User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/03/27

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Objective

Identify which fractions from the purification on 3/21/12 have the MBP-intein protein present.

Description

  1. 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.
  2. 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)
  3. 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.
  4. Remove the methanol with chromatography paper.
  5. 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
  6. 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.
  7. Remove the comb and transfer the gel within the plates to the Electrode Assembly with the short plates facing inward.
  8. Fill the Electrophoresis chamber with Tris-glycine electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS)
  9. Load 10μL of pre-mixed SDS-PAGE broad range ladder.
  10. Load 16μL of fractions 25-45 (collected on 3/21/12 FPLC) mixed with 4μL of 5x loading buffer (pre-mixed) into the wells.
  11. Run the gel at 200V for about a half hour.
  12. 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).
  13. Let stain for ~4 hours.
  14. 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.

Data

  1. Photos taken of the protein gels the next day.




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