I plan to do double cross-link for Chipping a non-DNA binding protein, and I wondered why people generally take off the first cross-linker and wash extensiveley before adding formaldehyde. Has anybody tried to just add formaldehyde for 10 min in the first cross-linking buffer?
I follow this protocol. Works well.
http://www.biotechniques.com/BiotechniquesJournal/2006/December/In-vivo-dual-cross-linking-for-identification-of-indirect-DNA-associated-proteins-by-chromatin-immunoprecipitation/biotechniques-40853.html
I did try the above protocol, but somehow my sonication profile was not as good as only with formaldehyde. Also, I followed the EGS cross-linking duration on the data sheet. How long did you cross-link with each? Also, if you do together then is 10 min enough for EGS to cross link? Sorry, its more of questions than answers.
I would think that washing off the first crosslinker avoids "over-crosslinking". that can cause problems when sonicating. for example if you over-crosslink you might need to sonicate longer to get the correct fragment sizes, however, sonicating longer will also affect the stability of the proteins and therefore their ability to be chipped. by washing off the first x-linker you might avoid that...i think you can easily test this by taking two flasks and x-linking one with washes in between and one without, then sonicate, run a gel and see the difference.
Thanks Seb. I never washed off EGS before adding HCHO. Maybe that lead to over cross linking. Also, in general do you have to sonicate more for double cross linking as compared to just HCHO?
I fix cells with EGS 20 minutes and then just add formaldehyde for additional 10 minutes. So cells have EGS 30 minutes in total.
I treat cells with MNase before sonication.
I don't wash EGS before adding formaldehyde. I followed this protocol : "Cells were washed twice with PBS, and a first cross-link was performed by adding PBS containing 1.5 mM final concentration of EGS (Ethylene Glycol bis, Succinimidysuccinate). Following 15 min incubation at room temperature with agitation, formaldehyde was added directly to the PBS-EGS at 1% final concentration and cells were incubated at room temperature for 15 min before addition of glycine solution (0.125M final concentration) to arrest the cross-linking reaction. After 10 min incubation at room temperature with agitation followed by washing twice with PBS, cells were scraped in 5 ml of cold PBS and were pelleted by centrifugation at 3,000 rpm for 5 min at 4°C." . Good luck
Arindam, the longer you sonicate the higher are the chances that you affect the epitopes, therefore protein stability is compromised (and ChIP results with it). I try to sonicate not more than 30 min (5min 30sec ON 30sec OFF) unless I have to. or you can try micrococcal nuclease digestion, it requires a few more "ingredients" to be added but I did it in the past and it works well, + I sonicate only 5 min at the end to solubilize everything.
You can also try different cross linkers with varying arm lengths as EGS may not give good results with all the proteins that indirectly associate with chromatin. For dual cross linking I do not wash off the first cross linker but just add formaldehyde for the last 10 minutes of incubation. Sonication conditions will vary according to the cell type and you will need to optimize them for your cell type and the sonicator that you are using. For dual cross linking I have been able to get similar fragment size as with single cross linking. Make sure that your cells are lysed well before sonication.
As stated by Ruchi Pandey, often different length crosslinkers need to be tried. Many people find success using DSG (proteochem.com/dsgcrosslinker100mg-p-45.html) in addition to EGS (proteochem.com/egscrosslinker100mg-p-153.html).
http://www.proteochem.com/dsgcrosslinker100mg-p-45.html
http://www.proteochem.com/egscrosslinker100mg-p-153.html
Hi Germain Rousselet, I had done such experiment previously.
I first crosslink adherent cells with 1.5mM EGS in PBS for 20 min and then add 37% formaldehyde to final concentration of 2% for annother 15 min. Then I preceded with beta-catenin ChIP. It did enhance the ChIP efficiency.
Maybe the people who remove the EGS protein-protein linker in consideration of over-crosslink and you may go check the concentration and incubation time they used.
Hope it helps!
has anyone tried formaldehyde crosslink first, followed by EGS crosslink? I am trying to capture a transient DNA-protein interaction after stimulation, and fear that proteins may dissociate during the 45 min EGS crosslink step (considering EGS is a slow step).
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