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Old 10-30-2012, 07:05 PM   #1
kylini
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Default Does Glycine actually quench Formaldehyde? No, but Tris does!

EDIT: See post #15. Glycine does not quench, but Tris does!
EDIT 2: Most figures removed in anticipation of publication.

Summary: 125 mM glycine, 333 mM glycine, and 750 mM glycine do not quench 333 mM (1%) formaldehyde. 750 mM Tris does.
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Old 10-30-2012, 09:08 PM   #2
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Old 10-31-2012, 04:59 AM   #3
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Are you using methanol-free formaldehyde from single use ampoules?
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Old 10-31-2012, 06:11 AM   #4
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Are you using methanol-free formaldehyde from single use ampoules?
Yes. I made the switch about a month ago. That said, I get the exact same "no difference between 20 sec and 10 min" with methanol-containing CH2O. My thought is that while the methanol increases permeability, the glycine is meant to quench the unused formaldehyde still in the media. If it's not quenching, it doesn't matter as CH2O will still get in just fine.

There has to be a condition of the quenching reaction that is obvious that I'm missing. It would make no sense for every lab in the world to use a method that doesn't work.
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Old 10-31-2012, 07:16 AM   #5
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Try quenching with 250 mM Tris just to see? Maybe your glycine is bad or something.. although thats improbable.
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Old 10-31-2012, 07:39 AM   #6
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I'll definitely give Tris a try. I also thought about a very real possibility:

Many protocols concentrate their cells prior to crosslinking (pellet and resuspend in PBS). They then use a small volume of CH2O to achieve the desired 1%. In short, large number of cells, small number of CH2O molecules.

I don't concentrate my cells and crosslink in media as I have data that suggests that transcription changes after such a perturbation. Therefore, I need a larger volume of CH2O to reach 1%, a much larger volume actually. Since the cell number is the same, I'm therefore drastically increasing the CH2O:cell ratio even if the concentration is technically the same. Concentrations don't crosslink; molecules do.

In any case, a crosslinking concentration assay never hurt anyone!
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Old 10-31-2012, 10:30 AM   #7
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Thats a pretty good idea actually, I think the rationale behind using less glycine in molar than formaldehyde was that most of it would be spent on actually fixing proteins / dna in the cells. Now if you have less cells but much more volume of Formaldehyde, I can see how its not sufficient to quench everything. How much volume for how many cells if I can ask?
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Old 10-31-2012, 11:11 AM   #8
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Old 10-31-2012, 12:23 PM   #9
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This is not the first time I have seen shearing like this using the Covaris. I think the glycine thing is a "red herring" here and I would suspect your shearing methodology is the problem. Have you tried any other sonicators? I would hope so after two years.

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Old 10-31-2012, 12:44 PM   #10
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This is not the first time I have seen shearing like this using the Covaris. I think the glycine thing is a "red herring" here and I would suspect your shearing methodology is the problem. Have you tried any other sonicators? I would hope so after two years.
I use a Fisher Sonic Dismembrator 550 and, recently, have confirmed that I am able to use the Covaris. Both sonicators result in the same profile and both were used for quenching experiments.

My sonication certainly isn't yet optimal, but it still doesn't change the result that adding 125 mM or 333 mM glycine BEFORE adding formaldehyde yields the SAME crosslinking efficiency as CH2O exposure without it. I have no reason to trust that glycine is actually quenching formaldehyde and this is why I need input.
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Old 10-31-2012, 12:57 PM   #11
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I don't think there is much doubt that glycine quenches formaldehyde. I would drop that line of thinking quickly if I was you. There is too much literature and too much evidence against you and it can't all be wrong. There is also no doubt that fixing time per se influences shearing efficiency. Something else is going on with your experiments. Either your glycine is off/concentration is wrong or something is up with your formaldehyde or your technique. It looks to me that you are massively over-crosslinking.

Generally, I have 10^8 cells per 200 ml, up to 6*10^8 cells per 1.2 L. I grow HeLa cells at a concentration of 5*10^5 cells/ml until I'm ready and then dump them into a vessel already containing the requisite 16% formaldehyde (final conc. 1%).

This is where I think your problems are. Something doesn't smell right. That is a ****-ton of cells and a lot of volume. Are you doing the quenching etc in suspension?
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Old 10-31-2012, 01:36 PM   #12
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That is correct. [omitted]

The reason I'm so concerned about the quenching is that 1% = 10 g per liter. Since CH2O has a molecular weight of about 30, it follows that there are 30 g per mol. 10g/L times mol/30g = 333 mM. How can something that reacts 1:1 to stop formaldehyde from crosslinking further possibly work at a 333 mM to 125 mM ratio?

Again, I added 125 mM glycine FIRST before formaldehyde and the cells went right on to over-crosslink (all of the material was crosslinked). I then added 333 mM glycine FIRST before formaldehyde and the cells STILL went right on to over-crosslink (again, all material was crosslinked). If I'm crosslinking in media, there should be a bit of extra quenching power from its components. Despite this, the amount of glycine I'm adding doesn't actually quench.

If quenching doesn't actually happen, that means my cells are crosslinking while I aliquot them into 50 ml tubes (another 5-10 minutes) and while they pellet (another 5 min). Instead of crosslinking for 30 seconds, I'm really crosslinking for 30 minutes, hence the reason my sonication sucks.

I can't drop the line of thinking that glycine isn't quenching formaldehyde in these conditions because I have reproducible, empirical evidence showing it doesn't and it explains my sonication failures despite two years of optimization using protocols directly from the labs of Richard Young and the ENCODE Consortium. That's why I think that there are other factors at play, such as the number of molecules (not the concentration) per cell.
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Old 10-31-2012, 02:29 PM   #13
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Quote:
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I can't drop the line of thinking that glycine isn't quenching formaldehyde in these conditions because I have reproducible, empirical evidence showing it doesn't and it explains my sonication failures despite two years of optimization using protocols directly from the labs of Richard Young and the ENCODE Consortium. That's why I think that there are other factors at play, such as the number of molecules (not the concentration) per cell.
Well, I don't know what career-stage you are at, but spending two years figuring out sonication and ChIP-Seq is far, far, far too long in my opinion. At the very least, I would have moved on to MNase a long time ago in your situation. Actually a compelling argument can be made for the preferential use of MNase over sonication these days, but that is a different discussion.

I would start thinking about optimization experiments in small dishes and then work up from there. Have you done this? I don't like the large number of cells and excessive volumes you are using. I typically do my x-linking in 10 cm dishes with 5 ml media at RT. I usually do multiple dishes at the same time but do not combine them into one massive container.

ENCODE ChIP-Seq data is far from perfect!!
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Old 10-31-2012, 03:56 PM   #14
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Well, I don't know what career-stage you are at, but spending two years figuring out sonication and ChIP-Seq is far, far, far too long in my opinion.
Don't I know it! I've definitely been pursuing other experiments on the side and this is definitely not the only thing my lab does. It'd just be nice to actually do these experiments in house and not rely on collaborators. Notably, I already have samples sequencing despite the sonication (horray for size selection after library prep) so this isn't holding me up. Other fun deep-sequencing projects are in the works too!

It's just that this glycine first experimental result goes completely against established protocols. I'm sure the number of molecules of formaldehyde vs. the number of cells (ignoring volume) is part of the problem. I was curious if anyone else knew what could be going on.
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Old 11-13-2012, 05:03 PM   #15
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Fun update!

Here's the edited experimental setup:

17.6 million HeLa cells were treated for 30 seconds with 1% methanol-free formaldehyde premixed for 3 minutes with a 3-fold molar excess of either glycine or Tris.

Here's the result:



In conclusion, glycine does not quench formaldehyde but Tris does.

[omitted]

I think this settles my problem. If you have quenching issues, give Tris a try! It's certainly been used in the literature; this just confirms that it works.
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Old 11-14-2012, 12:09 AM   #16
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Isn't there serum in the cell culture media that you're adding formaldehyde to?
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Old 11-14-2012, 05:36 AM   #17
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Isn't there serum in the cell culture media that you're adding formaldehyde to?
Yup. 10% I believe. I don't see the media "unquenching" the formaldehyde that's been pre-mixed with glycine, however. If anything, I would expect the media to help quench, but it doesn't do enough.

I can't stress this enough. I still have sonication issues. However, this settles my quenching issue.
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Old 11-14-2012, 05:45 AM   #18
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Did you check the pH of your glycine stock versus Tris?
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Old 11-14-2012, 05:46 AM   #19
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Did you check the pH of your glycine stock versus Tris?
Both were titrated to pH 7. Glycine was almost there to begin with.

[omitted]
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Old 05-07-2014, 02:20 PM   #20
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Quote:
Originally Posted by kylini View Post
Fun update!

Here's the edited experimental setup:

17.6 million HeLa cells were treated for 30 seconds with 1% methanol-free formaldehyde premixed for 3 minutes with a 3-fold molar excess of either glycine or Tris.

Here's the result:



In conclusion, glycine does not quench formaldehyde but Tris does.

[omitted]



I think this settles my problem. If you have quenching issues, give Tris a try! It's certainly been used in the literature; this just confirms that it works.


I'm a bit late to the party but what are those gels showing exactly... The Tris looks the same in both conditions...

I am doing ChiP-seq now myself
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