Standard protocols for protein estimation, for example, Lowry and Bradford's protocols, are available in any standard protein protocol book. With the number of buffers and chemicals used for the isolation and purification of different proteins, the researcher may decide the assay scheme depending on the compatibility of the purification buffers with the assay reagent. For every reagent there are certain components that interfere with the assay system. The BCA Protein Assay Kit from Pierce provides the list of chemicals that have compatibility with this assay and provide an easy methodology how to interpret the results of the assay. We found that the helpful point by means of using the BCA Protein Assay Kit ‘Pierce’ “is the compatibility with more buffers/salts solvents, and detergents than any other colorimetric protein assay”.
By using the 96 well plates for the protein estimation with BCA Protein Assay Kit “Pierce” and to avoid the error we used three wells for each sample and also for the standard. Following this way the kit can be used for large numbers of samples. The BCA Protein Assay Kit “Pierce” comes with 2 large bottles these are the reagent A and one small bottle called reagent B. These bottles used to prepare the working reagents by taking one part from reagent B and 50 parts from reagent A. to avoid the wastage of the reagent one should calculate how much required from working reagent then accordingly prepare the working reagent, because the working reagent is stable for one day. As it written in the instruction that provided with kit is that the “purple-colored reaction product of this assay is formed by the chelating of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range of 20 to 2000 µg/ml”.
This kit also provides 10 ampoules of Albumin standard (BSA) of concentration 2 mg/ml for the standard curve; we believe the kit should come with 20 ampoules of Albumin standard (BSA). This kit really is easy to use and with easily interpreted results. Also this kit will not take long time. Prepare your standard then prepare the working reagents fresh every time, then add 25 µl of the standard and use three wells for each standard and also for each sample, add 200 µl for each well and then incubate the plates for 30 minutes at 37ºC. After that keep the plate room temperature for 10 minutes and then get the reading using the micro-titer plate reader at OD 562 nm.