TITLE: Bradford protein assay – Protein concentration measurement (A590/A450 improved linearity)
AUTHORS: Daniel C Moreira

[DESCRIPTION]
This protocol describes how to measure the concentration of total protein in a sample performing the Bradford's assay using microtiter plates. Procedures are slightly modifications based on the method described in (1) and the linearization procedure described in (2) and (3).


(1) Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254.

(2) Zor & Selinger, Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies, Anal. Biochem. 236 (1996) 302–308.

(3) Ernst & Zor, Linearization of the Bradford protein assay, J. Vis. Exp. 38 (2010) 1918.



[STEPS]
SECTION: Bradford&#x27;s protein reagent preparation
1. 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.
SECTION: Bradford&#x27;s protein reagent preparation
2. Weight 100 mg of Coomassie Brilliant Blue G-250 (e.g., B0770, Sigma-Aldrich) and dilute it in 50 mL of ethanol using a magnetic stirrer at room temperature.
SECTION: Bradford&#x27;s protein reagent preparation
3. In a fume hood, slowly add 100 mL of 85% (w/v) phosphoric acid (e.g., W290017, Sigma-Aldrich) to the previous solution and homogenize.
SECTION: Bradford&#x27;s protein reagent preparation
4. Dilute the resulting solution to a final volume of 1,000 mL with distilled water1. Filter through a Whatman No. 1 paper (or equivalent). Store at room temperature protected from light.

1Alternatively, you can use commercially available ready-to-use reagents (e.g., 5000006, Bio-Rad).
SECTION: Protein standard solutions preparation
5. Prepare a ~4 mg/mL bovine serum albumin (BSA, e.g., A0281, Sigma-Aldrich) stock solution in PBS2.

Example: Dilute 6 mg in a final volume of 1.5 mL.

2If compatible3, use the same solvent/buffer in which the sample to be analyzed was prepared to make the BSA standard solutions.

3Check 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.
SECTION: Protein standard solutions preparation
6. 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:

   
SECTION: Protein standard solutions preparation
7. Prepare several BSA solutions at concentrations from 0.025 to 0.80 mg/mL4 using PBS (or the relevant buffer/solvent).

Example: If your stock solution is 3.94 mg/mL, first prepare a 0.8 mg/mL (100 µL to a final volume of 492.5 µL) and then dilute it serially to 0.40, 0.20, 0.10, 0.05 and 0.025 mg/mL (e.g., 100 µL of the previous solution + 100 µL of PBS).

4Under the conditions described in this protocol, the the ratio of absorbances at 590 nm over 450 nm is linear within the 0.025–1.00 mg/mL BSA range (see figure below). 

   
   

SECTION: Sample preparation
8. Dilute samples in appropriate buffer (e.g., PBS; see section/step 5) to achieve an expected concentration5 that lies within the concentration range of the standard curve (0.025–0.8 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. 

5If an estimate of protein concentration for your tissue/cell sample is not available, test several dilutions.
SECTION: Assay
9. 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').
SECTION: Assay
11. 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 (the ratio absorbance at 590 nm over absorbance at 450 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.
SECTION: Assay
10. 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.

SECTION: Calculation
15. Calculate the average of the ratio between absorbances (A590/A450) for each BSA standard and samples using the absorbance values of the triplicates.

Example data:

  
  
  
SECTION: Calculation
16. Build a standard curve. 

Plot the average A590/A450 ratio 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:



   
    


SECTION: Calculation
17. 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 112.5 mg/gww.


SECTION: Assay
12. 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. 
SECTION: Assay
13. Incubate the 96-well microtiter plate at room temperature for 5 min.
SECTION: Assay
14. Read the microplate at 590 nm and 450 nm. 

Read  before 1 h of incubation. At high protein concentrations, precipitation begins after 10–15 minutes.

Note: In case a microplate reader is not available, a picture taken with a smartphone camera can be used to acquire data and accurately calculate protein levels in samples (see the protocol '"Bradford protein assay – Protein concentration measurement using RGB data")