Since ELISA kits are very expensive, was wondering if I could measure that with western blot technique? and If yes, how to measure the protein concentration? For the cell lysate I use protease inhibitor cocktail in RIPA buffer to extract the protein and measure it with bradford assay. But for the proteins in conditioned media (DMEM+10%FBS), I dont have any protocols yet. Looking forward to your help. Thank you.
If you have access to an antibody that works with Western blotting, and it has sufficient sensitivity for your application, then you can use a Western blot. To quantify the protein on the blot, you will have to run standards with a known amount of the same protein on the same gel and use a quantitative imaging system.
Medium with 10% FBS is going to be a problem because there is an enormous amount of albumin protein in it, limiting the amount of protein you can run on the gel and distorting the bands.. There are columns that can be used to deplete the albumin, but it might be worth trying to use serum-free medium.
You can try Western Blot. Detection level depends upon the amount of secreted protein present in the sample. Load maximum possible amount in each well. At least 5-10ug of total protein required. You might have to use ECL-plus for detection.
Rubina please check your protocol again. Conditioned media mostly use without FBS or very less FBS concentration because FBS has protein concentration. For Elisa you can use in house ELISA rather than commercial kit which can reduce your cost.
thank you all for your suggestions..will try it with western blot first since we do have all the materials in our lab already for that..I have one more question regarding the protein precipitation with TCA,,,since we are dealing with secreted protein here in the media, what should be the internal control for this technique?
The internal control could be exogenous purified immunoglobulin G of the same species as your primary antibody added before the precipitation. The secondary antibody will recognize it and give you a ~50 kDa band.
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