Co-IP..... - What's important for the lysis buffer/washing buffer? (Dec/29/2006 )

Actually, I have many questions about co-IP, because I once saw positive result, when I repeated, however, the band disappear... several times already. It seems these 2 proteins (I am studying) interact on Tuesday and Wednesday, and say byebye after Thursday.. (near weekend!)....... Did any one meet similar situation of your proteins?

Anyway, I am looking for a buffer, which can give constant results. I alway use PBS with NP40 as both lysis and washing buffer. But I don't know what should I change and why.. For example, the sault concentration, does that mean the concentration of NaCl or all the sault including KCl and MgCl2.. .... Buffer system, for another example, should I use HEPEs or Tris, or something else, can I know why? ... DTT, should I add DTT in my lysis buffer? (I think it will destroy antibody..)... Glycerol, necessary?.. ..


Someone said that lysis buffer is not the optimal buffer for washing,. Then, what can I use for washing?

Sorry for so many questions, but any suggestion will be appreciated.

hi there
u have to try many times to optimize ur results by changing ur salt concentrations for example ( salt i mean her nacl)
and sometimes by changing detergent .
for using of lysis buffer as washing buffer its ok by me i used it and its ok but i read that sometimes u can lose ur protein if u use lysis buffer as washing buffer.
dont use DTT but make sure u use protease inhibitors in ur lysis buffer .
hope this help

I did immunoprecipitation like this:

i washed with lysis buffer and it was ok, my lysis buffer included:

50mM Tris-Hcl
150mM NaCl
1% TritonX-100

I Washed the G protein with this buffer, then add protenase inhibitor cocktail to the cell lysate and incubated it with washed G protein for around 60min
then add pre-cleared cell lysate to the Ab. Good luck!