3 replies to this topic

[memari]

I have read a lot of protocols about Chip(Chromatin Immunoprecipitation).
Why should I use glycine to a final concentration of 125mM ? why not more or less?

http://www.abcam.com...ip_protocol.pdf

I am sure that using 125mM is just an imitation with blind eye.
One molecule of Formaldehyde quenches one molecule of Glycine. so when we use 125mM glycine, it is good just for 125mM of Formaldehyde. 125mM formaldehyde is 0.375%.

http://www.trimen.pl...s/stezenia.html

One 1% of Formaldehyde is 0.33M.

But biologists always use 125mM glycine for all different concentration of Formaldehyde.
Do not you think that they (biologists ) are wrong?

125mM glycine is good just for 0.375 % of formaldehyde.

What is your idea?

-----
Babak

[Akdor]

Why do you think that they are wrong if this concentration is actually working for all the different formaldehyde concentrations? I suggest you to do a tritation and tell us the result, you will be surprise about it and about why we, biologists, cook in that way.

[memari]

I know what you mean maybe.
Glycine is viscous.

Finnaly I found a link with more concentration:

Quench formaldehyde by adding glycine to final concentration of 0.5 M
http://www.wadelab.c...tocol_chip.html

http://www.plosone.o...al.pone.0026217

glycine to a final concentration of 240 mM
http://www.personal...._preferred.html

Also some other links that I can not find them right now to past here.
Here claim this"Note: Do not quench with glycine (as in chromatin immunoprecipitation-ChIP).
Quenching with glycine will inhibit subsequent exonuclease digestion."
http://paul.phys.mcw...ng-genecapp.pdf


-----
Babak

Edited by memari, 05 September 2012 - 04:54 PM.

[Akdor]

That is why... not only the quenching. I never had problems with "exonuclease digestion".