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 SDS-PAGE? 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 Proteins are usually separated using polyacrylamide gels rather than agarose gels, which are used to separate DNA. This is because most proteins are much smaller than DNA fragments and polyacrylamide gels have pore sizes similar to sizes of proteins. The gel matrix formed by polyacrylamide is much tighter than agarose and resolves much smaller molecules. These gels are sometimes used to separate very small DNA fragments while agarose gels is used to separate very large proteins. But agarose gels will never resolve bands 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 Stacking gel – allow proteins to migrate rapidly and be compressed at edge of denser resolving gel – regardless of sizes. The samples of mixed proteins are thus concentrated into uniformly thin bands in each lane, before they move into denser resolving gel and begin to be separated according to 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 Comb is removed after polymerization is complete to create wells for sample loading. Although powdered or liquid unpolymerized acrylamide monomers are neurotoxins the precast readygels are already polymerized and safe to be used.

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 Main variable affecting migration rate of each protein are posttranslational modifications.

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 Mercaptoethanol and dithiothreitol are reducing agents

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 Stored at -20 degree, thawed at room temperature and heated to 37 degree for 5 minutes before use to dissolve any precipitated SDS.

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 Anionic detergent SDS, electrophoresis tracking dye and glycerol (increases density of samples so that they sink into wells as loaded)

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 Myosin light chains can be observed in results of western blot

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 White nitrocellulose membrane is packaged between two protective sheets of blue paper.

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 PBS provides the ideal pH and salt conditions for maintaining milk protein binding integrity. Tween 20 is a detergent that helps keep nonspecifically bound antibody to the membrane

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 Secondary goat-anti-mouse antibodies are purified from goat serum and chemically linked or conjugated to HRP. HRP enzyme catalyzes oxidation of colorimetric substrate in order to permit visualization of protein of interest

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). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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