I want to measure the expression levels of a particular cytokine level in plasma with western blotting. I have primary antibody from ELISA. Does it require any purification?
Please clarify my doubt.
Dear sarkar
why you dont use ELISa”? The western bloating process are so long . Also you can use ELI SPOT. You can not use the ELISA primary Antibody for western blot .
It is very difficult to standardize a western blot in such a way that you get reliable results every day, which is comparable between each of the runs. It would require you to sacrifice halve your blot for some sort of standard-curve (applying known amounts of cytokine, so you know which intensity of a dot belongs to which concentration of cytokine). ELISA is much better suited for quantification purposes.
It is not a good idea to use of WB to measure the expression levels of a particular cytokine level in plasma. However you can compare the density of blots in different groups with software like ImageJ. This comparison just gives you qualitative results and not quantitative. The primary Ab must be WB compatible.
Dear Siamak, Wesley and Reza.
I am glad for your response. Yes ELISA is the most easy procedure. Actually I am currently not having the recombinant protein to ensure the concentration present. And I need to identify the quantity in it.
I'm afraid that extremely low concentration of cytokines in plasma would not lead to nice results, if any :) Antibodies validated for ELISA might not work - sample processing might change the epitopes.
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