Inflammatory cytokine ELISA-kit on homeogenates from small biopsies? - (Jun/18/2012 )
I'm thinking about using an ELISA kit to measure the levels of inflammatory cytokines (e.g. TNF-alpha, IL-10, etc). We have small biopsi material from humans and wants to homeogenize with a RIPA buffer (that we use for our Western Blots).
Do any of you have experience with a kit that does this?
I found one but unfortunately it only work with serum/plasma, not homeogenates because of the detergents in RIPA buffer.
You can make extraction with Tris-HCl / NaCl with homogenizer and after centrifuge. Use the extracts in ELISA
I'm not sure that the TRIS/HCl approach will solubilize all of your molecules and might result in low and irreproducible extraction efficiencies. You may want to persist with the RIPA buffer type approach or include IGEPAL in a more simple buffer. Some detergent is not too alarming for many immunoassays but you may want to include a broad spectrum protease inhibitor cocktail. Spike in purified analyte prior to homogenization to demonstate the extraction efficiency and reproducibility thereof.
There are some literature reports from groups doing similar work to you for example :
We have used Bicine-CHAPS buffer and have good luck with both ELISA and Westerns from tumor biopsies.