RIPA as Extraction Buffer in ELISA - (Jan/25/2008 )

Can RIPA buffer be applied in wells to perform ELISA directly or should it be diluted in extraction buffer. My collegue has mentioned that RIPA will interfere with antibody binding and cannot be applied directly. Is this true?

---

Although I've only done one ELISA which was a horrible disaster, I am familiar with RIPA buffer and know some people who will use this buffer to lyse cells for an IP. Personally, I think this buffer contains a lot of detergent which can disrupt antibody binding (especially the SDS). However, it will disrupt the weakest and hopefully non-specific binding. It's up to you, how good your antibody is and how much antigen you need to be able to detect. Just know that RIPA is a pretty stringent buffer to use and you may loose signal. Generally it's best to start with a low stringency (little to no detergent, isotonic salt) and work your way up to clean up the results. At least this way you have the best chance of seeing what you are hoping for.

---

It should work. I ran successfully eNOS using lysis buffer supplied by manufacturer of the assay and RIPA buffer of my own, and results are comparable.

---

TMMF gave the answer,
in addition if u r not comfortable, dilute ur sample from 1:2 to 1:5.

---