Hello everyone,
I have a paper under review. For my project, I did cell culture for MCF-7 and gave inhibitors to some groups. Substrate of E1 was added. Then the estradiol of supernatant was measured with Elisa kit. The estradiol concentration of treatment groups were compared with control group. However, the reviewer said the estradiol concentration should be standardized to protein. Should I test the protein concentration in the supernatant or the protein concentration in the cells? Thanks so much.
Hi Xiaoye,
In my opinion, the big concern of the reviewer is probably the effects of the inhibitors on the cell growth. You need to make sure the total cell number or proteins after incubation before your Elisa assay. You can collect the supernatant first, and then collect the cells as well to detect either the cell number or the proteins. You need to calculate the estradiol concentration under the same cell number/proteins.
Hii Xiaoye,
For your doubt, I also have similar explanation like @Han Zhang. What I am understanding is that reviewer is talking about 'NORMALISATION OF PROTEIN LEVEL'. Its about protein concentration, that the treatment haven't caused any change in endogenous level of the protein in cells. you might got your reading using standard protein curve. But reviewer is looking for CELLS endogenous protein level which should be same under both control and treated condition (it is similar like GAPDH/Beta-actin for gene expression). It is somewhat to nullify the artifact.
You need to analyse the protein concentration in the cells.
Dear Xiaoye ,
Yes Indeed. You should normalize the estradiol concentration with total proteins or total steroids. Since it is difficult to quantify total steroids, the best bet is to go for total proteins. The idea of such a normalization is to confirm that whatever changes you find in the estradiol concentration among different treatment groups may not be because of factors like more no. of cells, more cellular protein investment etc, and is solely because of your treatment effect. You can also normalize by total no. of cells but we mostly do not know the exact number of cells that are lysed or used for the ELISA assay alone, so it yields an approximate figure for any quantitative measurement.
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