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Bradford Assay Troubleshooting

Introduction

The Bradford protein assay is an easy and simple method for protein quantification of your protein concentration, yet may still require troubleshooting occasionally.

The dye binds to both basic and aromatic amino acid residues, which results in an absorbance shift. It can be easily seen by a change in color from brown to blue. The concentration of your protein can be determined by referencing to a standard protein, most commonly BSA (Bovine serum albumin).

Factors such as; temperature, wavelength, detergents and even the type of cuvettes you use can influence the measurement and give you wrong results. If any of these problems are occurring, don’t worry! This ZAGENO troubleshooting guide will help you!

Below is a summary of common problems that occur when performing a Bradford protein assay.

The absorbance of the protein sample is lower than expected

  • Low molecular weight: The detection limit for the Bradford assay is approximately 3000-5000 Daltons. If your protein is smaller than this, then use a different quantification method such as the BCA assay.

  • Interfering substances: The sample may contain interfering substances, such as detergents (Table 1). Dilute your protein sample. Be sure your standards are diluted in the same buffer.

The absorbance of the protein sample is higher than expected

  • The concentration of your protein is too high: Dilute your protein sample and measure the protein concentration again.

  • Interfering substances: The sample may contain interfering substances, such as detergents (Table 1). Dilute your protein sample. Be sure your standards are diluted in the same buffer.

The absorbance of the standard is lower than expected

  • Dye reagents are too old or were improperly stored: The Bradford Reagent has an expiry time of approximately 12 months. If older, replace it with a new one and store it at 4°C.

  • Standard dilutions not prepared correctly: Follow the manufacturer's protocol for creating your protein standard dilutions.

  • Dye reagent may be too cold: Bring the Bradford Reagent to room temperature before starting the assay. Absorbance measured at incorrect wavelength: Measure the absorbance at 595 nm.

Samples are dark blue

  • High alkaline concentrations: This raises the pH above the Bradford reagent’s limit. Dilute or dialyze your sample

Precipitates in the sample

  • Detergents in your protein buffer: Dialyze your protein sample or dilute the sample to reduce the level of detergents.

The protein sample contains interfering substances:

Interfering substances in the buffer are one of the major problems in performing a Bradford assay. Table 1 summarizes a list of the most commonly used substances and their compatible concentrations. For detailed information, please refer to the manufacturers’ protocol. If the substance you are using is at an incompatible concentration or not listed at all, then take a look at the section below.

  • Concentration of interfering substance is incompatible with the Bradford assay: Dilute the sample to the point of no interference if your protein concentration is high enough or remove the substance by dialysis

  • Substance is not in the reagent compatibility list: Run two standard curves, one with your protein in your buffer, one with your protein in water and plot a graph of protein concentration versus absorbance. If the buffer is not interfering with your measurement, then two standard curves will have the same slope.

Table 1: Compatible substance concentrations in Bradford assay (modified after [1] Quick Start Bradford Protein Assay – Instruction Manual, #4110065, Bio-Rad; [2] Protein assay compatibility table, Tech tip #68, Thermo Scientific (2012))

Note: This is not a comprehensive list of compatible substances and the concentrations. There are many substances that can affect proteins in different ways. Reagents can change the pH of the assay, which will interfere with the Bradford assay as well.

Technical tips

  • The dye reagent can react with quartz cuvettes; it is better to use glass or plastic cuvettes

  • Contaminations on the glassware can influence your measurement, make sure they are clean before use

  • Temperature of Bradford reagent should be at room temperature

Problems in the lab? Learn about methods and experiments in our Knowledge  section or look up a specific Troubleshooting guide.

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