I did a glucose stimulated insulin secretion assay for MIN6 cells. I measured the released insulin in the Kreb's Ringer buffer containing certain concentrations of glucose. In the end I collected the cell lysates of the treated cells using TNE buffer with 1%SDS. I aim now at measuring the insulin content in these collected lysates by ELISA. My concern is about the TNE buffer with 0.1% SDS, I had a look over several articles I saw researchers extract proteins from cells using acid alcohol mixture or triton-X buffer. I wonder if I can use my cell lysates extracted in TNE buffer-0.1%SDS for determination of insulin content by ELISA.
The quick answer is no, it wouldn't work; do the acid acetone extraction like everyone else. In fact, you need not lyse cells in SDS. Simply freeze the cells and add pre-chilled (-20ºC) acid-acetone to extract and recover insulin.
It is unlikely that you will be able to get good binding of insulin to the primary antibody, and therefore accurate measurements of insulin by ELISA in the presence of 0.1% SDS.
Some antibodies can be more forgiving and at relatively low concentration (usually
Hi
I agree with Tausif Alam , kindly look at links here will help you
Good Luck
http://www.sciencedirect.com/science/article/pii/S0092867407003686
https://www.researchgate.net/post/Insulin_secretion_assay-can_anyone_help/2
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1751878/
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