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

• SDS-PAGE

• • Gel image indicates that there is a high degree of purity in the resulting fractionation of the cell lysate using the IMAC batch pull down method.
  • Gel lanes are in order from left to right: Ladder, Negative Control (laemmli sample buffer), Strip Fraction (400 mM EDTA nickel strip), 250 mM Imidazole elution, 100 mM Imidazole Elution, 50 mM Imidazole Wash, 40 mM Imidazole Wash, Unbound Lysate, Crude Lysate from Enzymatic Lysis, and a positive control (BSA - 750 ug/mL).
  • Gel indicates increasing purity of a protein sample at approximately 51 kDA as the washes and elutions are conducted, at the expense of critically lowering yield (this is expected using the quicker batch method, where the proteins are not uniformly collected)
  • Results indicate that fractions 5, 4, and 3 ought to be pooled together for concentration verification using BCA and Pierce Concentrator.

• BCA pooling:
  • 6 mL of Fractions 3 (40mM Imidazole) ,4 (100 mM), and 5 (250 mM) each were pooled into a 50mL conical for a final total of 16mL.
  • Pooled fractions were collected into a Pierce 20 kDA MWCO, and centrifuged for 30 minutes at 3,000x g at 22 degrees Celsius. 2mL of concentrated samples remained in the filter, and were resuspended and collected into micro centrifuge tubes.
  • Some of the protein aggregated in the tip of the filter. Samples were resuspended, but the protein may precipitate again. Most likely the protein needs to be resolubilized in a more stable buffer system, but for the purposes of co-spinning this is not critically necessary.

• Imidazole Dialysis
  • Suspended due to alternative scenario to remove contaminating substances using acetone precipitation; protocol provided by Thermo Scientific here: [[3]]

• BCA Assay:
  • Conducted post acetone precipitation (above). 9 BSA standard samples, 4 samples (fractions 3,4,5, and concentrated pool), and single replicates were only conducted today.

Protocol for BCA Protein Assay 1. Pipette 50 μl of each protein standard (including a blank) and sample into 1.5 ml microcentrifuge tubes in triplicate. 2. Add 200 μl of cold (-20°C) acetone to each tube. 3. Vortex and incubate 30 minutes at -20°C. 4. Centrifuge 10 minutes at maximum speed in a microcentrifuge. 5. Pour off the supernatants and allow the acetone to evaporate from the tubes at room temperature (RT) for 30 minutes. Note: If two precipitations are necessary to remove the interfering substance, repeat Steps 2-5 before proceeding. Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 2 6. Add 50 μl of ultrapure water to the protein pellets and vortex. 7. Add 1 ml of BCA Working Solution (50 parts Reagent A and 1 part Reagent B) to each tube and vortex. Incubate 30 minutes at 37°C. 8. Cool samples to RT and measure absorbance at 562 nm. 9. Plot a standard curve based on absorbance of the protein standards and determine the protein concentration of each unknown sample according to the BCA Protein Assay Kit instructions.