BRADFORD REAGENT 100ml

Introduction

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.

Key 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

1. Label eight test tubes from #1 to #8.

2. 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.

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

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Disclaimer: For laboratory research use only.