I would like to perform ELISA for insulin protein which secreted in the media.
I want to use a control as I don't have the cell number to normalize the ELISA results. Is there are any solutions other than going for western blott and using total protein as a control ?
What exactly do you want to do? Do you want to determine the amount of insulin in your supernatant or just the presence? If you only want to verify that your insulin is there, Western blot ist the better option, since it gives you also information about the size, which minimizes the likelihood of getting a false positive result.
However, if you want to quantify your insulin in a mixture of proteins like your supernatant, ELISA is the assay of choice, unless you have other fancy equipment in the lab. But then, you need to have recombinant insulin to prepare a standard curve. As a negative control, I would propose to use unconditioned medium.
In case I got it all wrong, I apologize.
I need to quantify the amount of insulin not only its presence.
Thank you very much for your help, but can you clarify more what do you mean by (unconditioned medium) ?
Unconditioned medium is just your cell culture medium that was not used for cell culture.
Actually, I can't think of any feasible way to determine the amount of insulin in your supernatant by ELISA or Western blot without recombinant insulin as a reference. How much insulin do these cells produce? Do you get a clear and distinct band in Coomassie or silver staining? Then you could use serial dilutions of any other protein as a standard and perform densitometric analysis of your SDS-PAGE at least for a rough estimation.
I used recombinant insulin to make a standered curve. But can I use the total secreted protein in the media as a reference between different cell treatments, as this will reflect the number of cells to some degree ?
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