Can someone explain what actually happens when washing beads in ChIP? Should I b - (Nov/23/2014 )
Hi folks,
I hope someone can explain this to me. Any time I ask somebody about this they just reply saying "It' just washes the beads"
I want to know what the effects of washing the ChIP'd DNA-Protein complexes attached to sepharose beads with different wash buffers.
Typical wash buffers:
Low Salt Wash Buffer
0.1% SDS
0.1% Triton X-100 150 mM NaCl
2mM EDTA
20mM Tris-HCl pH 8.0 dd H2O
0.22μm filtered
Store at 4oC
High Salt Wash Buffer
0.1% SDS
0.1% Triton X-100 500 mM NaCl
2mM EDTA
20mM Tris-HCl pH 8.0 dd H2O
0.22μm filtered
IP LiCl Wash Buffer
0.5 M LiCl
1% NP-40
1% deoxycholic acid 100mM Tris-Hcl pH 9.0 dd H2O
0.22μm filtered
Store at 4oC
As most of you know you wash with "low salt" first, then "high salt", then LiCl buffer... I want to know what exactly is supposed to happen/what is the function of each one of these wash steps?
Is it possible that any one of these wash steps can be to stringent for the protein-DNA-bead complexes thereby loosing immunoprecipitated sample??
Thank you
A wash buffer is just that. It washes off the dirty stains on the wall while retaining the paint. Each of these buffers have different stringency for washing, meaning they would remove non-specific junk attached to your beads depending upon how strongly it is bound. Some non-specific complexes are weakly bound and will go away with low salt buffer, some will survive that, so you step up your game with a higher stringency buffer.
Ultimately, each experiment is different. The precise three steps are just an ideal procedure that works most of the time, but not in all cases. So if you are dealing with something too softly bound or too tightly bound, you have to find your own process. It is not optional. That's why they pay us big bucks.
Wait, do they?