Polyacrylamide Gel Electrophoresis. Polyacrylamide Gel Electrophoresis. Polyacrylamide Gels. Polyacrylamide Gels. Polyacrylamide Gels

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1 Polyacrylamide Gel Electrophoresis Jeff Prischmann Diagnostic Lab Manager North Dakota State Seed Department Polyacrylamide Gel Electrophoresis Troubleshooting Applications Polyacrylamide gels are formed by the polymerization of acrylamide with a crosslinker (usually N, N methylene bisacrylamide). Polymerization is initiated by the introduction of a catalyst. Catalysts Ammonium persulfate with TEMED (N,N,N,N tetramethylethylene diamine) FeSO 4 with 3 % hydrogen peroxide Photochemical using riboflavin/temed Components Acrylamide: 30-40% solution Bisacrylamide: 2% solution Ammonium persulfate (APS): 10% solution. TEMED Buffer Water Other Crosslinkers DATD (N,N -diallytartardiamide) BAC (N,N -bisacrylylcystamine) PDA (piperazine diacrylate) 1

2 Composition T=% total concentration (w/v) of monomer (acrylamide plus crosslinker) in the gel. C=% total monomer represented by the crosslinker. Pore size is controlled by changing T and C. Composition When T is increased, gel pore size decreases. Small gel pores are used to separate smaller molecules. When C is decreased, it results in a more open gel pore structure. Gel Pore Size Composition 5% C (19:1 acrylamide/bis) is generally accepted for denaturing DNA/RNA separation. 3.3% C (29:1 acrylamide/bis) is generally accepted for native DNA/RNA separation. 2.6% C (37.5:1 acrylamide/bis) is generally used for SDS-PAGE of proteins. 2

3 Steps in Gel Preparation Prepare the gel casting cassette (wash and clean glass, treat with hydrophobic reagent if necessary, and assemble). Prepare the gel Pour the gel into the gel casting cassette. Allow a minimum of 2 hours prior to using the gel. Gel Preparation-Acrylamide/bisacrylamide For gels with separate acrylamide and bisacrylamide, calculate amounts needed separately. For gels with acrylamide and bisacrylamide together in the same solution, calculate amounts needed as a single amount. Stock acrylamide solutions are either 30% or 40%. Stock bisacrylamide solutions are usually 2%. Gel Preparation-Acrylamide/bisacrylamide Need to know total gel volume, concentrations of bisacrylamide and acrylamide stock solutions, % bisacrylamide (crosslinker) desired, and % acrylamide desired. Gel Preparation Example: For DNA gels using 19:1 or 29:1 acrylamide to bisacrylamide ratios: V = (X) (V 1 )/40 V = volume of 40 % needed (ml) V 1 = total gel volume (ml) X = % gel desired Gel Preparation-other components Buffer Water 10% (w/v) Ammonium persulfate (1ml per 100ml gel) or other catalyst TEMED (0.1ml per 100ml gel) or other catalyst PPE is required when handling and preparing gels, stains, and etc. (gloves, lab coat, goggles, etc.) Acrylamide is a neurotoxin and needs to be handled with caution. Gel stains can be toxic (DNA stains). 3

4 Gel and stain waste needs to be disposed of properly. Separate solid and liquid waste. Acidic /basic waste needs to be neutralized (TCA). Resolving gel only Stacking gels/resolving gels Gradient gels (% acrylamide increases from top of gel to bottom) Gradient Gel Maker 2 main types of gels As component A empties, the solution leaving the apparatus becomes more like component B. Slab gels Tube gels Gel Casting Cassette Glass plates Gel spacers Clamps Sample combs Gel Casting Stand Glass plates can vary in size (10cm wide up to 50cm; length from 52cm down to 8cm). Gel spacers/combs can vary in thickness (1.0mm to 2.0mm). Sample combs vary in the number of wells and size of wells. 4

5 Pre-made polyacrylamide slab gels are available from a number of manufacturers in different sizes or concentrations. Running Gels Appropriate amount of sample is added to sample wells. Tracking dyes used to monitor progress. Constant voltage typically used during electrophoresis. 5

6 Source: Bio-Rad Main Steps Prepare samples Prepare gel and buffers Load samples onto gel Run gel Stain gel Interpret/analysis of gel Archive (photograph, dry gel) Denaturing PAGE (SDS-PAGE) Native PAGE of seed protein (wheat and oat). Native PAGE of nucleic acids (barley). 6

7 SDS-PAGE (Laemmli method) Most commonly used type of PAGE for protein separation. Denaturing, discontinuous system (stacking/resolving gels). Samples separated by size Sharp band separation SDS-PAGE SDS-PAGE uses a stacking gel and resolving gel combination. SDS-PAGE SDS-PAGE Proteins are stacked between chlorine ions and glycine. Proteins are separated in the separation/resolving gel. SDS Gel Electrophoresis Source: Abcam Native PAGE (wheat seed protein) ISTA standard reference acid PAGE method for the separation of gliadins. 10% acrylamide gels, ph 3.2 Gliadin proteins extracted from seed using 2-chloroethanol (single seed or bulk seed). Buffer components: Acetic Acid and glycine. 7

8 Native PAGE (wheat seed protein) Native PAGE-Wheat Methyl green used as a tracking dye in the sample. Gels run 5 hr at 500V, 20C. Gels stained with coomassie blue G Visualization in approx 2hr. Native PAGE-Wheat Native PAGE-Wheat Native PAGE (oat seed protein) ISTA standard reference acid PAGE method for the separation of gliadins. 10% acrylamide gels, ph 3.2 Avenin proteins extracted from oat seed using 2-chloroethanol (single seed or bulk seed). Native PAGE (oat seed protein) Methyl green used as a tracking dye in the sample. Gels run 2 hr at 500V, 20C. Gels stained with coomassie blue G Visualization in approx 2hr. 8

9 Native PAGE-Oat Native PAGE-Barley DNA Barley genomic DNA isolated from leaf/seed. Barley PCR products 6% acrylamide gels 1.5 hr at 275V Buffer: TBE Native PAGE-Barley DNA Native PAGE-Barley DNA Gels stained with SYBR gold or ethidium bromide. Visualize in minutes Native PAGE-Barley DNA Native PAGE-Barley DNA 9

10 Results Results Variety ID or genetic purity tests. Tests conducted with known standards. Most varieties have unique banding patterns or fingerprints. Gels photographed, archived, dried. Troubleshooting PAGE PAGE Advantages Smiling gel front (center of gel running faster than either end; heat problem, buffer problem). Vertical streaking of protein (sample overload; dilute samples, reduce voltage). Sample does not settle into well bottom (sample density problem). Dye front does not migrate (buffer problem, power supply problem). Skewed of distorted bands (poor polymerization around sample wells). Allows pore sizes to be altered easily. Superior resolution with small pore size (a 0.1% difference in size can be detected, i.e. 1 base in a 1 Kb DNA fragment) Virtually no batch to batch differences in acrylamide or bisacrylamide. References References Cooke, R.J Gel Electrophoresis for the Identification of Plant Varieties. J. Chromatography 698: C.W. Wrigley Identification of Food Grain Varieties. Chapter 4. Variety Identification by Electrophoretic Analysis. Cooke, R.J ISTA Handbook of Variety Testing. Sambrook, J Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis. Chapter 5 in Molecular Cloning: A Laboratory Manual. 3 rd Editon. National Diagnostics ( 10

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