RIPA for DNA Isolation - Can you do simultanous isolation of protein and DNA with RIPA (Mar/24/2008 )
I just started using RIPA to lyse C2C12 myotubes and was wondering if it is possible to isolate DNA at the same time as isolating the protein?
I'm not sure if RIPA leaves the DNA in any condition to be analyzed but after lysing your cells, when you spin the lysate the majority of the pellet at the bottom of the tube is genomic DNA. However, if you are looking for chromatin-bound proteins, RIPA may be too strong and disrupt these interactions.
Is RIPA too strong even if you have cross linked, like in ChIP? What is another buffer I could use during my ChIP protocols besides RIPA?
Honestly, I'm not sure about the use of RIPA in ChIP assays. I've never had to do ChIP. I just know that RIPA is a very strong lysis buffer that I use only for making samples for simple western blots. Never for an IP or chromatin binding assays, and especially not if you want to isolate an active protein (kinase, phosphatase, ect.).
As for which buffer you should use, there are a few protocols here on this website which I'm sure you've checked out but also, if you search the internet you can see if there is a buffer commonly used. Just check out the methods/materials of papers that did ChIP. Compare different groups to see what they did the same and what is different. Also, many of the companies have protocol sections on their websites. Granted they are going to highlight their products such as Santa Cruz has a ChIP protocol featuring a lysis buffer you can buy from them but the recipe (which they give) is very straight forward: 5mM PIPES pH 8.0, 85mM KCl, 0.5% NP-40 and Protease Inhibitor Cocktail (1 tablet/50 ml).