diagnostics of crosslinking in ChIP - complete or incomplete (Jun/14/2005 )

Hi,all,
could you share your diagnostics of crosslinking in ChIP :
complete or incomplete ?

For fly ChIP, "the presence of DNA in the fractions at higher density
(bottom of the gradient) is
diagnostics of incomplete crosslinking in ChIP .
nice day!

ZY

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I would say incomplete, this all has to be optimised for your antibody,

crosslinking with formaldehyde can mask the epitope your antibody recognises and you will not get efficient IP. We use centromere and chromatin modification antibodies and routinely crosslink in 1% formaldehyde for 5mins and then wash the cells throuoghly 3 times in pbs. We use cell lines which i suppose is exposed to the fix more readily than fly mush.

Nick

hi, Nick,
thanx a lot .

how about the diagnostics of over -crosslinking in ChIP ?

To check the size of just sonicated DNAs ( i.e. HMW smear ,longer ) ?

or after IP , check them ?

since crosslinking mask the epitope antiboday recognise, over -crosslinking
cause less IP efficiency?


nice day!

ZY

zy101,

if you over cross link, this increases the liklihood of the epitope being masked and you have inefficent IP or no IP at all.

are you IP'ing a histone? or a chromatin protein? Histones don't require much cross linking as they are "packaged" as nucleosomes, however other chromatin proteins that bind transiently will require cross linking.

Nick

hi, Nick,
thanx again.

It's quite meaningful (at least to me, maybe also for other guys) to discuss
ChIP with you guy in this forum .

I did histone ChIP ,which enrichment( anti-histone IP against mock IP)
is always high; even I have difficulty to find one region that has less enrichment
(i.e.negative genomic region control); when shift to chromatin protein ChIP,
sometimes the enrichment is still goood,but problem in reproducibility,getting me in
hot-water. I 'm in troubleshooting .

As you know, with the cuticle of animals, I dont know how to measure exactly what
extent of cross linking take place, i.e. complete,incomplete, or over-crosslinking,
so general diagnostics of crosslinking in ChIP become useful & important
to my work now.

Besides, coud you like to be so kind :
how you know the lysis is complete or incomplete,e.g. checking by microscope?

Again, with the cuticle, even I treated it with trypsin,collengase, I still need some
diagnostics of the lysis i.e complete/incomplete.
nice day!

ZY

zy101

I am not not familiar with working with cuticles, we routinely use cell lines and we have an abundant source of cells.

I might suggest you go check out the abcam website, they have set themselves up as a ChIP company and have good tips about ChIP (again with cell lines).

I could only suggest you do a titration of time for cross linking and then perform ChiPs for all fractions.

As for lysis, again you will have to do the same over titrate over time. This leads to alot of ChIP and usage of a lot of your antibody!

Otherwise check out Abcam Website for more ideas.

Good luck!

Nick