My ChIP product Western is dirty - (Oct/05/2006 )
Hi guys,
After my ChIP, I ran an western to see whether my protein had been pulled down. I used the same antibody both for IP and for western, so I knew the antibody in IP was gonna show up (as a heavy chain and a light chain) in my western. However, the western film turned out much more dirty than I expected. Although I see my protein in WT, not in KO sample, the background was so high that it was just some black strip runing from the top to the bottom. Anybody met this problem before? What are those backgrounds? Thanks a lot!
Also, on the blot, the IP band of my protein is faint. So I think the reason was not that I used too much antibody. This antibody was made in house, and it works very specificaly on my Input sample (the lysate before IP). So what gave me such a big black strip on my IP sample lanes?
After my ChIP, I ran an western to see whether my protein had been pulled down. I used the same antibody both for IP and for western, so I knew the antibody in IP was gonna show up (as a heavy chain and a light chain) in my western. However, the western film turned out much more dirty than I expected. Although I see my protein in WT, not in KO sample, the background was so high that it was just some black strip runing from the top to the bottom. Anybody met this problem before? What are those backgrounds? Thanks a lot!
Hi guys,
After my ChIP, I ran an western to see whether my protein had been pulled down. I used the same antibody both for IP and for western, so I knew the antibody in IP was gonna show up (as a heavy chain and a light chain) in my western. However, the western film turned out much more dirty than I expected. Although I see my protein in WT, not in KO sample, the background was so high that it was just some black strip runing from the top to the bottom. Anybody met this problem before? What are those backgrounds? Thanks a lot!
You reversed the crosslinking on the sample you ran on the gel, right?
Yes. Before loading the gel, I incubated my samples with loading buffer (containing BME) on 95 C for 15 mins. Is that enough to reverse the crosslink?
Also, on the blot, the IP band of my protein is faint. So I think the reason was not that I used too much antibody. This antibody was made in house, and it works very specificaly on my Input sample (the lysate before IP). So what gave me such a big black strip on my IP sample lanes?
Hi guys,
After my ChIP, I ran an western to see whether my protein had been pulled down. I used the same antibody both for IP and for western, so I knew the antibody in IP was gonna show up (as a heavy chain and a light chain) in my western. However, the western film turned out much more dirty than I expected. Although I see my protein in WT, not in KO sample, the background was so high that it was just some black strip runing from the top to the bottom. Anybody met this problem before? What are those backgrounds? Thanks a lot!
You reversed the crosslinking on the sample you ran on the gel, right?
Also, on the blot, the IP band of my protein is faint. So I think the reason was not that I used too much antibody. This antibody was made in house, and it works very specificaly on my Input sample (the lysate before IP). So what gave me such a big black strip on my IP sample lanes?
Hi guys,
After my ChIP, I ran an western to see whether my protein had been pulled down. I used the same antibody both for IP and for western, so I knew the antibody in IP was gonna show up (as a heavy chain and a light chain) in my western. However, the western film turned out much more dirty than I expected. Although I see my protein in WT, not in KO sample, the background was so high that it was just some black strip runing from the top to the bottom. Anybody met this problem before? What are those backgrounds? Thanks a lot!
You reversed the crosslinking on the sample you ran on the gel, right?
In our Fast ChIP method we incubate the DNA for 10-20min at 95-100C so I would say that the 95C for 15 min is sufficient. The only difference is that in our protocol the solution is very basic (around pH 10-11). This may be significant in catalyzing the breakage of the methylene bridge crosslink (we don't know).
Hi Sure,
I met the same problem, and I still don't know the solution.
Try to increase the washing time of the beads. It helped in my case to some extent.
Anyway, what kind of buffer do you use for washing? And do you apply a pre-clearing step during immunoprecipitation?
If you could find out something, please let me know it.
Thanks.
I met the same problem, and I still don't know the solution.
Try to increase the washing time of the beads. It helped in my case to some extent.
Anyway, what kind of buffer do you use for washing? And do you apply a pre-clearing step during immunoprecipitation?
If you could find out something, please let me know it.
Thanks.
Have you tried a control using only beads (no chromatin). THis would at least narrow the problem down to either the beads or the chromatin sample.
I haven't tried that, thank you for the idea.