Objectives
- Create two calibration curves for Adenosine Deaminase(ADA) using the Bradford assay technique.
- Prepare 5 standard gold solutions for AA testing
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 from 10mg/2mL stock solution
Sample Number
|
Concentration (μg/mL)
|
ADA (mL)
|
Buffer (mL)
|
Coomassive Blue(μL)
|
1 |
10 |
0.0056 |
0.794 |
200
|
2 |
9 |
0.005 |
0.795 |
200
|
3 |
7 |
0.0039 |
0.7961 |
200
|
4 |
5 |
0.0028 |
0.7972 |
200
|
5 |
3 |
0.0017 |
0.7983 |
200
|
- Note: 1μg/mL was too small of a volume to prepare from the 10mg/mL stock. Another stock solution of 20μg/μL was prepared(with buffer and ADA from the stock solution) and a dilution to 1μg/μL was performed from the 20μg/μL solution.
Table 2. Preparation of 20μg/μL ADA standard solution
ADA (mL)
|
Buffer(mL)
|
0.0111 |
0.9889
|
Table 3. Preparation of 1μg/μL ADA standard solution from 20μg/μL ADA standard solution
Sample number
|
Concentration(μg/μL)
|
ADA (mL)
|
Buffer(mL)
|
Coomassive Blue(μL)
|
6 |
1 |
0.05 |
0.75 |
200
|
|