User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/04/04

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Objective

Run a protein gel to confirm the MBP-intein eluted off of the column, and in which fractions, and to check its purity.

Description

  1. Make the resolving gel:
    • 3.3mL H2O
    • 4.0mL 30% acrylamide mix
    • 2.5mL 1.5M Tris (pH 8.8)
    • 0.1mL 10% SDS
    • 0.1mL 10% ammonium persulfate (add this right before pouring the gel)
    • 0.004mL TEMED (add this right before pouring the gel)
  2. Pour the gel between the short plate and spacer plate (after they have been secured in the casting frame on the casting stand). Squirt a layer of methanol on top of the resolving gel for a straight, level layer of gel.
  3. Remove the methanol with chromatography paper.
  4. Make the stacking gel:
    • 3.4mL H2O
    • 0.83mL 30% acrylamide mix
    • 0.63mL 1.0M Tris (pH 6.8)
    • 0.05mL 10% SDS
    • 0.05mL 10% ammonium persulfate
    • 0.005mL TEMED
  5. Pour this layer of gel on top of the resolving layer between the two glass plates. Put in a 15-well comb. Wait until it is polymerized.


Notes

The gel never polymerized. I will have to make another gel later on.



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