Hi!
I'm new to ELISA, and I'm confused about the methods for analyzing the results.
I'm doing a very easy experiment. I'm going to check the amount of one secreted protein in the cell culture supernatant in 3 different experimental groups.
So I wonder if I need to do the total protein assay to normalize the results of ELISA, if I want to compare the protein concentration in different groups?
In the protocols, total protein assay is only suggested when using tissue samples or cell extracts, but not when using supernatant. So I don't know if I need to do the total protein assay, and is BCA kit suitable for it?
Although, theoretically, I think a total protein assay should be done...
Normalizing supernatants from cell culture on the protein content of the medium doesn't make much sense, as your medium usually contains a lot of serum proteins, which is practically constant over time.
Rather, use the number of cells, volume of medium and incubation times for normalizing, whatever is appropriate. Within such a set of conditions defined, the number of cells in a dish (or per area) or the total amount of protein or DNA per dish may be used as reference.
Depends a bit on what you're after and what is the physiological basis of the signal you are measuring.
As there are several dependencies, small errors in each parameter will accumulate and can distort the results quite much, thus careful planning and minimizing variations is essential.
I would propose to normalize results in relation to not affected control proteins (structural components, chaperones etc.)
You can use any factor/gene/protein which is not affected regarding its expression by your experimental conditions/stimulus. I would propose to use different controls, maybe GAPDH, beta-actin (what ever).
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