Run a protein gel to confirm the MBP-intein eluted off of the column, and in which fractions, and to check its purity.
- 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)
- 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.
- Remove the methanol with chromatography paper.
- 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
- 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.
The gel never polymerized. I will have to make another gel later on.