The Bradford Protein Assay Tips - Preparing For Western Blotting

The Bradford Protein Assay is a simple spectroscopic technique which is used in laboratory research to measure the total concentration of protein in a particular sample. The principle of the procedure revolves around the concept that the maximum absorbance of acidic Coomassie Brilliant Blue G-250 alters when protein binding occurs. The anionic state of the reagent is stabilised through ionic and hydrophobic interactions, resulting in a noticeable colour change. Consequently, the Bradford Protein Assay is often a vital step of sample preparation before comprehensive SDS-PAGE and Western Blot analysis in antibody research. Factors such as loading time, cuvette material and dye temperature can all affect the results which are drawn from the process.

Below are some useful tips to assist in calculating an accurate protein concentration and allow you to get the most out of the Bradford Protein Assay.

Is there a way to optimise accuracy when loading multiple samples?

The use of an 8-channel pipettor is recommended when loading multiple samples and this is advised to reduce sample evaporation as a result of loading delays. By extension, we also recommend diminishing evaporation and increasing the accuracy of results by reducing any delays between loading the sample and taking measurements. In all Bradford Protein Assays, it is vital to suitably load the cuvettes with enough sample solution to ensure that the light path is transecting the meniscus.

What type of cuvette provides optimal results?

We advise using either glass or plastic cuvettes as quartz cuvettes can interact with the dye reagent. Additionally, it is essential that the cuvettes are clean and free of residual matter, if they are not then the reagent could interact with any lingering protein deposits. Specific cuvette cleaning solutions are available from certain retailers.

Can the Bradford Protein Assay be used on proteins of all sizes?

No, this particular assay is only useful in the detection and quantification of proteins which are at least 3000 Daltons in mass. Therefore, you may wish to reconsider using this particular technique with smaller proteins, where methods such as the Bicinchoninic Acid Assay may be more appropriate.

Is there an optimal amount of time to incubate the sample?

The optimal incubation time will vary between samples and kits. Incubating a sample for too long can increase the possibility of interfering aggregates. Likewise, a sample with a higher protein concentration could escalate the chances of dye-protein aggregates causing light scattering. Consequently, we advocate always following the manufacturer’s recommended guidance.

Can the dye reagent conditions affect the accuracy of protein quantity calculations?

Yes, the conditions in which the dye is stored can influence experimental results. If the dye has been incorrectly stored or is too cold, it can cause the absorbance of your standard to appear lower than expected. This can be avoided by allowing your dye to reach room temperature prior to experimentation.

Can the age of the dye reagent influence the results?

Yes, if the dye is too old, it can cause the absorbance level of the standard to appear lower than its true value. The Bradford Reagent has an expiry date of 12 months, after which time it may become unstable and form particulates. We therefore recommend replacing the dye reagent after a year.

 

The Bradford Protein Assay is a rapid process to perform which, unlike some assays, is compatible with reducing agents and within the right conditions is a highly useful technique in protein quantification. When used correctly, the method represents a cost-effective means to achieve comprehensive sample preparation. The utilisation of these tips in antibody research should provide the foundation for precise protein loading calculations and optimal preparation for the SDS-PAGE and Western Blot processes.