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Normal IgG background


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#1 yg_eagle

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Posted 13 May 2009 - 09:41 AM

Hi, all, I am doing chip assay on some transcriptional factors and I use normal rabbit IgG as negative control. I uesd all the methods I knew, but unfortunately, the normal IgG always gave a band in following PCR check. If compared to my positive control, anti-acetylated H4, the band is very weak; if compared to my transcriptional factors antibodies, the differences are not very obvious. I know maybe my transcriptional factors antibodies are not very good. But on the other hand, I do need decrease the normal IgG background. I noticed that in many papers using normal IgG as chip negative control, they all showed clear and perfect backgroud. How did they do that? Does anyone know? I really appreciated your kind help!

#2 KPDE

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Posted 14 May 2009 - 01:39 PM

Hi, all, I am doing chip assay on some transcriptional factors and I use normal rabbit IgG as negative control. I uesd all the methods I knew, but unfortunately, the normal IgG always gave a band in following PCR check. If compared to my positive control, anti-acetylated H4, the band is very weak; if compared to my transcriptional factors antibodies, the differences are not very obvious. I know maybe my transcriptional factors antibodies are not very good. But on the other hand, I do need decrease the normal IgG background. I noticed that in many papers using normal IgG as chip negative control, they all showed clear and perfect backgroud. How did they do that? Does anyone know? I really appreciated your kind help!


If you want to get rid of the band all together I would suggest diluting your DNA 2 fold or more before running PCR or using fewer cycles. It can be difficult to eliminate any background such that it is undetectable by PCR if you run many cycles (like 35 or more).

To help reduce background, the most effective thing we've found is to reduce the amount of chromatin you use (try a two fold and a four fold dilution). In our experience the background decreases proportionately with the decrease in chromatin but the specific IP doesn't (even though I know we are far below the point where we could be saturating the antibody).

#3 yg_eagle

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Posted 17 May 2009 - 10:09 PM

Hi, KPDE, thank you so much for your kind reply. I just talked to a chip expert yesterday and she almost gave the same suggestions as you. That is, reduce the amount of chromatin for IP first, then adjust template amount and cycle number of PCR later. Sounds really good ideas! I will try them in my next round chip assay.

Many thanks again!

Ge




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