Team:UCLA/Notebook/Recombinant Expression/29 July 2015
BCA Assay for Protein Concentrations of the Tamura Batches
Fractions 1-Strip for the dual Tamura purifications were acetone precipitated and BCA Assay was conducted to determine concentration of samples before concentration. Fresh BSA standards are additional precipitated for assaying purposes.
- Protocol for BCA Protein Assay
- 1. Pipette 100 μl of each protein standard (including a blank) and sample into 1.5 ml microcentrifuge tubes in triplicate.
- 2. Add 400 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.
- 6. Add 100 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.
21 samples - 21 mL of working solution. 25 mL of Reagent A and 0.5 mL of Reagent B. Mix to combine. Use immediately.