Bca Protein Assay - Find Proteins

BCA Protein Assay – Find Proteins

By: - Science - September 15, 2011
bca protein assay %E2%80%93 find proteins

There are many ways to determine protein levels in biochemistry and one of the ways is to use BCA protein assay. Also known as Smith assay, it is used to determine exact levels of protein by showing color change to purple from green. The amount of protein in the sample is determined at that point by colorimetric techniques.

The BCA protein assay is a highly alkaline solution with a pH of 11.25. The solution is made up of cupric sulfate pentahydrate, sodium bicarbonate, sodium carbonate, sodium tartrate and bicinchoninic acid. Within the solution and in practice there will be two separate reactions. First through reaction, the Cu2 is reduced to a proportional amount of the protein present in the sample. Next, molecules form with Cu1 and form a purple product that strongly absorbs light. This is important as this is how the protein will eventually be measured. The solution should then be heated, as the reaction rate will increase and additionally, the temperature will increase assay sensitivity. To complete the procedure, the amount of protein present in the sample is measured by absorption spectra.

Another comparable protocol to BCA protein assay is called Bradford assay. There are two main Bradford assay protocol types, a standard and micro. The way to do the standard Bradford assay protocol, prepare protein standards with final concentrations of BSA and 0.15 M NaCl ranging from 0 to 1500 micrograms BSA/mL. You should also prepare the unknown sample for comparison. In a test tube you will add 100 microliters of each preparation. To each tube at 5 microliters of Coomassie blue and mix by inversion.

You will now be using a spectrophotometer to measure a wavelength of 595 nm then blank using the 0 concentrated test tube. After 5 minutes, read each tube using the 595 nm spectrophotometer. Plotting the absorbance of standards and concentration will help you see the concentration of the unknown.

The micro Bradford assay protocol has the participant prepare 5 test tubes. One is blank NaCl, the others are 1, 5, 7.5 and 10 microliters of BSA and NaCl. As in the previous example, add 100 microtliters of each into a test tube then add 1 mL Coomassie blue. Use the spectrophotometer at 595 nm and blank the 0 concentrated solution. Wait for two minutes and read the test tube absorbency with the spectrophotometer. At that point you can plot your findings and calculate the concentrations of any unknown samples.


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