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abcam 209 GFP antibody for CHIP- is there an alternative GFP antibody useful for

CHIPGFP antibody CHIP-PCR CHGFP IP C.elegans

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#1 michele.xyz

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Posted 25 September 2012 - 06:01 AM

Hi all,
I am working on setting up CHIP using a GFP-tagged protein in C.elegans. I found that the ab209 Abcam rabbit polyclonal antibody is widely used for CHIP so I thought I'd give that a go. But: this antibody only comes in 50ul aliquot for about £200 and I have to optimise the CHIP before I can even start thinking about testing my genes. And I have already used half of it for Western Blotting with moderate success: I didn't get clean blots with this antibody. It is sensitive but not very specific I found on ordinary SDS-PAGE using the abcam protocol for blotting.

Hence my questions:
Does anybody have experience with another GFP antibody for CHIP-PCR? What are your recommendations?
Has anybody used this antibody for CHIP in C.elegans- what concentrations worked well?

I am using the protocol according to the Tissenbaum lab, A Mukhopadhyay - 2008

Any insight-expertise-suggestions would be greatly appreciated!

Thanks!

#2 chabraha

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Posted 25 September 2012 - 11:08 AM

I will be ChIP'ing with this exact antibody O/N tonight............I will have the qPCR results in a day or so.............my GFP tagged protein of interest is in very low abundance within MEFs, so I will let you know how it goes..............if your looking for a good WB antibody I'd recommend Roche's anti-GFP mouse monoclonal............works well in ICC as well.
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#3 michele.xyz

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Posted 26 September 2012 - 01:25 AM

thanks- i am looking forward to your report Posted Image

btw, how much antibody do you use for what concentration of cell lysate? do you CHIP in triplicate?

#4 chabraha

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Posted 27 September 2012 - 08:20 AM

I will do my 1st test run only once...........but then if it works I will always do it at least in triplicate.........especially if enrichments are lower. I decided to scrap my GFP ChIP till next week..........I noticed that the ab290 is unpurified serum so they dont actually give you the concentration of specific serum. However, they do give you the overall concnetration.............and it's huge 5ug/ul.........so I think I'm going to only add 1ul of antibody in 1ml of diluted chromatin (~2x10^6cells)..........the reason im only going to add 1ml is that my protein A beads only bing 5-6ug of IgG per 20ul..........which is what I add to each IP............so long story short, I will hopefully have something good to report next week.
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#5 michele.xyz

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Posted 28 September 2012 - 12:32 AM

Thanks for the heads up! I like!
I have just heard of another new generation GFP- antibody coupled to agarose beads which apparently is very good for CHIP (link below), and somebody else has recommended not to do CHIP with GFP antibody over night, as this increases the background.

http://www.chromotek...apr/gfp-trap-a/)

Another anti GFP I have come across in the literature is this one: 3E6 mouse monoclonal from Invitrogen, which comes as 100ug lyophilised.

I am a little bit unsure about what to make of the concentration of the rabbit antibody. I'll start off using 2mg resuspended in 500ul lysis buffer per tech rep, and I have no idea how much antibody to use so I'll have to establish that. If I get away using little, that would be great, but if I need 10ul/tech rep (which some people in our lab have used but for other antibodies), I need to think of something else because that's going to be so expensive!

Looking forward to your progress update!
Cheers

#6 chabraha

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Posted 23 October 2012 - 08:57 AM

Just wanted to post that the abcam 290 antibody worked just fine for me..........I added 1ul of antibody into ~1ml of diluted chromatin, incubated it overnight and then added protein A beads for 2hrs.
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