same time • Stacking gel – allow proteins to migrate rapidly and be compressed at edge of denser resolving gel • Separating gel – begin to separate as per their molecular weights How are protein bands formed? • Two ion fronts sandwich protein bands • SDS-PAGE running buffer – Tris and glycine (pH 8.3) • Gel – Tris-HCl buffer (pH 8.8) • Chloride ions migrate rapidly than glycine ions in an electric field - When electrophoresis begins - proteins have intermediate mobility - trapped between two fronts - How does polymerization start? • Acrylamide and bis acrylamide monomers – initiators – Ammonium persulfate (APS) and catalyst – Tetramethylethylenediamine (TEMED) • Higher concentration of resolving gel is poured – lower concentration stacking gel is poured – sample comb is inserted into unpolymerized stacking gel solution Molecular weight of proteins • Proteins – composed of 20 aa – 89 to 204 daltons • Kilodalton (KD) – used for protein molecular masses – 10 KD and 220 KD Factors affecting mobility • Electrical charge + mass = mobility • SDS added in sample buffer and gel running buffer - Strong anionic (negatively charged) detergent - Binds and coats proteins – denatured in linear chains - Mass determines migration Denaturation of four structures of proteins • Primary, secondary, tertiary and quaternary • β-mercaptoethanol (BME) or dithiothreitol (DTT) – breaks disulfide bonds • Heat, reducing agent and ionic detergent – disrupt 2°, 3° and 4° results in linear chains of aa How to identify proteins in polyacrylamide gel? • Complex mixture of proteins - Appear as distinct blue- stained bands(stained gel) - Positions and intensities – determine size and abundance of proteins - Specific proteins – Western blots – positive identification by antibodies Sources of errors • Identical migration may be different proteins of same sizes • Proteins of similar composition, function and evolutionary origin may be different in molecular weight because of post translational modifications What are the different reagents that will be used? • Molecular weight markers – Precision plus protein kaleidoscope prestained protein standards • Proteins genetically engineered to be specific molecular weights then bound to dye molecules – visible on gel • Heated to 37 ° for 5 minutes • Load 5 µl per gel What are the different reagents that will be used? • Laemmli sample buffer – used to solubilize proteins in fish muscle samples - Mixture of Tris buffer + SDS + tracking dye (bromophenol blue) + Glycerol • Actin and Myosin standard – used to identify conserved muscle proteins – positive control – rabbit skeletal muscle consisting myofibrils - Actin, myosin heavy chain, three myosin light chains and tropomyosin- on destained gel Actin and myosin standard • Bands at 210 KD (myosin heavy) and 43 KD (actin) – present in all of muscle samples - Biologically active myosin has quaternary structure - both heavy and light chains - Myosin light chains (15-25 KD) vary in molecular weight between fish species What are the different reagents that will be used? • Dithiothreitol (DTT) or β-mercaptoethanol (BME) – added to Laemmli buffer to ensure complete breakage of disulfide bonds. - Reduces formation of background bands • Ready Gel precast gels – 15% Tris-HCl gels - Resolves smaller molecular weight proteins What are the different reagents that will be used? • Electrophoresis running (1X TGS) buffer – Tris-glycine-SDS (TGS) running buffer contains Tris to buffer the pH, glycine to provide ions to transmit current, and SDS to maintain denaturation of proteins in gel. • Bio-Safe Coomassie protein stain – High affinity between dye and proteins - Wash gel 3 times with DI H2O before staining What are the different reagents that will be used? • Bio-Safe Coomassie protein stain – Optimal staining time is 1 hour – gentle shaking - Destaining of gel – protein bands become intense - Rinse the stained gel with several changes of a large volume of deionized water - And complete destaining overnight in water What are the different materials needed for each workstation? • Fish samples, Blade/knife, water bath set to 95 degree, Prot/Elec pipet tips for gel loading, Mini-protean 3 electrophoresis module (gel box), Power supply, gel comb, staining trays, DI H2O, etc Steps • Protein extraction from various fish samples (March 18) – 25 minutes- March 18 • Run SDS-PAGE (March 25) – 30 minutes - Stain one gel with Coomassie blue (March 25 + 26) • Another gel – western blot (March 25)- 2.5 hrs • Immunodetection with antibodies(March 27) Western Blot reagents and equipment • Mini Trans-Blot Apparatus : Passes electric current horizontally through gel – forcing negatively charged proteins to migrate out of gel onto nitrocellulose membranes • Nitrocellulose membranes: Proteins bind to positive charge - Durable membrane can undergo multiple washing and incubation steps - White background to visualize color development of proteins Western Blot reagents and equipment • Blotting paper – supports gel and nitrocellulose - Protects filter pads during assembly and electrophoresis - Facilitates uniform flow of buffer and current through gel - 100% cotton fiber – does not contain any additives that may interfere with blotting process Western Blot reagents and equipment • Filter pads: Press the gel and nitrocellulose tightly and uniformly - Eliminate bubbles – allows transfer of proteins out of gel onto the membrane - Should be cleaned in distilled water before use Western Blot reagents and equipment • Blotting buffer: Should be diluted to 1X • Blocker: 5% nonfat dried milk powder in phosphate buffered saline (PBS) and 0.025% tween 20 • Surface area unoccupied by proteins- blocked by incubating milk solution – before incubation with primary antibody Western Blot reagents and equipment • Primary antibody: recognizes or bind protein under study - Injecting chicken myosin protein into mice • Secondary antibody: binds to primary antibody - Polyclonal goat anti-mouse antibody conjugated to horseradish peroxidase enzyme - Injecting goats with primary mouse antibodies • • • This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). 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