Objectives
- Create two calibration curves for Adenosine Deaminase(ADA) using the Bradford assay technique.
Bradford Assay General Protocol
- The following protocol was described here. (*Note: use section 2.3, page 5).
- Prepared a stock solution that is roughly 10mg ADA in 2mL buffer using a 2mL volumetric flask(Note the actual concentration prepared).
- Prepare 6 standard solutions (1μg/mL, 3μg/mL, 5μg/mL, 7μg/mL, 9μg/mL, 10μg/mL) in 1mL (prepared these solutions in an eppendorf tube.)
- Add 200μL of the Bio-Rad Protein Assay reagent to the eppendorf tube.
- Add necessary amount of protein from previously prepared 10mg in 2mL stock solution.
- Add necessary amount of buffer to fill eppendorf tube to 1mL.
- Shake the eppendorf tube vigourously for ~3min.
- Prepare 1mL of a blank solution (800μL buffer and 200μL Bio-Rad Protein Assay reagent.
- Take UV-vis spectra after preparation (no more than an hour after they were prepared).
- Prepare a calibration curve.
- Repeat Steps 1-7.
- Note: All spectra were taken using a plastic cuvette.
- Note: The UV-vis spectra was taken with the blank automatically subtracted out.
Table 1. Preparation of ADA standard solutions
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