1.2.1. The Bradford assay
The Bradford assay for protein quantification is a popular protein assay because it is simple, rapid, inexpensive, and sensitive. It is based on the direct binding of Coomassie Brilliant Blue G-250 dye (CBBG) to proteins at arginine, tryptophan, tyrosine, histidine, and phenylalanine residues.
- Prepare the protein reagent (Bradford reagent) by dissolving 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95 % ethanol.
- Add 100 ml of 85 % (w/v) phosphoric acid.
- Dilute to 1l with distilled water
- Let the solution stir overnight to assure maximal dissolution.
- Filter (e.g. through Whatman #1 paper) and store in a dark bottle.
- Prepare a standard curve from a 1 mg/ml of bovine
serum albumin (BSA fraction V) stock solution by pipetting 1, 2, 5, 7, 10, 15
and 20 µl of this solution into glass test tubes.
It is of importance not to extend the range of the standard curve beyond 20 µg/µl when using BSA to guarantee that measurements are within a linear range.
- Complete each tube with distilled water to a final volume of 20 µl.
- Prepare these in triplicates.
- Prepare blank samples containing 20 µl of distilled water.
- Also prepare triplicates from 20 µl of each unknown sample, that must be adequately diluted (this must be done empirically) to give measurements within the range of the standard curve.
- Add 1 ml of Bradford reagent to blanks, standards and samples, vortex, leave for 2 min at room temperature and transfer the solution to disposable plastic cuvettes.
- Set spectrophotometer to a wavelength of 595 nm.
- Absorbance should be measured after 2 min and before 1h from the moment that Bradford reagent was added.
- Average the absorbance readings of each of the triplicates and subtract blanks from standards and samples.
- Plot absorbance values of the standard curve samples against their protein concentration (µg/µl).
- Determine the concentration of the unknown samples by linear regression.