Synonym(s):

Coomassie dye binding protein assay, Coomassie dye binding protein assay, Protein dye reagent, Protein dye reagent

Description

Bradford method utilizes Coomassie Brillant Blue G-250 dye binding to an unknown protein and forming a complex which can be detected spectophotometrically at 595 nm.

Specifications

Item	Value
Chemical Name	Bradford Reagent
Appearance	Clear Blue Liquid
Abs.@ Peak A	Report
Abs.@ Peak Abs.@595nm	>1.80
Characteristic Pattern	Pass
Peak A	463-467nm
Hazard Class	Class 8
Hazard UN	UN185
Hazard PG	PGIII
Storage	2~8℃. Keep Dry. Warm to Room Temperature Before Opening.

Features

Rapid and easy-to-use: Entire assay (10–20 samples) takes only ~10 minutes (excluding sample prep)
High sensitivity: Detects as little as 0.2 µg of protein in 1–20 µL
Excellent linearity: 10–150 µg/mL linear detection range
Versatile: Compatible with both cuvette-based and 96-well plate assays
Cost-effective alternative to more expensive protein quantification kits

Application

Quantitative measurement of total protein concentration in biological or biochemical samples
Protein estimation in cell lysates, column fractions, purified protein preparations, etc.

Assay Protocol Overview

Tube-Based Protocol

Prepare BSA standard curve (125–0 µg/mL) via serial dilution
Add 100 µL of each standard or sample to test tubes
Add 1 mL Bradford Reagent, mix and incubate at RT for 10 min
Measure absorbance at 595 nm

96-Well Plate Protocol

Add 20 µL of each sample or standard into wells
Add 200 µL of Bradford Reagent
Shake plate 30 sec, incubate 10 min at RT
Read at 595 nm with a microplate reader

Procedures

Protocols for Measurements in Test Tubes A

Preparation of BSA Standards and Sample Dilutions

Label eight test tubes from #1 to #8.
Prepare serial dilutions of the BSA standard as follows:

Tube #1: Add 20 µL of BSA standard (5 mg/mL) to 780 µL of PBS.
Tube #2: Mix 100 µL of PBS with 400 µL from Tube #1.
Tube #3: Mix 100 µL of PBS with 400 µL from Tube #2.
Tube #4: Mix 120 µL of PBS with 360 µL from Tube #3.
Tube #5: Mix 120 µL of PBS with 240 µL from Tube #4.
Tube #6: Mix 150 µL of PBS with 150 µL from Tube #5.
Tube #7: Mix 100 µL of PBS with 100 µL from Tube #6.
Tube #8: Add 100 µL of PBS only (blank control).

This is a serial dilution of the BSA standard.

	1	2	3	4	5	6	7	8
BSA final concentration (μg/ml)	125	100	80	60	40	20	10	0

Dilute the sample of interest to a desired concentration (10–125 μg/mL) and note the dilution factor (X).

Label Tubes #9 to #11

In Test Tube #9, transfer 20 µL of the diluted protein sample.
In Tube #10, transfer 20 µL of the diluted protein sample at a different dilution factor.
In Tube #11, transfer 20 µL of PBS only.

B. Measure the concentration of BSA standards and the sample of interest.

Transfer standards #1 to #8 into designated wells of a 96-well plate in duplicate to ensure reliable results.
Add 200 µl of Bradford Reagent to each well.
Shake the plate for 30 seconds to mix thoroughly.
Incubate at room temperature (RT) for 10 minutes.
Measure absorbance at 595 nm using a microplate reader.
Subtract the average absorbance of blank replicates from all standard and sample readings.
Plot a standard curve: average blank-corrected absorbance vs. BSA concentration (µg/ml).
Determine the protein concentration of your diluted samples using the standard curve, then calculate the original concentration by applying the dilution factor.

Notes

Mix reagent thoroughly before use; bring to room temperature
Avoid detergents (use BCA assay instead if sample contains detergents)
(SK3021, SK3051)
For best results, prepare a standard curve using the same protein as the sample
Duplicate measurements and fresh standard curves are strongly recommended

Comparable To:

Sigma-Aldrich Bradford Reagent (B6916), but more economical

For Research Use Only. Not for human or animal therapeutic use.

Documents

INFO

COA

SDS

Bradford Reagent 100ml