User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/04/10

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Objective

Remove the His-tag and enterokinase-tag from GFP. Prepare MBP-intein for synethesis with gold nanoparticles.

Description

MBP-intein Dialysis

  1. Dilute 1M Tris, pH 6.8, to 300mL 50mM Tris, pH 6.8 (15mL 1M Tris, pH 6.8 + H2O).
  2. Combine fractions 17-19 from the affinity chromatography MBP-intein purification on 4/3/12.
  3. Hydrate the membrane of the Slide-A-Lyzer® Dialysis Cassette by submerging it in the 300mL of 50mM Tris, pH 6.8.
  4. Remove the cassette from the buffer and take 3mL of the protein and inject into a Slide-A-Lyzer® Dialysis Cassette.
  5. Suspend the cassette with protein in the 300mL of 50mM Tris, pH 6.8.
  6. Place at 4°C overnight.

GFP Digestion of Tags

  1. Dialyze the previously purified GFP in a 500 mM Tris-HCl, pH 8.0, 10 mM CaCl2 buffer for approximately 1.5 hours.
  2. Mix 0.8mL of the dialyzed protein with 0.1mL of EKMax™ Buffer, 1μL of EKMax™, and .099mL H2O.
    • See [[1]] for more info on this enzyme.
    • Make three of these reaction mixtures.
  3. Place these mixtures on a heat block at 37°C overnight.

Notes

When we went to remove the dialysis tubing with GFP from the buffer, we noticed that all of the GFP had leaked from the tubing and was now dispersed throughout the buffer. We will need to try to find a way to extract it tomorrow. The digestion of the tags was not completed.



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