3 replies to this topic

[Lars Koch Hansen]

Dear all

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.

Best regards
L

[Sotirios]

You can make extraction with Tris-HCl / NaCl with homogenizer and after centrifuge. Use the extracts in ELISA

[Ben Lomond]

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 :  KZ Matalka - 2005
Neuroendocrinology Letters No.3 June Vol.26, 2005  

This is available full text and would be a good starting point for you.  

Good luck

[indyslim]

We have used Bicine-CHAPS buffer and have good luck with both ELISA and Westerns from tumor biopsies.