[Joann]

Isn't there serum in the cell culture media that you're adding formaldehyde to?

[kylini]

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.

[austinso]

Did you check the pH of your glycine stock versus Tris?

[kylini]

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]

[TheSeqGeek]

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

[kylini]

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

I do suppose a good experimental explanation is in order!

For the limited number of lanes shown, I added a 3-fold molar excess of glycine or Tris directly to formaldehyde and mixed (a so-called "prequench" since if it works, formaldehyde should be totally dead). I then introduced that mixture to HeLa cells to crosslink (or not ideally), spun down, washed, and lysed the cells, and then sonicated. The resulting sheared chromatin samples were either heated to 65 overnight with RNase and Proteinase K to reverse crosslinks, or instead kept at 4 degrees (RNase-treated only) to keep crosslinks intact. The resulting samples were phenol:chloroform:IAA extracted and ethanol precipitated (meaning that the crosslink-reverse-minus lanes represent UNCROSSLINKED DNA ONLY while crosslink-reverse-plus lanes represent TOTAL DNA).

It's obvious the two Tris-prequenched lanes are identical. This is because crosslinking never occurred and, thus, sonication was very efficient (naked chromatin). Recovery was high in both lanes despite skipping crosslink reversal (implying no loss to phenol due to protein-DNA linking). Tris quenches formaldehyde.

On the other hand, the two glycine-prequenched lanes are drastically different. Sonication was very inefficient (crosslinked chromatin) in the de-CH2O-plus lane and only uncrosslinked DNA (de-CH2O-minus) had good efficiency. Further, a huge amount of sample disappeared from the crosslink-reversal-minus lane compared to the total DNA lane. This is because crosslinking occurred despite prequenching and the resulting DNA-protein complexes were trapped in the phenol interphase. Glycine, thus fails to quench formaldehyde.

Take home message: if you want to control crosslinking time, use Tris. This will allow you to go *very short* with your times if desired (30 seconds, 1 min, etc.) and see differences in sonication and ChIP signal. However, I do not recommend such short times, since the reason ChIP works is longer crosslinking times! If you go that short, you'll only get Pol II and histone sequencing to work! (Yeah... oops)

[TheSeqGeek]

I do suppose a good experimental explanation is in order!

For the limited number of lanes shown, I added a 3-fold molar excess of glycine or Tris directly to formaldehyde and mixed (a so-called "prequench" since if it works, formaldehyde should be totally dead). I then introduced that mixture to HeLa cells to crosslink (or not ideally), spun down, washed, and lysed the cells, and then sonicated. The resulting sheared chromatin samples were either heated to 65 overnight with RNase and Proteinase K to reverse crosslinks, or instead kept at 4 degrees (RNase-treated only) to keep crosslinks intact. The resulting samples were phenol:chloroform:IAA extracted and ethanol precipitated (meaning that the crosslink-reverse-minus lanes represent UNCROSSLINKED DNA ONLY while crosslink-reverse-plus lanes represent TOTAL DNA).

It's obvious the two Tris-prequenched lanes are identical. This is because crosslinking never occurred and, thus, sonication was very efficient (naked chromatin). Recovery was high in both lanes despite skipping crosslink reversal (implying no loss to phenol due to protein-DNA linking). Tris quenches formaldehyde.

On the other hand, the two glycine-prequenched lanes are drastically different. Sonication was very inefficient (crosslinked chromatin) in the de-CH2O-plus lane and only uncrosslinked DNA (de-CH2O-minus) had good efficiency. Further, a huge amount of sample disappeared from the crosslink-reversal-minus lane compared to the total DNA lane. This is because crosslinking occurred despite prequenching and the resulting DNA-protein complexes were trapped in the phenol interphase. Glycine, thus fails to quench formaldehyde.

Take home message: if you want to control crosslinking time, use Tris. This will allow you to go *very short* with your times if desired (30 seconds, 1 min, etc.) and see differences in sonication and ChIP signal. However, I do not recommend such short times, since the reason ChIP works is longer crosslinking times! If you go that short, you'll only get Pol II and histone sequencing to work! (Yeah... oops)

In bacteria I don't think you can get away with those short times. Takes time to pass through all the barriers.

What was your final quench concentration for Tris?


Quote from a this paper----Brent Sutherland, et al., "Utility of formaldehyde cross-linking and mass spectrometry in the study of protein–protein interactions" 2008 Journal of Mass Spectrometry, 43, 699

"The addition of the buffer, Tris, appears much more efficient in stopping formaldehyde cross-linking reactivity. Even at its lowest concentration (1.2- fold), the degree of modification is significantly lower than that obtained for glycine at the same concentration"


Glycine does work to a degree and maybe quite well in certain conditions but I don't think people check as to just HOW effective it is in quenching. Someone used it and then it became a protocol and then a standard in biology and when something doesn't work for slightly different condition your PI may say "He's not as good as this other kid". Anyway, Tris does seem to better quencher than glycine.

I can even kind of see how it became a trend... "Well formaldehyde cross links proteins and proteins are made of amino acids and we have a lot of glycine in our lab which is an amino acid and there's this chem paper that says this reaction works so let's go with that" -- Conditions, they do matter.

[kylini]

In bacteria I don't think you can get away with those short times. Takes time to pass through all the barriers.

What was your final quench concentration for Tris?


Quote from a this paper----Brent Sutherland, et al., "Utility of formaldehyde cross-linking and mass spectrometry in the study of protein–protein interactions" 2008 Journal of Mass Spectrometry, 43, 699

"The addition of the buffer, Tris, appears much more efficient in stopping formaldehyde cross-linking reactivity. Even at its lowest concentration (1.2- fold), the degree of modification is significantly lower than that obtained for glycine at the same concentration"


Glycine does work to a degree and maybe quite well in certain conditions but I don't think people check as to just HOW effective it is in quenching. Someone used it and then it became a protocol and then a standard in biology and when something doesn't work for slightly different condition your PI may say "He's not as good as this other kid". Anyway, Tris does seem to better quencher than glycine.

I can even kind of see how it became a trend... "Well formaldehyde cross links proteins and proteins are made of amino acids and we have a lot of glycine in our lab which is an amino acid and there's this chem paper that says this reaction works so let's go with that" -- Conditions, they do matter.

Regarding concentration: 1% formaldehyde is really 333 mM. I calculate however many mols is present in my given final volume, multiply it by 2.5, and then add an appropriate amount of 3 M Tris in the neutral-basic range. 750 mM final concentration happens to be a decent ballpark for that number. That said, it's overkill and it does make spinning cells down require a ton of 50 ml conicals (unless you're adherent, then SCORE). Feel free to titrate down as desired.

Regarding that paper: yuuuuuup! Most of my literature searching involved crystallographic sources and, strangely, some industry whitepapers on embalming. That paper had the clearest "cite me" figure.

Regarding tradition: I do love how 2/3 of protocols are Young Lab clones and the remainder follow ENCODE. I'm not saying they're bad, but I do really wonder what those washes truly do.

Regarding blind assumptions and crosslinking length: sometimes, tradition isn't a bad thing. I assumed that lowering crosslinking times would "magically" improve signal:noise by eliminating non-specific binding. I was right! *NOTHING* but Pol II bound to chromatin after 30 sec of CH2O and ChIP-Seq. It was the cleanest Pol II dataset I've ever seen, but my other 20 multiplexed samples were very very devoid of useful information (thank goodness I only paid for 1 lane, hoping to add more for depth after checking the result). Increasing crosslinking time has since re-enabled capture of those "accessory" factors. The terrible quenching of glycine probably played a role in ChIP-Seq working for everyone else!

[JVGen]

Just found this post while searching google about Glycine quenching. It's quite annoying to see prior posts have been edited to remove information in anticipation of publication.

Have your results been published since? This thread would be useful, but all the good bits and pieces have been removed.