Bradford Protein Assay for Protein Concentration Measurement
By Daniel C Moreira
Protocol Steps
- 1Prepare a solution containing 0
Prepare a solution containing 0.01% (w/v) Coomassie Brilliant Blue G-250 (e.g., B0770, Sigma-Aldrich), 4.7% (v/v) ethanol, and 8.5% (v/v) phosphoric acid as described in the next steps.
- 2Dilute the resulting solution to a...
Dilute the resulting solution to a final volume of 1,000 mL with distilled water 1 . Filter through a Whatman No. 1 paper (or equivalent). Store at room temperature protected from light. 1 Alternatively, you can use commercially available ready-to-use reagents (e.g., 5000006, Bio-Rad).
- 3Prepare a 4 mg/mL bovine serum albumin (BSA, e
Prepare a 4 mg/mL bovine serum albumin (BSA, e.g., A0281, Sigma-Aldrich) stock solution in PBS 2 . Example : Dilute 6 mg in a final volume of 1.5 mL. 2 If compatible 3 , use the same solvent/buffer in which the sample to be analyzed was prepared to make the BSA standard solutions. 3 Check buffer components incompatibility with the Bradford reagent. There are several available online. For example: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6852.pdf and https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf .
- 4Check the BSA concentration in the stock solution
Check the BSA concentration in the stock solution. Read it at 280 nm in an UV-transparent cuvette and use the equation below to calculate the actual BSA concentration. Example : If you diluted 50 µL of the BSA stock solution in a final volume of 1,000 µL and it resulted in a absorbance of 0.130, you have:
- 5Dilute samples in appropriate buffer (e
Dilute samples in appropriate buffer (e.g., PBS; see section/step 5) to achieve an expected concentration 5 that lies within the concentration range of the standard curve (0.05–0.04 mg/mL; see section/step 7). Example : Considering that rat liver contains ~100 mg protein/g wet weight, if you have a rat liver extract/homogenate that was prepared diluting the tissue sample 1:20 (resulting in ~5 mg protein/mL), you need to further dilute by a factor of least 15 (total dilution 1:300, resulting in ~0.333 mg/mL) before proceeding with the assay. 5 If an estimate of protein concentration for your tissue/cell sample is not available, test several concentrations.
- 6Add 260 µL of water to...
Add 260 µL of water to at least three wells of a 96-well microtiter plate. These will be the microplate reader blanks (also known as 'auto zero' or 'reference wells').
- 7Add 10 µL of each sample...
Add 10 µL of each sample to at least three wells of a 96-well microtiter plate. Ideally, you should use several dilutions of the sample and check whether there is a linear response between signal (absorbance at 595 nm) and amount of sample. One convenient assay of doing it is to pipet several volumes (e.g., 1, 2, 5, 10 µL) and add it up to 10 µL with the appropriate buffer. This also maximizes the chance of getting at least one dilution of the sample within the dynamic range of the standard curve for unknown samples. Example : • Dilution 1 – 1 µL sample + 9 µL buffer; • Dilution 2 – 2 µL sample + 8 µL buffer; • Dilution 3 – 5 µL sample + 5 µL buffer; • Dilution 4 – 10 µL sample.
- 8Add 10 µL of each BSA...
Add 10 µL of each BSA standard solution to at least three wells of a 96-well microtiter plate. Remember to prepare a zero/blank standard, which is the buffer used to prepare the standards but with no BSA.
- 9Calculate the average absorbance for each...
Calculate the average absorbance for each BSA standard and samples using the absorbance values at 595 nm of the triplicates. BSA standard solutions. Sample example: 5 µL of rat liver homogenate (1:500, w/v) + 5 µL PBS.
- 10Build a standard curve
Build a standard curve . Plot the average absorbance value at 595 nm on the Y- axis versus BSA concentration in mg/mL on the X-axis. Calculate a linear regression (Y = a × X + b; e.g., 'add a linear trendline' in Microsoft Excel). Example:
- 11Calculate protein concentration in samples using...
Calculate protein concentration in samples using the equation generated in the previous step. Interpolate unknown protein concentration from the standard curve and multiply by all dilutions. Example : For this example (see step 15), the dilution factors are 500 (a rat liver homogenate was prepared at 1:500, w/v) and 2 (5 µL were added to the microplate and added up to 10 µL with buffer). For tissues, the final result is often expressed as mg protein per gram of wet weight (mg/gww) assuming 1 g/mL. Thus, this rat liver sample used as example would have 113.09 mg/gww.
- 12Add 250 µL of Bradford's protein...
Add 250 µL of Bradford's protein reagent to each well containing BSA standards or samples. If available, use a positive displacement pipette device (e.g., Multipette M4, Eppendorf) to avoid bubbles. Do not add Bradford's protein reagent to wells containing 260 µL water. These are used just as blanks for the microplate reader.
- 13Incubate the 96-well microtiter plate at...
Incubate the 96-well microtiter plate at room temperature for 5 min.
- 14Read the microplate at 595 nm
Read the microplate at 595 nm. Read before 1 h of incubation. At high protein concentrations, precipitation begins after 10–15 minutes. Alternatively, also acquire absorbances at 450 nm and 590 nm (see step 7).