Hi everyone! I am used to doing BCA test while doing protein extraction from cells or tissue, but do you usually use it while working with serum, i.e. mouse serum? Or do you just consider your results from ELISA or dot blot according to 1 ul of serum?
Hello Iuliia Eriukova
When serum is used for ELISA, I usually use a number of dilutions to determine the best dilution for the sample that lie within the linear range of the standard curve. For instance, I use undiluted sample(A) and diluted sample(A) (at 1:10, 1:50 and 1:100 dilutions) because the concentration of the analyte in the blood to be analyzed is not known. By using these dilutions I will get an idea of how much analyte is present in the sample. You may also run a negative serum control to minimize any variation from outside factors.
On the other hand, after protein extraction from cells or tissue, you mentioned that you perform the BCA assay, which is right. For lysates from cells or tissues you should carry out the total protein assay as you are likely to get variable amounts of protein in each sample and this would affect the level of detection. For instance, more the weight of tissue or greater the number of cells, more will be the concentration of protein present and hence higher the probability of detection. Thus, there is a need to perform the BCA assay on the lysate before carrying out ELISA or dot blot so that the volume of each sample can be normalized to deliver the same amount of total protein for the assay.
Best.
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