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CHIP questions


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#1 rick2000

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Posted 30 January 2009 - 01:38 AM

Hello again....I have some questions regarding CHIP assays.


First of all, is it wrong to have a mock control only with the beads or do I have to put an unspecific antibody? Some researches use beads and some others an antibody.

A second question : Is it safe to compare the results between different antibodies, because, as far as I know, each antibody has different specificity.

And a third question : Do you use an antibody for the core region of histone H3 or H4 to check nucleosome density?

Thanks very muck in advance

#2 Davo

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Posted 01 February 2009 - 01:59 PM

A mock control is better if you use a non-specific antibody. I think I got mine from Santa Cruz - Normal Rabbit IgG, and its cheap.

When you say compare antibodies, do you mean the same kind of antibody or two antibodies for two different targets? I think it a little different to compare different antibodies, however the use of +ve and -ve controls may make it easier to interpret.

I can't help you with the 3rd questions sorry...

Dave

#3 rick2000

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Posted 01 February 2009 - 11:55 PM

A mock control is better if you use a non-specific antibody. I think I got mine from Santa Cruz - Normal Rabbit IgG, and its cheap.

When you say compare antibodies, do you mean the same kind of antibody or two antibodies for two different targets? I think it a little different to compare different antibodies, however the use of +ve and -ve controls may make it easier to interpret.

I can't help you with the 3rd questions sorry...

Dave


Thanks Dave

I mean two antibodies for two different targets. I see a lot of articles where they compare the results from two different atibodies, and I am not very sure that I agree with this.

#4 KPDE

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Posted 02 February 2009 - 07:02 PM

Hello again....I have some questions regarding CHIP assays.


First of all, is it wrong to have a mock control only with the beads or do I have to put an unspecific antibody? Some researches use beads and some others an antibody.

A second question : Is it safe to compare the results between different antibodies, because, as far as I know, each antibody has different specificity.

And a third question : Do you use an antibody for the core region of histone H3 or H4 to check nucleosome density?

Thanks very muck in advance


I've used beads alone and non-immune IgG both multiple times and haven't seen a difference. This may not be the case for every region of the genome but at least for where I've looked it doesn't seem to matter which one you use.

I don't see how it would be possible to compare the level of enrichment for two different antibodies. The enrichment will be heavily affected by the affinity of the antibody, the level of epitope masking of the antigen, and a few other factors completely unrelated to the density of binding of your factor of interest.

I've used Abcam ab1791 antibody for H3 as a proxy for nucleosome density. The results correlated well with Dnase I hypersensitivity data.




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