Bradford Protein Assay

Bradford Protein Assay


Protein Concentration Test Tube Microplate
High-concentration assay 0.2–1.4 mg/ml protein 0.05–0.5 mg/ml protein
  100 µl sample 10 µl sample
  5 ml diluted reagent 200 µl diluted reagent
Low-concentration assay 1.25–25 µg/ml protein 8–80 µg/ml protein
  800 µl sample 160 µl sample
  200 µl reagent concentrate 40 µl reagent concentrate
Compatible Reagents
Reagents Compatible With Bio-Rad's Protein Assay Standard Procedure*
Acetate 0.6 M KCI, 1 M
Acetone Malic acid, 0.2 M
Ammonium sulfate, 1 M Mercaptoethanol, 1 M
Amino acids MES, 0.7 M
Adenosine, 1 mM Methanol
Acidic pH MgCl2, 1 M
Boric acid MOPS, 0.2 M
BES, 2.5 M NAD, 1 mM
Barbital NaCl, 5 M
ATP, 1 mM NaSCN, 3 M
Ampholytes, 0.5% Peptones
Deoxycholate, 0.1% Phenol, 5%
Citrate, 50 mM Phosphate, 1 M
CDTA, 0.05 M PIPES, 0.5 M
Cacodylate-Tris, 0.1M Polyadenylic acid, 1 mM
DNA, 1mg/ml Polypeptides (MW <3,000)
DTT, 1 M Pyrophosphate, 0.2 M
Eagle's MEM rRNA, 0.25 mg/ml
Earle's salt solution tRNA, 0.4 mg/ml
EDTA, 0.1 M Total RNA, 0.3 mg/ml
EGTA, 0.05 mM SDS, 0.1%
Ethanol Sodium phosphate
Formic acid, 1 M Streptomycin sulfate, 20%
Fructose Thymidine, 1 mM
Glucose Tricine
Glutathione Tris, 2 M
Glycerol, 99% Triton X-100, 0.1%
Glycine, 0.1 M Tyrosine, 1 mM
Guanidine-HCl Urea, 6 M
Hank's salt solution Vitamins
HEPES, 0.1 M  
*The reagent compatibility table lists chemical reagents that do not directly affect the development of dye color. Since every chemical-protein reagent combination has not been assayed, it is possible that some of the listed reagents produce interference in combination with certain proteins. The reagent concentrations given in the reagent compatibility table are for the standard assay procedure (0.2–1.4 mg/ml protein). Concentrations compatible with the microassay procedure (1.25–25 µg/ml protein) are 1/40 of those listed. For example, 5 M NaCl in a sample assayed by the standard assay is equivalent to 0.125 M NaCl in a sample assayed by the microassay procedure. This is due to the differences of sample-to-dye ratios between the standard and microassay procedures.
Typical Standard Curve
Typical standard curve for the Bio-Rad protein assay standard assay procedure bovine serum albumin and bovine γ-globulin standards.
Protein Assay Selection Guide

Chart RC DC Protein Assay DC Protein Assay Quick Start Bradford Assay Assay Bio-Rad Protein Assay

  * Nonidet P-40 >0.25%, Triton C-100 >0.05%, Tween 20 >0.01%, SDS >0.025%
** DTT >1 mM, EDTA >1 mM

Protein Assay Comparison
Characteristics of Bio-Rad's Protein Assays
  Protein Assay
Quick Start™ Bradford Bio-Rad DC RC DC™
Adapted from method of Bradford (1976) Bradford (1976) Lowry et al. (1951) Lowry et al. (1951)
Standard concentration assay
Sample volume 100 µl 100 µl 100 µl 100 µl
Linear range 0.125–1.5 mg/ml 0.2–1.5 mg/ml 0.125–1.5 mg/ml 0.125–1.5 mg/ml
Low-concentration assay
Sample volume 1 ml 800 µl 200 µl 200 µl
Linear range 1.25–25 µg/ml 1.25–25 µg/ml 5–250 µg/ml 5–250 µg/ml
Microplate assay sample volume 5 µl 10 µl 5 µl *
Minimum incubation time 5 min 5 min 15 min 15 min
Assay wavelength 595 nm 595 nm 650–750 nm 650–750 nm

All assays are easy to use, require little reagent preparation, and give accurate and reproducible results.

* To adapt the RC DC assay to a microplate format, follow the micro test tube (microfuge tube) assay protocol in the RC DC instruction manual up to the centrifugation step where the supernatant is discarded. The pellet can then be transferred to the microplate, and the microplate assay protocol in the DC protein assay manual can be followed.

In a typical protein assay, a chemical reagent is added to a protein sample, producing a visible result, such as a color change in the sample solution. This color change is quantitated with a spectrophotometer or microplate reader, and compared to a standard curve of known concentrations of protein versus their absorbance after reaction with the reagent. The amount of protein in the unknown sample is determined by interpolation, reading the concentration of protein on the standard curve that corresponds to its absorbance.

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The Bio-Rad protein assay is a simple colorimetric assay for measuring total protein concentration and is based on the Bradford dye-binding method (Bradford 1976). Using standard procedure, the assay is used with samples having protein concentrations between 200 and 1,400 µg/ml (20–140 µg total). It is easy to adapt the assay from the standard-concentration range to a low-concentration (<25 µg/ml; 1–20 µg total) microassay or for rapid determinations in 96-well microplates.

The Bio-Rad protein assay is:

  • Based on the color change of Coomassie brilliant blue G-250 dye in response to various concentrations of protein — the dye binds to primarily basic (especially arginine) and aromatic amino acid residues
  • Useful for measuring proteins and polypeptides, depending on the charged groups, with molecular weights >3,000–5,000

Many detergents and basic protein buffers interfere with the assay; interference may be caused by chemical-protein or chemical-dye interactions. Refer to the list of compatible reagents for more information.

Bio-Rad protein assay kits offer either bovine serum albumin or bovine γ-globulin standards.

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

Sample volume 100 µl
Linear range 0.2-1.5 mg/ml
Sample volume  1 ml
Linear range 1.25-20 µg/ml
Assay wavelength 595 nm
Minimum incubation time 5 min
Adapted from method of Bradford
Number Description Options
1123 Protein Assay Applications Bibliography Click to download
LIT33 Instruction Manual, Bio-Rad Protein Assay, Rev C Click to download
1069 Colorimetric Protein Assays, Rev D Click to download
6836 Bio-Rad Protein Assay Quick Guide, Ver A Click to download
6852 Reagent Compatibility Chart for Bio-Rad Protein Assays Quick Guide,
Ver A
Click to download

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