4 replies to this topic

[HP1b]

Hi everybody,
during a conversation with some chemists, I´ve been told that formaldehyde reversing X-linking (high temperature and high salt concentration) may result in a loss of histone acetylation levels due to some similarities between the nature of bond that formaldehyde and the acetyl-group have. In other words, reverse X-linking might determine a sort of "chemical deacetylation".

Is anybody aware of such issue?

I´ve been working on ChIP for a long time and this issue doesn´t really matter as the IP is already done at the stage of reverse X-linking, but now, since I´m planning to perform a specific assay which is not a conventional ChIP, such issue might be critical to be addressed.

Basically, what I´m planning to do is a sort of Co-IP assay, by pulling down of a specific histone modification and then performing a western blot detection against acetylated residues. Since I think I need either to perform X-linking to keep the complexes or to perform (by mere assumption) reverse X-linking to properly run a PAGE, I´m wondering wheather the reverse X-linking itself might somehow affect the outcome by "chemically deacetylating" my targets.

Any suggestion?

I´m sorry for the quite weird question and I hope to read your feedback.

Edited by HP1b, 23 June 2009 - 02:18 AM.

[KPDE]

HP1b, on Jun 23 2009, 03:17 AM, said:

Hi everybody,
during a conversation with some chemists, I´ve been told that formaldehyde reversing X-linking (high temperature and high salt concentration) may result in a loss of histone acetylation levels due to some similarities between the nature of bond that formaldehyde and the acetyl-group have. In other words, reverse X-linking might determine a sort of "chemical deacetylation".

Is anybody aware of such issue?

I´ve been working on ChIP for a long time and this issue doesn´t really matter as the IP is already done at the stage of reverse X-linking, but now, since I´m planning to perform a specific assay which is not a conventional ChIP, such issue might be critical to be addressed.

Basically, what I´m planning to do is a sort of Co-IP assay, by pulling down of a specific histone modification and then performing a western blot detection against acetylated residues. Since I think I need either to perform X-linking to keep the complexes or to perform (by mere assumption) reverse X-linking to properly run a PAGE, I´m wondering wheather the reverse X-linking itself might somehow affect the outcome by "chemically deacetylating" my targets.

Any suggestion?

I´m sorry for the quite weird question and I hope to read your feedback.

We have done the reversal of crosslinking two different ways: one being the traditional method, 65C in 500mM NaCl with a high pH (probably around 10 or so) for 4-6 hours, the second being boiling (100C) for 10 minutes in a high pH (10-11) chelating (EDTA or chelex) buffer.  We get consistent ChIP results for H3K9Ac with either method.  I would think that, if heat were chemically deacetylating histones, then ChIP results for acetylated histones would be fairly inconsistent.  Also, do your chemistry friends know if the methylene bridge of the crosslink and the amide bond in acetylated lysines behave differently at high pH (~10), as this is the pH that reversal of crosslinking usually takes place in.

[HP1b]

KPDE, on Jun 24 2009, 01:03 PM, said:

We have done the reversal of crosslinking two different ways: one being the traditional method, 65C in 500mM NaCl with a high pH (probably around 10 or so) for 4-6 hours, the second being boiling (100C) for 10 minutes in a high pH (10-11) chelating (EDTA or chelex) buffer.  We get consistent ChIP results for H3K9Ac with either method.  I would think that, if heat were chemically deacetylating histones, then ChIP results for acetylated histones would be fairly inconsistent.  Also, do your chemistry friends know if the methylene bridge of the crosslink and the amide bond in acetylated lysines behave differently at high pH (~10), as this is the pH that reversal of crosslinking usually takes place in.

Thanks for the answer KPDE,
the point, though, is that you perform the reversal of X-linking after the IP and at that stage you wouldn´t care anymore of protein as the thing you´re interested in is DNA and you basically want to detach it from histones (in a ChIP experiment). So, you IP H3K9Ac histone and then perform the reversal of X-linking in the IPed fraction containing DNA of interest.

My case is different, for such an assay I´m not interested in DNA at all, not at this stage at least. Rather, I would need to collect, let´s say, the other histones that are IPed together with my target and then try and visualize them on a PA gel. In this case, I feel the second method you propose could fit, as I actually should "boil" my samples before loading them. Do You think I could use Lämmli sample buffer directly to the sample? Would the pH be too low to properly perform the reversal of X-linking? I would really need to have the reversal of X-linking done, otherwise I think the gel run wuold be likely compromised.

In other words, if I made myself clear, what I look for is a way to collect IPed proteins which are suitable for subsequent Western Blot analysis from a sample that has been X-linked.

Thanks again for your answer!

edit: Oh, I will ask my collegues for sure about the possible different behaviours of methylene bridge of the crosslink and the amide bond in acetylated lysines at high pH.

Edited by HP1b, 25 June 2009 - 05:55 AM.

[KPDE]

HP1b, on Jun 25 2009, 06:49 AM, said:

KPDE, on Jun 24 2009, 01:03 PM, said:

We have done the reversal of crosslinking two different ways: one being the traditional method, 65C in 500mM NaCl with a high pH (probably around 10 or so) for 4-6 hours, the second being boiling (100C) for 10 minutes in a high pH (10-11) chelating (EDTA or chelex) buffer.  We get consistent ChIP results for H3K9Ac with either method.  I would think that, if heat were chemically deacetylating histones, then ChIP results for acetylated histones would be fairly inconsistent.  Also, do your chemistry friends know if the methylene bridge of the crosslink and the amide bond in acetylated lysines behave differently at high pH (~10), as this is the pH that reversal of crosslinking usually takes place in.

Thanks for the answer KPDE,
the point, though, is that you perform the reversal of X-linking after the IP and at that stage you wouldn´t care anymore of protein as the thing you´re interested in is DNA and you basically want to detach it from histones (in a ChIP experiment). So, you IP H3K9Ac histone and then perform the reversal of X-linking in the IPed fraction containing DNA of interest.

My case is different, for such an assay I´m not interested in DNA at all, not at this stage at least. Rather, I would need to collect, let´s say, the other histones that are IPed together with my target and then try and visualize them on a PA gel. In this case, I feel the second method you propose could fit, as I actually should "boil" my samples before loading them. Do You think I could use Lämmli sample buffer directly to the sample? Would the pH be too low to properly perform the reversal of X-linking? I would really need to have the reversal of X-linking done, otherwise I think the gel run wuold be likely compromised.

In other words, if I made myself clear, what I look for is a way to collect IPed proteins which are suitable for subsequent Western Blot analysis from a sample that has been X-linked.

Thanks again for your answer!

edit: Oh, I will ask my collegues for sure about the possible different behaviours of methylene bridge of the crosslink and the amide bond in acetylated lysines at high pH.

That's what I get for reading a post just before going home.  I was a little slow on the uptake.

As far as the pH of sample buffer being too low for reversal of crosslinking, I can't say.  I'm not sure if the pH is important for the reversal or not.  I've always assumed that the main importance of the pH was in protecting the DNA at high temperature, however, it may aid in the crosslinking reversal as well.  I think giving it a try with some non-precious chromatin might be the way to go.

[daying6001]

KPDE, on 25 June 2009 - 12:05 PM, said:

HP1b, on Jun 25 2009, 06:49 AM, said:

KPDE, on Jun 24 2009, 01:03 PM, said:

We have done the reversal of crosslinking two different ways: one being the traditional method, 65C in 500mM NaCl with a high pH (probably around 10 or so) for 4-6 hours, the second being boiling (100C) for 10 minutes in a high pH (10-11) chelating (EDTA or chelex) buffer.  We get consistent ChIP results for H3K9Ac with either method.  I would think that, if heat were chemically deacetylating histones, then ChIP results for acetylated histones would be fairly inconsistent.  Also, do your chemistry friends know if the methylene bridge of the crosslink and the amide bond in acetylated lysines behave differently at high pH (~10), as this is the pH that reversal of crosslinking usually takes place in.

Thanks for the answer KPDE,
the point, though, is that you perform the reversal of X-linking after the IP and at that stage you wouldn´t care anymore of protein as the thing you´re interested in is DNA and you basically want to detach it from histones (in a ChIP experiment). So, you IP H3K9Ac histone and then perform the reversal of X-linking in the IPed fraction containing DNA of interest.

My case is different, for such an assay I´m not interested in DNA at all, not at this stage at least. Rather, I would need to collect, let´s say, the other histones that are IPed together with my target and then try and visualize them on a PA gel. In this case, I feel the second method you propose could fit, as I actually should "boil" my samples before loading them. Do You think I could use Lämmli sample buffer directly to the sample? Would the pH be too low to properly perform the reversal of X-linking? I would really need to have the reversal of X-linking done, otherwise I think the gel run wuold be likely compromised.

In other words, if I made myself clear, what I look for is a way to collect IPed proteins which are suitable for subsequent Western Blot analysis from a sample that has been X-linked.

Thanks again for your answer!

edit: Oh, I will ask my collegues for sure about the possible different behaviours of methylene bridge of the crosslink and the amide bond in acetylated lysines at high pH.

That's what I get for reading a post just before going home.  I was a little slow on the uptake.

As far as the pH of sample buffer being too low for reversal of crosslinking, I can't say.  I'm not sure if the pH is important for the reversal or not.  I've always assumed that the main importance of the pH was in protecting the DNA at high temperature, however, it may aid in the crosslinking reversal as well.  I think giving it a try with some non-precious chromatin might be the way to go.

Hi Have you solve the problem. I am doing a qualification exam proposal.IS it reasonable to say I use ChIP to pull down my target DNA, and reverse cross link to use western with histon acetylation antibody to detect increase of acetylation?

Thank you