DSG in addition to formaldehyde? - (Oct/05/2006 )
Since the efficiancy of my ChIP experiments is rather poor, I look for alternatives to improve the pull-down.
Has anyone of you experience with DSG, in combination with formaldehyde fixation?
I found a paper that describes, that it works with NFkB & STAT-3.
I tried it once now but had the problem, that it was quite hard to dissolve (as it is described in the literature) - so I'm not sure, how the final concentration was that I used for the cross-linking.
Second problem that I faced was, that the pellet became quity jelly and I lost a big amount of it during the following washing steps 
Has anyone of you any suggestions??
Thanks in advance 
what is DSG?
ah, sorry: DSG = disuccinimidyl glutarate, a NHS ester, used to link proteins with proteins
In my case, I hope to achieve better ChIP results, when I first link my protein of interest to the other proteins, that are in the big protein complex, when it binds to target promoter regions, and than use formaldehyde, in order to link the proteins with DNA.
Has anyone tried this?
Can't say I have tried it myself.
I am aware with ChIP it is entirely possible to overcrosslink proteins that would lead to complete masking of the epitope your antibody targets. I was under the impression formaldehyde crosslinks proteins to DNA as well as other proteins but i could be wrong.
You would have to play around with fixation times with formaldehyde and DSG to obtain an optimal time of fixation whereby you are able to then IP with your antibody. It's a finicky process but worth the effort in the end.
I am aware with ChIP it is entirely possible to overcrosslink proteins that would lead to complete masking of the epitope your antibody targets. I was under the impression formaldehyde crosslinks proteins to DNA as well as other proteins but i could be wrong.
You would have to play around with fixation times with formaldehyde and DSG to obtain an optimal time of fixation whereby you are able to then IP with your antibody. It's a finicky process but worth the effort in the end.
Methylnick, You're right, that formaldehyde crosslinks protein to protein as well as protein to DNA, but apparently DSG is better for crosslinking protein complexes. Or possibly is has something to do with doing the crosslinking of proteins and DNA in two discreet steps. At least in this reference (E. Nowak et al., 2005. Biotechniques 39(5) 715-725) they are able to get more efficient pull down of NF-kb bound comlexes.
Positive Thinking, when you say your pellet got jelly like and you lost a lot, are you refering to the nuclear pellet which you are trying to sonicate. Is it not breaking up upon sonication. If this is the case, it's possible that you're overcrosslinking. I know of a few people who have had this problem when crosslinking too long or at too high of a temperature using formaldehyde.
Can't say I have tried it myself.
I am aware with ChIP it is entirely possible to overcrosslink proteins that would lead to complete masking of the epitope your antibody targets. I was under the impression formaldehyde crosslinks proteins to DNA as well as other proteins but i could be wrong.
You would have to play around with fixation times with formaldehyde and DSG to obtain an optimal time of fixation whereby you are able to then IP with your antibody. It's a finicky process but worth the effort in the end.
Methylnick, You're right, that formaldehyde crosslinks protein to protein as well as protein to DNA, but apparently DSG is better for crosslinking protein complexes. Or possibly is has something to do with doing the crosslinking of proteins and DNA in two discreet steps. At least in this reference (E. Nowak et al., 2005. Biotechniques 39(5) 715-725) they are able to get more efficient pull down of NF-kb bound comlexes.
Positive Thinking, when you say your pellet got jelly like and you lost a lot, are you refering to the nuclear pellet which you are trying to sonicate. Is it not breaking up upon sonication. If this is the case, it's possible that you're overcrosslinking. I know of a few people who have had this problem when crosslinking too long or at too high of a temperature using formaldehyde.
KPDE: yes, I refer to the nuclear pellet. Anyhow, when I repeated the 2-step-fixation procedure, I didn't have this problem anymore. So it seems to be a random or seldom problem.
What I surely had to adjust (compared to the one-step fixation protocol with formaldehyde only) was the sonication conditions --> longer/stronger sonication intervals!
Now I will perform the first ChIPs comparing 1-step vs. 2-step fixation ... let's wait, if I see a difference!
Greetings,
Can't say I have tried it myself.
I am aware with ChIP it is entirely possible to overcrosslink proteins that would lead to complete masking of the epitope your antibody targets. I was under the impression formaldehyde crosslinks proteins to DNA as well as other proteins but i could be wrong.
You would have to play around with fixation times with formaldehyde and DSG to obtain an optimal time of fixation whereby you are able to then IP with your antibody. It's a finicky process but worth the effort in the end.
Methylnick, You're right, that formaldehyde crosslinks protein to protein as well as protein to DNA, but apparently DSG is better for crosslinking protein complexes. Or possibly is has something to do with doing the crosslinking of proteins and DNA in two discreet steps. At least in this reference (E. Nowak et al., 2005. Biotechniques 39(5) 715-725) they are able to get more efficient pull down of NF-kb bound comlexes.
Positive Thinking, when you say your pellet got jelly like and you lost a lot, are you refering to the nuclear pellet which you are trying to sonicate. Is it not breaking up upon sonication. If this is the case, it's possible that you're overcrosslinking. I know of a few people who have had this problem when crosslinking too long or at too high of a temperature using formaldehyde.
KPDE: yes, I refer to the nuclear pellet. Anyhow, when I repeated the 2-step-fixation procedure, I didn't have this problem anymore. So it seems to be a random or seldom problem.
What I surely had to adjust (compared to the one-step fixation protocol with formaldehyde only) was the sonication conditions --> longer/stronger sonication intervals!
Now I will perform the first ChIPs comparing 1-step vs. 2-step fixation ... let's wait, if I see a difference!
Greetings,
This will be good to know. I'm going to be doing ChIP for Smads so I might have to try a few crosslinking protocols. Do you think you could post the results of the 1 step vs. 2 step comparison.
Cheers