I'm a first year graduate student and for my project I am attempting to do a chromatin immunoprecipitation, but am having some trouble. I'm using primary neonatal rat ventricular myocytes infected with a virus that overexpresses a g-protein coupled receptor kinase. I have cells in an untreated group and a treated group (a drug that induces a hypertrophic phenotype). I am using the millipore chip protocol. For the sonication step, I have done 3 ten-second pulses of samples on ice at a 4-Watt output, 20,000Hz to generate DNA fragments in the 200-1000bp range. I have had a very hard time getting a reasonable concentration of DNA after reversing the crosslinkes. My concentrations have ranged from 1ng/ul to about 15ng/ul. I'm not sure what I can do to increase the yield in the protocol itself. I'm currently doing a random hexamer amplification to get a higher amount of DNA, but I'd prefer to be able to get a higher concentration from the technique itself. Also, I have been able to detect the protein I'm targeting with my IP antibody, but for some reason am having a hard time getting a signal for acetylated lysine or total histone. Is it possible to pull down a DNA-binding protein without the chromatin? Any help would be greatly appreciated.
1) For the crosslinking, I've been following the protocol's suggestion of 10 minutes at 37 degrees. I add formaldehyde to a final concentration of 1% in the culture medium.
2) I have been decrosslinking in a water bath at 65 degrees for 4 hours.
3) I'm using a probe sonicator directly in the samples while the samples are on ice. I might have to optimize this step more. I'm curious as to your thoughts on enzyme digestion. It seems like it would require less optimization and be more consistent.
6) I'm using primary cells (myocytes) and a proprietary 1% SDS buffer for lysis. Is it possible this is not enough or too much SDS?
7) The case I gave you was sadly the best case I've experienced. I'm basically not even sure I'm getting DNA. My concentrations are ranging from around 0.5ng/ul to maybe 2ng/ul on a Nanodrop. This is such a small amount of DNA (after the immunoprecipitation). Each sample originally contained 1E6 cells, so it seems like a very small amount to recover, even after ChIP. I've been eluting with 250 uL of a provided elution buffer followed by adding EDTA and Proteinase K and digesting for an hour. Following this, I've been following my lab's phenol/chloroform DNA extraction and resuspending the resulting pellet in 100uL water. Do you think this purification step is necessary ,or it it is do you think I could find a kit for purification of the DNA?
8) I really appreciate the assistance. This protocol is very technically demanding, and is going to require a lot of fine tuning to suit my experimental setup.