Thermo Fisher Scientific (Pierce)
BCA Protein Assay Kit
23227
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Measurement of protein concentration is important in many biological applications, e.g. protein analysis by western blotting where equal amounts of total protein should be loaded per lane of an SDS-PAGE gel. The BCA method is a convenient and easy assay to determine protein concentration in cell lysate or tumor extract samples. The assay is based on the measurement of the absorbance of known concentrations of BSA (a standard protein) and using the generated standard curve to calculate protein concentration in the sample.
Measurement of protein concentration
Protein samples can originate from any mammalian species. We usually measure protein concentration in the lysates of human cells or extracts of human tumor xenografts. The kit contains all key components to generate a standard "BSA" curve and measure protein concentration in your samples, namely, BSA solution at 2 mg/ml and "A" and "B" reagents, which are mixed at 1:50 ratio, and added to BSA dilutions and samples.
The manufacturer's protocol is straightforward, but could be simplified further. BSA solution should be serially diluted (preferentially in the diluent of your samples). 6-7 serial 1:2 dilutions is enough to produce a standard curve. Diluent should be used as "0" protein to provide a "blank" value. The samples should be diluted to fit the middle of the standard curve.
BSA soultion is supplied in sterile 1-mL ampules and could be sterilly aliquoted to prevent drying out and contamination. The volumes of "A+B" mixture pe volume of your samle are more flexible than indicated by the company and are easily adapted to fit your experiment. For example, the voumes of the samples and BSA dilutions per well of 96-well plate could vary from 5 to 50 microliters, whereas volumes of "A+B" solution could vary from 100 to 150 microliters per well (obviously, larger sample volumes would require more developing solution). Time of plate incubation also is more flexible than indicated: the plate can be read as soon as 10-15 minutes (if you are in a hurry) and as long as 1 hr (if you were late).
After adding "A+B" solution (premixed at 1:50 volume ratio) to the samples and incubation at 37oC for approximately 30 minutes, blue color precipitate develops in the wells containing protein and absorbance levels are proportional to protein concentration. Protein concentration in the samples is determined by reference to a standard BSA curve.
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The image shows the standard BSA curve produced using BCA kit from Pierce/Thermo Fisher Scientific.
The kit is very reliable, easy to use, the results are consistent and reproducible.get reproduc
None detected.
The Thermo Scientific/Pierce BCA kit is an excellent tool to determine protein concentration in biological samples.