Misc problems from ChIP - (Jul/06/2009 )
Hi guys,
It is me again. =)
1) I found somewhere in this forum regarding a blocking step.
Blocking step
1) Incubate the Salmon Sperm DNA Agarose in 1% BSA in ChIP dilution buffer (to block the agarose matrix)
2) Incubate for 30 minutes mixing at room temperature
3) After incubation, spin agarose and remove solution
4) Wash once in ChIP assay buffer and continue assay as normal
The problem is the beads. After washing with ChIP dilution buffer, the beads somehow clump together and somehow solidify. Thus the amount of beads for every ChIP is different. Did I do something wrong here?
2) I did 3 times for ChIP. 1st time, unspecific binding. 2nd time, with pre clearing over 4 hours and blocking step, I have nice enrichment of my antibody. 3rd time with the same condition as 2nd time, it is not reproducible. The only difference here is I did not check my shearing for the 3rd time. What can go wrong here?
Please help and thank you very much.
Cheers
i used to block but now now used magnetic beads in the invitrogen ChIP kit. it's better for what i'm doing especially since i want to do ChIP-seq and don't want to sequence up salmon sperm dna by chance.
hope this helps
timjim on Jul 6 2009, 01:58 PM said:
It is me again. =)
1) I found somewhere in this forum regarding a blocking step.
Blocking step
1) Incubate the Salmon Sperm DNA Agarose in 1% BSA in ChIP dilution buffer (to block the agarose matrix)
2) Incubate for 30 minutes mixing at room temperature
3) After incubation, spin agarose and remove solution
4) Wash once in ChIP assay buffer and continue assay as normal
The problem is the beads. After washing with ChIP dilution buffer, the beads somehow clump together and somehow solidify. Thus the amount of beads for every ChIP is different. Did I do something wrong here?
2) I did 3 times for ChIP. 1st time, unspecific binding. 2nd time, with pre clearing over 4 hours and blocking step, I have nice enrichment of my antibody. 3rd time with the same condition as 2nd time, it is not reproducible. The only difference here is I did not check my shearing for the 3rd time. What can go wrong here?
Please help and thank you very much.
Cheers
I would think the best thing to do (aside from switching to magnetic beads) is to do your IP in the presence of the blocking reagents rather than washing the beads off (i.e. do the ChIP in dilution buffer containing 5% BSA and 100ug/ml sssDNA).
Thanks for the input. My ChIP is working now by using dynabeads. I am still quite confused with Protein A or Protein G but I guess both works as well.
I do use BSA for my IP and now I still have some slight IgG background. I dont know how to get rid of them! =)
I have another question. I have two samples: treated and non treated and I would like to check on ChIP seq or ChIP PCR. Could I do WGA or LM PCR to obtain more ChIP DNA so that I do not have to repeat my ChIP again. Is it feasible? Or is the WGA will interfere with the downstream application for quantitative purposes.
Thanks alot!