User:Elizabeth Ghias/Notebook/Experimental Biological Chemistry/2011/10/25

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Objective

To run a protein gel (SDS-PAGE) to determine if the MBP-intein fusion has been expressed and purified.

Description

  1. The stacking gel was prepared by miing the following in an Erlenmeyer flask: 17 mL of H2O, 4.15 mL of 30% arylamide, 3.15 mL of 1.0M Tris (pH 6.8), 0.25 mL of 10% SDS.
  2. Once the pre-made resolving gel solution had set (after 20 minutes), 0.25 mL of 10% ammonium persulfate and 0.025 mL of TEMED were added to the stacking gel solution.
  3. The stacking gel solution was the poured on top of the resolving gel and allowed to set for 20 minutes.
  4. The four samples to be loaded into the the protein gel were prepared as shown in the box below.
  5. Tube 1 was loaded into lane 1, tube 2 into lane 2, tube 3 into lane 3, and tube 4 into lane 4.
  6. Electrophoresis was run for 40 minutes.
     Tube 1 - 10 μL of ladder mixed with 4 μL of orange dye
     Tube 2 - 16 μL of protein from Peak 1 with 4 μL of orange dye
     Tube 3 - 16 μL of protein from Peak 2 with 4 μL of orange dye
     Tube 4 - 16 μL of protein from Peak 3 with 4 μL of orange dye

Data

Image:Group2.JPG

Image:AllGels.JPG

Notes

  • Protein was present, but the sample was impure. Purification was not successful.

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