Several methodologic questions concerning phospho-ELISA. - (Jan/29/2008 )

First of all thanks for the input for the topic "RIPA as extraction buffer".

I am running TAS - ELISA (or trying to introduce a such) on a phosphorylated protein. I have encountered several problems. My experience is mainly WB and i use the expreince gained from that technique to ELISA.

The questions are:

1. If the phosphorylation of my protein is very dynamic (read volatile), should I use antiphosphatases such as Na-orthovanadate only in RIPA/extraction buffer or in all buffers used in all steps of my ELISA (eg.: wash buffers, blocking buffers, conjugate buffers?)

2. Could it be that antibodies that are applicable in WBs are useless in ELISAs? Is there a possibility that the denaturation of proteins in WBs with application of reducing agents (eg Beta Mercapto Ethanol) exposes domains in the protein that antibodies can recognize but not in ELISA where such denaturation is not possible.

3. In regards to question 2 - can or should the cellular extraction on which the ELISA is to be done be processed in some way. In other words: is there any rationale in for instance boiling my lysate prior to use so that the proteins denature?

hi,

1-u need phospho stbilizers in ur lysis buffer alone, once the phopho protien bind to the antibody u donot need any more stabilizers in the further steps.

2- yes some times, if the antibody is recognizing a confirmational epitope, if u use denatured antigen for immunization then i wud suggest u to denature the sample before the ELISA.

3- denaturing the protein, again entirely depends on the antibody, findout about ur antibody if it is recongnizing linear epitope, if that epitope is not hidden, then u donot see much difference with native and denatured antigens. some times boiling can lead to the precipitation.

gud luk