10 replies to this topic

[nigelcr]

What's the general consensus as to what constitutes a ChIP grade antibody?  What experiments do I have to perform to prove that my antibody is ChIP grade?  Thanks!

[KPDE]

nigelcr, on Mar 10 2010, 08:26 AM, said:

What's the general consensus as to what constitutes a ChIP grade antibody?  What experiments do I have to perform to prove that my antibody is ChIP grade?  Thanks!

A ChIP grade antibody is considered ChIP grade if it works in ChIP .

An experiment you can try is perform ChIP, reverse crosslinks but do not add proteinase K and run the eluted protein on Western to see if you pulled it down.  It's not enough to just do a straight IP as the crosslinking can sometimes mask epitopes.

[epigenius]

KPDE, on Mar 11 2010, 04:42 PM, said:

nigelcr, on Mar 10 2010, 08:26 AM, said:

What's the general consensus as to what constitutes a ChIP grade antibody?  What experiments do I have to perform to prove that my antibody is ChIP grade?  Thanks!

A ChIP grade antibody is considered ChIP grade if it works in ChIP .

An experiment you can try is perform ChIP, reverse crosslinks but do not add proteinase K and run the eluted protein on Western to see if you pulled it down.  It's not enough to just do a straight IP as the crosslinking can sometimes mask epitopes.

yes, make sure to use a polyclonal Ab. Polyclonal will help reduce the chance of masked epitopes during crosslinking

[Dukey]

KPDE, on Mar 11 2010, 02:42 PM, said:

A ChIP grade antibody is considered ChIP grade if it works in ChIP .

My thoughts exactly!

[nigelcr]

KPDE, on Mar 11 2010, 02:42 PM, said:

nigelcr, on Mar 10 2010, 08:26 AM, said:

What's the general consensus as to what constitutes a ChIP grade antibody?  What experiments do I have to perform to prove that my antibody is ChIP grade?  Thanks!

A ChIP grade antibody is considered ChIP grade if it works in ChIP .

An experiment you can try is perform ChIP, reverse crosslinks but do not add proteinase K and run the eluted protein on Western to see if you pulled it down.  It's not enough to just do a straight IP as the crosslinking can sometimes mask epitopes.

Thanks for the reply.  I've done the experiment that you suggested and I do see my protein at the appropriate molecular weight using a polyclonal antibody on a western.  However I do see a number of accessory bands, all of which are smaller than the protein of interest.  I'm assuming these are one of 2 things: 1) a combination of degradation products and alternative isoforms; or 2) non-specific crap.

It gets a little more tricky when I look at actual ChIP data where I'm immunoprecipitating with my polyclonal antibody.  Here's what I see for a gene that I expect to be regulated by my protein of interest:

Protein of interest ChIP % input = 0.3486%
PolII ChIP % input = 0.5147%
IgG ChIP % input = 0.0077%

Oh wow, my polyclonal antibody works for ChIP right?  Well, I'm not so convinced.  Here is what I see for the housekeeping gene PP1A:

Protein of interest ChIP % input = 0.6542%
PolII ChIP % input = 2.7785%
IgG ChIP % input = 0.031%

So I'm getting ~ 20 fold enrichment in the ChIP with my protein of interest compared to the IgG control in the housekeeping gene where my protein of interest has no business binding.  I'm using a pretty generic ChIP protocol as far as I can determine.  I am a novice at this and any constructive comments would greatly be appreciate.  Thanks!

[NemaToStella]

nigelcr, on Mar 15 2010, 04:23 PM, said:

KPDE, on Mar 11 2010, 02:42 PM, said:

nigelcr, on Mar 10 2010, 08:26 AM, said:

What's the general consensus as to what constitutes a ChIP grade antibody?  What experiments do I have to perform to prove that my antibody is ChIP grade?  Thanks!

A ChIP grade antibody is considered ChIP grade if it works in ChIP .

An experiment you can try is perform ChIP, reverse crosslinks but do not add proteinase K and run the eluted protein on Western to see if you pulled it down.  It's not enough to just do a straight IP as the crosslinking can sometimes mask epitopes.

Thanks for the reply.  I've done the experiment that you suggested and I do see my protein at the appropriate molecular weight using a polyclonal antibody on a western.  However I do see a number of accessory bands, all of which are smaller than the protein of interest.  I'm assuming these are one of 2 things: 1) a combination of degradation products and alternative isoforms; or 2) non-specific crap.

It gets a little more tricky when I look at actual ChIP data where I'm immunoprecipitating with my polyclonal antibody.  Here's what I see for a gene that I expect to be regulated by my protein of interest:

Protein of interest ChIP % input = 0.3486%
PolII ChIP % input = 0.5147%
IgG ChIP % input = 0.0077%

Oh wow, my polyclonal antibody works for ChIP right?  Well, I'm not so convinced.  Here is what I see for the housekeeping gene PP1A:

Protein of interest ChIP % input = 0.6542%
PolII ChIP % input = 2.7785%
IgG ChIP % input = 0.031%

So I'm getting ~ 20 fold enrichment in the ChIP with my protein of interest compared to the IgG control in the housekeeping gene where my protein of interest has no business binding.  I'm using a pretty generic ChIP protocol as far as I can determine.  I am a novice at this and any constructive comments would greatly be appreciate.  Thanks!

This is probably a stupid question, but how sure are you that your protein of interest is not binding to the housekeeping gene (more specifically, what region are you looking at - intron, exon, 5' UTR etc...?). Genomw-wide we often see binding of specific transcription factors to regulatory regions of supposed housekeeping genes, often within the 5' UTR/promoter (and you do have a high PolII enrichment!). I am not too surprised about your results.

[nigelcr]

NemaToStella, on Mar 16 2010, 10:50 PM, said:

nigelcr, on Mar 15 2010, 04:23 PM, said:

KPDE, on Mar 11 2010, 02:42 PM, said:

nigelcr, on Mar 10 2010, 08:26 AM, said:

What's the general consensus as to what constitutes a ChIP grade antibody?  What experiments do I have to perform to prove that my antibody is ChIP grade?  Thanks!

A ChIP grade antibody is considered ChIP grade if it works in ChIP .

An experiment you can try is perform ChIP, reverse crosslinks but do not add proteinase K and run the eluted protein on Western to see if you pulled it down.  It's not enough to just do a straight IP as the crosslinking can sometimes mask epitopes.

Thanks for the reply.  I've done the experiment that you suggested and I do see my protein at the appropriate molecular weight using a polyclonal antibody on a western.  However I do see a number of accessory bands, all of which are smaller than the protein of interest.  I'm assuming these are one of 2 things: 1) a combination of degradation products and alternative isoforms; or 2) non-specific crap.

It gets a little more tricky when I look at actual ChIP data where I'm immunoprecipitating with my polyclonal antibody.  Here's what I see for a gene that I expect to be regulated by my protein of interest:

Protein of interest ChIP % input = 0.3486%
PolII ChIP % input = 0.5147%
IgG ChIP % input = 0.0077%

Oh wow, my polyclonal antibody works for ChIP right?  Well, I'm not so convinced.  Here is what I see for the housekeeping gene PP1A:

Protein of interest ChIP % input = 0.6542%
PolII ChIP % input = 2.7785%
IgG ChIP % input = 0.031%

So I'm getting ~ 20 fold enrichment in the ChIP with my protein of interest compared to the IgG control in the housekeeping gene where my protein of interest has no business binding.  I'm using a pretty generic ChIP protocol as far as I can determine.  I am a novice at this and any constructive comments would greatly be appreciate.  Thanks!

This is probably a stupid question, but how sure are you that your protein of interest is not binding to the housekeeping gene (more specifically, what region are you looking at - intron, exon, 5' UTR etc...?). Genomw-wide we often see binding of specific transcription factors to regulatory regions of supposed housekeeping genes, often within the 5' UTR/promoter (and you do have a high PolII enrichment!). I am not too surprised about your results.

No, that definitely isn't a stupid question, and it is possible that my protein interacts with the PP1A promoter (the primers amplify the 5'-UTR and TSS of PP1A).  I've shown in earlier experiments that my protein interacts with several general chromatin components, so it could interact with the promoter of this gene.  However, I probably should have shared a little more data and details of my experimental setup, which may make my concerns a little clearer.

I initially did these experiments by transiently transfecting 293 cells with constructs encoding either HA-tagged protein of interest or lacZ-V5, and then running ChIPs in both cell cultures using either the protein-specific antibody or a supposedly ChIP-grade anti-HA antibody from Abcam (ab9110).  These are the ChIP-qPCR data that I got for PP1A in both sets of cells:

293/HA-Gene of Interest:
Anti-HA ChIP % input = 0.8527%
Anti-protein of interest ChIP % input =  1.2289%
Anti-PolII ChIP % input = 4.9938%
IgG ChIP % input = 0.0085%


293/lacZ-V5:
Anti-HA ChIP % input = 0.4148%
Anti-protein of interest ChIP % input = 0.4307%
Anti-PolII ChIP % input = 7.6386%
IgG ChIP % input = 0.0058%

So, the ChIPs in the cells transfected with the HA-tagged protein look okay to me, with the protein-specific antibody behaving pretty much as I'd seen before.  However, the concerning results are the ones for the cells transfected with lacZ-V5.  As you can see, the ChIP-grade anti-HA antibody is ChIPing as much PP1A promoter DNA as the protein-specific antibody.  Given that the HA-antibody shouldn't be detecting *anything* in this experiment, it leads me to conclude that either I'm doing something wrong (I'm currently trying other protocols and conditions) or the ChIP-grade anti-HA antibody isn't actually ChIP-grade.

I guess this brings me back to my original question:  is there any uniform consensus as to what a 'ChIP-grade' antibody is?  Clearly, the Abcam anti-HA antibody "works for ChIP" until I used it in a cell line where no HA was being expressed.  Also (and probably more importantly for me  ), if anyone has any ideas as to what is going on here and how I can get this experiment working I'd really appreciate it.  Thanks in advance!!

[Mighty Mouse]

For what it's worth I've had some luck using primers for LINE1 as a negative control. It seems to me the best way to determine if you have successfully performed a ChIP is to compare your %input from your gene of interest versus a negative control region (as suggested by KDPE in these forums). I've been using LINE1 and I've seen others use the 28srRNA gene. I've also had a little bit of success using an intergenic region ~40kbps upstream from my promoter of interest, although I have some difficulty getting clean qPCR data from such regions as there is very little binding in general...

MM

[nigelcr]

Mighty Mouse, on Mar 17 2010, 06:08 PM, said:

For what it's worth I've had some luck using primers for LINE1 as a negative control. It seems to me the best way to determine if you have successfully performed a ChIP is to compare your %input from your gene of interest versus a negative control region (as suggested by KDPE in these forums). I've been using LINE1 and I've seen others use the 28srRNA gene. I've also had a little bit of success using an intergenic region ~40kbps upstream from my promoter of interest, although I have some difficulty getting clean qPCR data from such regions as there is very little binding in general...

MM

Thanks mighty mouse.  If you have been doing ChIP with human cells would you be willing to share the sequence of the LINE1 primers?

[Mighty Mouse]

Well I don't do ChIP with human cells, I do it with mouse brain. But here are the primer sequences, perhaps they'll be of some use to you:

LINE1-F 5’-AAA CGA GGA GTT GGT TCT TTG AG-3’
LINE1-R 5’-TTT GTC CCT GTG CCC TTT AGT GA-3’

MM

[nc03]

I am trying to do ChIP with HA tagged protein. Anyone knows which company has good quality anti HA antibody (I mean ChIP grade that has worked for you).
Thanks in advance
nc03