Matt Gethers/CRI, Thailand/Labwork/Bradford Assay
- Get the Bradford staining solution from the fridge in the adjacent bay
- Get the appropriate number of cuvettes for samples, blanks, and standards.
- Use the pump on the staining solution to aliquot 1 ml to each cuvette. Save two cuvettes for the blanks.
- If generating a standard, reserve four cuvettes for dilutions of BSA. The BSA is at 10 mg/ml, so make a 1/10 dilution into water, then aliquot various amounts into the four different cuvettes. A good set is 1, 2, 4, and 8 μl of the 1/10 dilution (1, 2, 4, and 8 μg respectively). This should be within the linear range of the assay.
- Use a piece of parafilm over the top of the cuvette to mix.
- When adding sample to the 1 ml aliquot of dye, try to stay within the range of the standard, i.e. a very concentrated sample may require only 1 μl (or a dilution thereof) while a less concentrated sample may require 10 μl. In each case, be sure to mix the sample well before adding to the dye.
- Allow the reaction to proceed for 5 minutes at room temperature.
- Use the spectrophotometer and the SP program to measure the OD 595 of each sample.
- Paste the OD data into Excel and make a mass vs. OD graph using the standard. Using the set described above, the masses will be 1, 2, 4, and 8 μg. Fit a linear model to the data. If of the form OD = m*Mass, then divide the OD of the samples by the slope to get the mass.
- After calculating the masses in each sample, divide by the volume of sample that was added to each cuvette. You can then determine the volumes necessary to add approximately the same mass of protein to each lane of the SDS-PAGE.
|Sample||μl for 3 μg (pellet) or 30 μg (supernatant)||μl 6x loading dye (with βME)||H20 to bring up to 18 μl|
|pellet culture 1 - IPTG||8.5||3||6.5|
|pellet culture 1 + IPTG||5||3||10|
|pellet culture 2 - IPTG||9||3||6|
|pellet culture 2 + IPTG||8||3||7|
|supernatant culture 1 - IPTG||5||3||10|
|supernatant culture 1 + IPTG||13.5||3||1.5|
|supernatant culture 2 - IPTG||5.5||3||9.5|
|supernatant culture 2 + IPTG||11||3||4|
I added 1 μl of each supernatant and 10 μl of each pellet sample to 1 ml of Bradford stain.
- Note: there is an excel file under "pIs001" on the computer associated with the spec.
I generated a standard using 1 mg/ml BSA dilution: 1, 2, 4, and 8 μl. I added 1 μl purified HmgR to 1 ml of Bradford mix and used a piece of parafilm to mix each sample. HmgR Concentration: 2.545 μg/μl = 2.545 g/L. HmgR is ~28 KDa, so 2.545g/L divided by 28,000 g/mol = 90.7 μM.
I generated a standard using 1 mg/ml BSA dilution: 1, 2, 4, and 8 μl. I added 1 μl purified GpxR (p'So) to 1 ml of Bradford mix for lots 1-4 and used a piece of parafilm to mix each sample. GpxR Concentrations (assuming MW of 18,500):
- Lot 1: 2.08 μg/μl; ~112 μM for monomer, 56 μM for dimer
- Lot 2: 1.99 μg/μl; ~107.5 μM for monomer, 53.75 μM for dimer
- Lot 3: 2.02 μg/μl; ~109 μM for monomer, 54.5 μM for dimer
- Lot 4: 1.95 μg/μl ~105.4 μM for monomer, 52.7 μM for dimer