Identify which fractions from the purification on 3/21/12 have the MBP-intein protein present.
- Do a "Bradford-by-eye" to visualize protein in the fractions collected on 3/21/12. (20μL Bio-Rad Protein Assay + 20μL of fraction + 60μL distilled water
- After testing about half of the fractions, it looked like most had protein, so I decided it was unnecessary to continue based on the repetitive procedure and the graph produced by the FPLC on 3/21/12.
- 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.
- Remove the comb and transfer the gel within the plates to the Electrode Assembly with the short plates facing inward.
- Fill the Electrophoresis chamber with Tris-glycine electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS)
- Load 10μL of pre-mixed SDS-PAGE broad range ladder.
- Load 16μL of fractions 25-45 (collected on 3/21/12 FPLC) mixed with 4μL of 5x loading buffer (pre-mixed) into the wells.
- Run the gel at 200V for about a half hour.
- Remove the gel from in-between the glass plates and immerse it in the staining solution (0.25% Coomassie Brilliant Blue R250 (w/v) in 40% (v/v) methanol and 10% (v/v) glacial acetic acid).
- Let stain for ~4 hours.
- Remove from stain, rinse with destain solution (50% methanol, 10% acetic acid). Immerse the gels in destain solution, microwave for 30 seconds, and let destain overnight.
- Photos taken of the protein gels the next day.