ChIP on peripheral blood samples.... - anyone done it?? (Nov/12/2007 )
Hi again everyone,
Just a quick question....for my ChiP-on-chip experiments I will be using patient material that has been frozen (Blood > ficoll > etc). Has anyone used this or similiar material for ChIPs? I am wondering whether I culture the cells for a short time (eg: ON?) or just use them straight away. Any suggestions/experiences with this would be greatly appreciated 
Clare
-Clare-
QUOTE (Clare @ Nov 12 2007, 05:48 AM)
Hi again everyone,
Just a quick question....for my ChiP-on-chip experiments I will be using patient material that has been frozen (Blood > ficoll > etc). Has anyone used this or similiar material for ChIPs? I am wondering whether I culture the cells for a short time (eg: ON?) or just use them straight away. Any suggestions/experiences with this would be greatly appreciated
Clare
Just a quick question....for my ChiP-on-chip experiments I will be using patient material that has been frozen (Blood > ficoll > etc). Has anyone used this or similiar material for ChIPs? I am wondering whether I culture the cells for a short time (eg: ON?) or just use them straight away. Any suggestions/experiences with this would be greatly appreciated
Clare
Our lab has done ChIP from liver, kidney, lung, PAP smears, and cells from urine (though I can't say anything about the success of the last two). The main key is to get enough material. If it's likely you'll have less than 1 million cells then it would likely be helpful if you pretreat your protein A or G beads with carrier DNA (e.g. salmon sperm DNA) and BSA and, as well, do the IP in the presence of both of these.
-KPDE-
QUOTE (KPDE @ Nov 13 2007, 04:22 AM)
Our lab has done ChIP from liver, kidney, lung, PAP smears, and cells from urine (though I can't say anything about the success of the last two). The main key is to get enough material. If it's likely you'll have less than 1 million cells then it would likely be helpful if you pretreat your protein A or G beads with carrier DNA (e.g. salmon sperm DNA) and BSA and, as well, do the IP in the presence of both of these.
Hi Hi
I have been preclearing my protein g with rabbit IgG. Are you suggesting I switch to BSA/ssDNA instead? At this stage I have no idea how many cells I will get from each patient....I did quite a bit of reading yesterday (which is driving me insane as I wait for samples etc) and many people seem to grow their PBMCs in media for 24hours before use in experiments...but I haven't found any papers where people use these cells for ChiP. Perhaps I will try both methods as see how I go...
Clare
-Clare-