some questions about fast ChIP - (Mar/27/2008 )
i have some questions about the fast ChIP from the nature protocol
this is what i did in my experiment.
i just incubated antibody and chromatin O/N , and before adding them into protein G sepharose Beads, i wash beads three times with IP buffer(inhibitor inside) at 4 C,and then dilute beads with 200ul IP buffer(inhibitor,100ug/ml salmon sperm DNA and 5%BSA ), incubate for 1h before mix them with beads.
i just want to ask why does the protocal say that beads are washed at 20-25C, can non-specific thing binded on bead be washed off more easily/
and i also want know if 1h is enough for blocking beads.
and the isolation step, can i use tris to resuspend beads ,you know this can make DNA more stable and can easily stored longer,cause i'm always afraid that DNA will be degraded before i run it on PCR again.
many thx!
wish anyone could help me about this. 
best wishes
this is what i did in my experiment.
i just incubated antibody and chromatin O/N , and before adding them into protein G sepharose Beads, i wash beads three times with IP buffer(inhibitor inside) at 4 C,and then dilute beads with 200ul IP buffer(inhibitor,100ug/ml salmon sperm DNA and 5%BSA ), incubate for 1h before mix them with beads.
i just want to ask why does the protocal say that beads are washed at 20-25C, can non-specific thing binded on bead be washed off more easily/
and i also want know if 1h is enough for blocking beads.
and the isolation step, can i use tris to resuspend beads ,you know this can make DNA more stable and can easily stored longer,cause i'm always afraid that DNA will be degraded before i run it on PCR again.
many thx!
wish anyone could help me about this.
best wishes
It doesn't really matter at what temp the beads are washed. I think the 22-25C is in there because Nature Protocols was particular about making sure we put a temperature down for each centrifugation step.
As for blocking the beads, I'm sure 1 hour is sufficient.
Regarding the elution step: I don't think using tris buffer could cause any problems and is probably even an improvement. In our Matrix ChIP protocol we store the DNA in 25mM Tris, 1mM EDTA at pH 9.5-10. This pH may seem a little high but, in our experience, is even better at storing DNA of very low concentrations. And since Tris is such a weak buffer at that high of a pH we don't have as many problems with PCR as we would likely have with a buffer which is nearer its buffering range at pH 10 (like a bicarbonate buffer).
Anyways, good luck.
Joel
thanks a lot
wish i could get a good result 