User:Nader A. Khamis/Notebook/Experimental Biological Chemistry AU Fall 2011/2011/10/25

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Objective

  • Perform gel electrophoresis on MBP fusion protein to determine if group 2 and group 3's purification and protein expression were successful.

Description

  • In this experiment, SDS-Page was used with 12% discontinuous polyacrylamide gel.
  • The Gel is composed of the Stacking Gel and the Resolving Gel.

The Stacking Gel, which has a lower concentration of acrylamide and pH compared to resolving gel, concentrates proteins into tight more easily visible bands.

The Resolving Gel seperates proteins according to their molecular weight.

The following manual was used to carry out the experimental procedure: [1]


1. Preparation of Stock Solutions:

A. 30% Acrylamide Mix, 10% SDS , and 1ml of 10 % Ammonium Persulfate (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. Approximately 0.1 mL APS and 0.004 mL TEMED were pipetted to a 10 mL resolving gel solution

B. The gel plates was set up.

C. The gel comb was placed into plates and a mark was made 1 cm below the teeth using a permanent marker

D. The resolving gel solution was poured into plates until the mark.

E. Ethanol was added above the resolving gel solution until the top.

F. Allow gel to polymerize (20 minutes)

G. Excess Ethanol was removed.

H. Approximately 0.05 mL APS and 0.005 mL TEMED were pipetted to 5 mL stacking gel solution

I. The gel solution was poured into plates until it was full

J. The comb was inserted into the solution and gel was left for 20 minutes to polymerize.


3.Preparation of samples


  • In Tube 1, approximately 10µl of ladder (blue) and 4µl of loading buffer (orange) were pipetted into it.
  • In Tube 2, approximately 16µl of protein 1 and 4µl of loading buffer (orange) were pipetted into it.
  • In Tube 3,approximately 16µl of protein 2 and 4µl of loading buffer (orange) were pipetted into it.
  • In Tube 4, approximately 16µl of protein 3 and 4µl of loading buffer (orange) were pipetted into it.


4. Following the preparation of samples, SDS - Gel Electrophoresis was carried out.


  • The comb was removed, the samples were loaded into the wells, and the gel was run at 200V for a period of 35 minutes.
  • Following Gel Electrophoresis, the gel was stained with Coomassie blue and was left on the shaker overnight

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


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