Does Glycine actually quench Formaldehyde?

[theduke]

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.

[kylini]

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[FiReaNG3L]

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?

[kylini]

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!

[FiReaNG3L]

Try quenching with 250 mM Tris just to see? Maybe your glycine is bad or something.. although thats improbable.

[kylini]

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.

[FiReaNG3L]

Are you using methanol-free formaldehyde from single use ampoules?

[kylini]

..........

[kylini]

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.