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T lymphocyte ChIPs are not working...


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6 replies to this topic

#1 Clare

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Posted 26 March 2009 - 07:28 AM

Hi everyone :(

I'm stuck. For a while we have been trying to ChIP human T cells (sorted from peripheral blood using CD3 MACS beads) and it's not working :rolleyes: When we ChIP cells from AML patients (not sorted) the ChIP works fine. We have tried ~6 different primer sets and the result is always the same. The T cell ChIPs do not work! ARG! I am going crazy! Why would all other chips work and not T cells? There must be something I have overlooked and it's driving me batty.

Any ideas??

Please???

I've always said "B cells are brilliant, T cells are trouble"

Clare

#2 KPDE

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Posted 26 March 2009 - 01:48 PM

Hi everyone :P

I'm stuck. For a while we have been trying to ChIP human T cells (sorted from peripheral blood using CD3 MACS beads) and it's not working :wacko: When we ChIP cells from AML patients (not sorted) the ChIP works fine. We have tried ~6 different primer sets and the result is always the same. The T cell ChIPs do not work! ARG! I am going crazy! Why would all other chips work and not T cells? There must be something I have overlooked and it's driving me batty.

Any ideas??

Please???

I've always said "B cells are brilliant, T cells are trouble"

Clare


Have you tried ChIP on any other sorted cells and had it work?

Edited by KPDE, 26 March 2009 - 01:49 PM.


#3 Clare

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Posted 27 March 2009 - 02:14 AM

Hey there :)

Yes we have. We sorted human CD34+ cells using MACS beads and they work fine as well.
Hmmm..

Clare

PS: Happy Friday!


Have you tried ChIP on any other sorted cells and had it work?
[/quote]

#4 KPDE

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Posted 27 March 2009 - 04:52 PM

Hey there :rolleyes:

Yes we have. We sorted human CD34+ cells using MACS beads and they work fine as well.
Hmmm..

Clare

PS: Happy Friday!


Have you tried ChIP on any other sorted cells and had it work?



So when you say the ChIP isn't working, what isn't working? Low signal, very little difference between mock and IP at your region of interest, enrichment at a negative control region?

#5 Clare

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Posted 06 April 2009 - 03:40 AM

Hi again,

Sorry for the late reply - I have been in Belgium enjoying many beers :)

With our T cell chips we are not seeing any difference between the mock and the IP :( And that's with quite a few different sets of primers too.

Clare


So when you say the ChIP isn't working, what isn't working? Low signal, very little difference between mock and IP at your region of interest, enrichment at a negative control region?
[/quote]

#6 JPchip

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Posted 29 April 2009 - 05:29 PM

I'm curious what it is you're trying to ChIP in your T-cells and whether the target is the same in B-cells that do work.

My first though was that you might be trying to ChIP a transcription factor on chromatin which might have been dissociating from DNA during time it takes to do the sorting which I assume occurs before fixation, but if CD34+ sorted cells work then I guess that rules that out.

Could the thing you've overlooked be that this site is simply not occupied by whatever it is you're ChIPing for in T-cell but it is in B-cells. Is there any reason it should be occupied in both, since the function of T-cells and B-cell is very different. Is there a suitable positive control you could use to be sure there is nothing wrong with the ChIP protocol for T-cell preparations (e.g. another site known to bind the factor of interest, or another antibody to a generic protein like a histone or RNA Polymerase II)?

#7 Clare

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Posted 05 May 2009 - 02:12 AM

I'm curious what it is you're trying to ChIP in your T-cells and whether the target is the same in B-cells that do work.

My first though was that you might be trying to ChIP a transcription factor on chromatin which might have been dissociating from DNA during time it takes to do the sorting which I assume occurs before fixation, but if CD34+ sorted cells work then I guess that rules that out.

Could the thing you've overlooked be that this site is simply not occupied by whatever it is you're ChIPing for in T-cell but it is in B-cells. Is there any reason it should be occupied in both, since the function of T-cells and B-cell is very different. Is there a suitable positive control you could use to be sure there is nothing wrong with the ChIP protocol for T-cell preparations (e.g. another site known to bind the factor of interest, or another antibody to a generic protein like a histone or RNA Polymerase II)?


Hi there :)

We are looking at histone mods and since I posted this, our ChIPs are actually working now :) Turns out our cells like to be cultured for 24h after isolation!
C




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