Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/13 July 2015"

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Agenda

• SDS-PAGE Verification of Fractions
  • Verify purity and presence of protein at correct MW on SDS.
    • If pure, proceed to BCA pooling.
    • If impure, proceed to BCA pooling only with the most purified fractions.

• BCA pooling
  • Pool the most pure fractions together in a 50 mL conical to concentrate the amount of proteins in solution possible for the Co-Spinning Procedure.
    • Use the Protein Concentrators purchased to centrifuge and concentrate the solutions completely.

• Imidazole removal via dialysis of pooled fractions
  • Imidazole interferes at the 280nm necessary for excitation during the BCA colorimetric assay, protocol used here [1]
  • Save 4 mL of the pooled fraction for dialysis using dialysis cassettes following the Teule (Nature 2009) protocol here [http://www.ncbi.nlm.nih.gov/pubmed/19229199].
    • The following is the protocol (using tubing -- will be adjusted for use in cassettes):

Take a piece of prepared dialysis tubing and wash it under running deionized water. Clip one end of the tubing to make a bag. Transfer 4 ml of the eluted fractions into the dialysis bag (fractions 4–7 from Steps 51 and 52) and close the bag with another clip. Place the closed bag in a large beaker filled with deionized water (4 liters) over a stirring plate to dialyze the samples at room temperature. Change the dialysis buffer (water) at least 10 times, at 2 h intervals over a period of 24 h. The dialysis is important to remove the components of the elution buffer. For freeze-drying58, dialyze the sample extensively against 5 mM ammonium bicarbonate.

• BCA Assay
  • Conduct BCA Assay using the protocol and reagents found here [2].
    • It is necessary to pool samples and dialyze samples to remove imidazole for this assay to work. We will try this assay with the imidazole concentration still intact, using the elution buffer as a blank int he spectrophotometer, but this may not work well.
    • either way, we will try this before our advisor meeting at 1600 hours.

• Logistics
  • We will attempt to conduct all of this in Boelter, to reduce strain on Boyer.
  • In Boyer: Image SDS-PAGE, take all fractions, BCA reagent kit, protein concentrators, prepared albumin standards, and dialysis cassettes to Boelter.
  • In Boelter: Dialyze solution is imidazole. Conduct the BCA Assay. Record and upload all notes for today underneath in this wiki no later than 1500 hours for the advisor meeting.

Results