Nuclear extraction of brain tissue for ChIP - (Mar/21/2008 )
So, I have been having a hard time with ChIP (tried different forms of traditional, fast, magnetic).
After analyzing the protein that I am trying to pull down in the IP part of the ChIP, I have found it to be present in the cytoplasm and nucleus. After our treatment we see a significant change in the nucleus, but no real change in the cytoplasm.
Actually, the protein is hard to even do a western blot on if you just extract all the protein together - the cytoplasm seems to have more non specific binding to the antibodies (or break down products, or something).
Okay - What I want to do is, after cross-linking, quenching, and PBS washing, I want to maybe use a hypotonic solution let the cells swell, dounce homogenize with the (A) pestle, spin it hard, discard supernatant, then add RIPA buffer, dounce with the ( B ) pestle, then continue on by sonicating, etc.
Do you think this will work? Do you think this will help in the ChIP? Is there a better way to do this? Most nuclear extract buffers seem to include glycerol, do I need that in my RIPA (should I not use RIPA)?
What about just doing what I did before, just add RIPA right after PBS washes and dounce with ( B ), then instead of sonicating, spin real hard. At this point the long strands of DNA(crosslinked to proteins) should pellet, right? I could get rid of the supernatant, resuspend in more RIPA and then sonicate. Would this work?
Another question - after sonication I would spin hard and only use the supernatant for ChIP, once sonicated the pellet shouldn't have the dna-protein complexes, right? It would have unsheared maybe.
Looks like you need an entire course in molbio 
So, I have been having a hard time with ChIP (tried different forms of traditional, fast, magnetic).
NOT ALONE
After analyzing the protein that I am trying to pull down in the IP part of the ChIP, I have found it to be present in the cytoplasm and nucleus. After our treatment we see a significant change in the nucleus, but no real change in the cytoplasm.
Actually, the protein is hard to even do a western blot on if you just extract all the protein together - the cytoplasm seems to have more non specific binding to the antibodies (or break down products, or something).
DON'T BE BOTHERED ABOUT CYTOPLASM WHEN YOU ARE DOING CHIP. YOU ARE WASHING AWAY ALL SOLUBLE COMPONENTS AFTER LYSIS, INSOLUBLE WILL LIKELY GO AWAY IN PELLET AFTER SONICATION. BUT YES, IF YOU DO KNOW THAT THERE IS A REAL STRONG NON-SPECIFIC BINDING IN CYTO, YOU MAY WANT TO MAKE SURE YOUR NUCLEAR EXTRACTION IS REAL GOOD.
Okay - What I want to do is, after cross-linking, quenching, and PBS washing, I want to maybe use a hypotonic solution let the cells swell, dounce homogenize with the (A) pestle, spin it hard, discard supernatant, then add RIPA buffer, dounce with the ( B ) pestle, then continue on by sonicating, etc.
GOOD SO FAR.
Do you think this will work? Do you think this will help in the ChIP? Is there a better way to do this? Most nuclear extract buffers seem to include glycerol, do I need that in my RIPA (should I not use RIPA)?
GLYCEROL IS NECESSARY IF YOU WANT FUNCTIONAL NUCLEAR EXTRACT. NOT FOR CHIP. I WOULD DEFINITELY TRY WITH LESS STRINGENT BUFFER THAN RIPA.
What about just doing what I did before, just add RIPA right after PBS washes and dounce with ( B ), then instead of sonicating, spin real hard. At this point the long strands of DNA(crosslinked to proteins) should pellet, right? I could get rid of the supernatant, resuspend in more RIPA and then sonicate. Would this work?
I LOST YOU HERE.
Another question - after sonication I would spin hard and only use the supernatant for ChIP, once sonicated the pellet shouldn't have the dna-protein complexes, right? It would have unsheared maybe.
ONCE SONICATED, IF PELLET HAS CHROMATIN, YOUR SONICATION NEEDS IMPROVEMENT.
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THERE ARE MANY OTHER THINGS THAT MAY BE GOING WRONG, YOU NEED TO DESIGN YOUR EXPERIMENTS WITH CONTROLS ALL OVER THE PLACE, AND FIRST SHOW THAT YOU CAN DO SOME COMMONPLACE ChIPS, THEN GO TO YOUR SPECIFIC PROTEIN. PERHAPS YOU NEED A BETTER AB.
HTH/
I have had many courses in molecular biology. Just trying to get others' exertise.
Thanks for the help.
What kind of less stringent buffer would you suggest?
DON'T BE BOTHERED ABOUT CYTOPLASM WHEN YOU ARE DOING CHIP. YOU ARE WASHING AWAY ALL SOLUBLE COMPONENTS AFTER LYSIS, INSOLUBLE WILL LIKELY GO AWAY IN PELLET AFTER SONICATION.
What steps do you take when lysing the cells?
I have had many courses in molecular biology. Just trying to get others' exertise.Thanks for the help.
What kind of less stringent buffer would you suggest?
DON'T BE BOTHERED ABOUT CYTOPLASM WHEN YOU ARE DOING CHIP. YOU ARE WASHING AWAY ALL SOLUBLE COMPONENTS AFTER LYSIS, INSOLUBLE WILL LIKELY GO AWAY IN PELLET AFTER SONICATION.
What steps do you take when lysing the cells?
If you have not used upstate (now millipore) ezchip kit and protocol, I would strongly advice to do so. Protocol works like a charm every time. Comes with some controls, and precise info regarding which steps should be controlled for etc. Kit is worth buying cause it comes with some ab controls and primers and etc; all buffers that come with the kit are easily makable in the lab, so there would be no recurring cost. I found upstate buffer combination to work best for all my specific proteins (low-amount proteins, not the histones and stuff that works even if my son were to do it.)