Question

Comment on the reliability of each of the protein concentrations in a set of test tubes where the absorbances are know but the unknown concentration were determine from a standard curve.

Answer 1

In our experiments, we generally measure the unknown concentration of protein sample by comparing it to set standards of known protein. Thus, for making a proper estimation of concentration of our protein of interest, we need to select those proteins as standards/ reference points which have similarities with our protein of interest in their properties.

But, it is quite difficult every time to find out a protein standard which has similar properties like our protein of interest. So, in assays, we use some readily available standard proteins such as Bovine Serum Albumin (BSA) and Bovine Gamma Globulins (IgG). But, as the properties of BSA and IgG might vary from the protein sample being tested, so this experiment will lead us to get just an ‘Estimation’ of concentration of unknown sample.

As the reference protein and the test protein might not be identical in their properties, so we need to be very careful regarding the use of buffers for both. The buffers for both Standard and Tests must be identical. To maintain the accuracy and precision of the experiment and to avoid random error, we need to make replicates of both standard and test samples. We generally run the standards in triplicate to avoid making mistakes in estimating test sample concentration.