I am conducting a study in which I have to measure the concentration of different hormones in cell supernatant samples by means of ELISA assays. Although the seeding conditions are always identical, in terms of number of cells, medium, concentration of the drug to be tested, etc., the values derived from the ELISA are very variable. Certainly no matter how precise one tries to be there can be variations in the actual number of cells/well. This is why I would like to normalise the results to the total protein content. I had thought of normalising for DNA content but as the cells are not synchronised I thought it might not be accurate enough. I still have all the supernatant samples but I don't know if I can get any useful data from those for normalisation. Any advice?
Thank you very much for your help.
Do you have the data for the number of cells? If so, that may be your best option for normalization. The second best option may be to measure protein levels of housekeeping genes and use that data for normalization. Total protein vs your measured hormones will also be better than no normalization at all, but total protein might be affected by your treatment so that would lead to another set of variables.
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