User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/10/25
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Objective
Description
1. Stacking Gel: Lower concentration of acrylamide, and pH compared to resolving gel. Concentrates proteins into tight more easily visible bands. 2. Resolving Gel: Allows for the separation of proteins, according to their molecular weight. Protocol was taken from the following manual: [1]
A. 30% Acrylamide Mix: 10% SDS , 10 % Ammonium Persulfate (APS) (1 mL): 100 mg solid APS with 1 mL H2O B. Resolving Gel Solution (10 mL) : 3.3 mL H2O, 4.0 mL 30% Acrylamide mix, 2.5 mL Tris buffer (1.5M pH 8.8), 0.1 mL 10% SDS C. Stacking Gel Solution (5 mL) : 3.4 mL H2O, 0.83 mL 30% Acrylamide mix, 0.63 mL Tris buffer (1.0M pH 6.8), 0.05 mL 10% SDS
A. Add 0.1 mL APS and 0.004 mL TEMED to 10 mL resolving gel solution B. Prepare gel plates C. Place gel comb into plates and mark 1 cm below teeth with permanent marker D. Pour resolving gel into plates until the mark E. Add ethanol to evenly level gel in plate F. Allow gel to polymerize (20 minutes) G. Remove excess ethanol H. Add 0.05 mL APS and 0.005 mL TEMED to 5 mL stacking gel solution I. Pour gel solution into plates until full J. Insert comb and let gel polymerize (20 minutes)
A. Tube 1: 10 μL ladder (blue) , 4 μL loading buffer (orange) B. Tube 2: 16 μL protein 1 , 4 μL loading buffer (orange) C. Tube 3: 16 μL protein 2 , 4 μL loading buffer (orange) D. Tube 4: 16 μL protein 3 , 4 μL loading buffer (orange)
A. Remove the comb B. Load samples into wells C. Run gel for 35 minutes at 200 Volts
A. Coat gel with Coomassie Blue stain B. Leave overnight in shaker DataNotes |