User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/10/25

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Contents

Objective

  • Perform gel electrophoresis to determine if protein expression and purification was successful for group's 2 and 3.

Description

  • SDS-Page was used for this experiment with 12% discontinuous polyacrylamide gel.
  • Gel is composed of two parts:

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]


  • 1. Prepare Stock Solutions:

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



  • 2. Pour Discontinuous Gel:

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)



  • 3. Prepare SDS-Page Samples

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)



  • 4. Run the SDS-PAGE Electrophoresis:

A. Remove the comb

B. Load samples into wells

C. Run gel for 35 minutes at 200 Volts


  • 5. Stain Polyacrylamide Gel:

A. Coat gel with Coomassie Blue stain

B. Leave overnight in shaker

Data

Notes

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