Scrape vs. TrplyE pre-ChIP - (Nov/17/2008 )
Hello all, new to this world. I will soon be cross-linking cells for use in a ChIP assay, most of the protocols suggest scraping the cells off after fixation. I have never done this but have used TryplE to detach cells, is there any reason why I couldn't get them off this way?
Appreciate your time,
Stephen
Use of Trypsin (or anything similiar to it) should be avoided in ChIP experiments because it attacks proteins, which is how Trypsin realeases cells - by breaking down the proteins used to attach the cells to the flask. Therefore to minimise any degradation of your target protein which you hope to IP, scraping is the safer option.
Dave
Dave
I agree. In addition, I don't even know if trypsinization will work after the proteins have been fixed.
thanks for the replies
tried cell scraping today for the first time, seems like a simple technique but got very low number of cells off a 10cm plate, will work on it
i can see how trypsin-like action would be hindered by fixation
you could trypsinize your cells first, inactivate with medium, resuspend in D-PBS and crosslink in solution...
tried cell scraping today for the first time, seems like a simple technique but got very low number of cells off a 10cm plate, will work on it
i can see how trypsin-like action would be hindered by fixation
One thing I've found in transferring fixed cells is that, if there is a low amount of soluble protein in the medium that the cells are in (e.g. PBS as opposed to media with FBS) they tend to stick to the inside of the pipet. I do my fixation in medium containing FBS so it's not a problem for me but if you remove the media and do fixation in PBS, this could be part of the reason for low recovery. If that's the case you might want to try adding the formaldehyde directly to the media. If you're worried about the formaldehyde getting quenched by the media you can try a higher concentration (I use 1.4-1.5% or 40µl of 37% formaldehyde per ml of media). Alternatively you could try treating your pipets to prevent the cells from sticking.
tried cell scraping today for the first time, seems like a simple technique but got very low number of cells off a 10cm plate, will work on it
i can see how trypsin-like action would be hindered by fixation
One thing I've found in transferring fixed cells is that, if there is a low amount of soluble protein in the medium that the cells are in (e.g. PBS as opposed to media with FBS) they tend to stick to the inside of the pipet. I do my fixation in medium containing FBS so it's not a problem for me but if you remove the media and do fixation in PBS, this could be part of the reason for low recovery. If that's the case you might want to try adding the formaldehyde directly to the media. If you're worried about the formaldehyde getting quenched by the media you can try a higher concentration (I use 1.4-1.5% or 40µl of 37% formaldehyde per ml of media). Alternatively you could try treating your pipets to prevent the cells from sticking.
thanks for the info
i do add the formaldehyde directly to the media but then wash in PBS + inhibitors a couple of times after that and tried scraping them in the PBS, it is possible all my cells never came back out of the pipette, that would explain the low yield on what should be a pretty simple technique
how do you pre-treat the pipettes to prevent sticking?
Good question. I don't know if it's sticking to more hydrophilic stuff or more hydrophobic stuff.
In any case, if you are crosslinking in media already, you might as well transfer the cells while they're still in media and then wash the pellets with PBS afterwards (you don't have to vigorously resuspend the pellet with PBS either; I think the most important aspect of the wash is to get rid of the volume of media remaining above the pellet after aspirating the first time).