Jul 01, 2022
Bradford protein assay – Protein concentration measurement (A590/A450 improved linearity)
- 1Universidade de Brasília
Protocol Citation: Daniel C Moreira 2022. Bradford protein assay – Protein concentration measurement (A590/A450 improved linearity). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3pd9ql25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 01, 2022
Last Modified: July 01, 2022
Protocol Integer ID: 65792
Keywords: Protein, Protein determination, Colorimetric assay, Bradford, Microtiter plate, Microplate
Abstract
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.
Materials
MATERIALS
Whatman #1 paperCatalog #1001150
Bio-Rad Protein Assay Dye Reagent ConcentrateBIO-RADCatalog #5000006
Bovine Serum Albumin lyophilized powder essentially fatty acid free and essentially globulin freeSigma AldrichCatalog #A0281
Ultrapure water (Type 1)
Corning® 96 well NBS™ MicroplateSigma AldrichCatalog #CLS3651
UV-transparent cuvettesBIO-RADCatalog #1702511
Coomassie Brilliant Blue GSigma AldrichCatalog #B0770
EthanolFisher ScientificCatalog #10538071
Phosphoric acid solutionSigma AldrichCatalog #W290017
Equipment
SpectraMax M3 Multi-Mode Microplate Reader
NAME
Microplate Reader
TYPE
Molecular Devices
BRAND
8002482
SKU
LINK
Multi-mode microplate readers (Absorbance, Fluorescence (top/bottom read), and Luminescence (top/bottom read)). Ranges: Abs, 200–1000 nm; FL, 250–850 nm; Lumi, 250–850 nm. Light source: Xenon flashlamp. Detector: Silicon photodiode, Photomutiplier tube. Readtypes: Endpoint, Kinetic, Spectrum scan, and Well scan.
SPECIFICATIONS
Bradford's protein reagent preparation
Bradford's protein reagent preparation
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.
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.
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.
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).
Protein standard solutions preparation
Protein standard solutions preparation
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.
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:
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).
Sample preparation
Sample preparation
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.
Assay
Assay
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').
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.
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.
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.
Incubate the 96-well microtiter plate at room temperature for 5 min.
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")
Calculation
Calculation
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:
BSA standard solutions.
Sample example: 5 µL of rat liver homogenate (1:500, w/v) + 5 µL PBS.
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:
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.