DNAse and NaF in lysis buffer...a good idea? - Does NaF or Na3VO4 inactivate DNAse I? (Aug/15/2005 )
I want to look at ser/thr-phosphorylated proteins via Western blot (immunoblotting with a phospho-specific antibody). Our usual cell-lysis protocol calls for DNAse I and RNAse in the lysis buffer (which gets rid of the snotty gooey cellular DNA & RNA). Since I was looking for changes in phosphorylation, I decided to add sodium fluoride (NaF) and sodium orthovanadate (Na3VO4) to the lysis buffer as well, in the hopes of preserving the phosphorylation state (though I'm not sure if they work for ser/thr phosphorylation...I was always taught Na3VO4 was good for tyrosine, but I figured they couldn't hurt). Every time I add these, the samples are really snotty and have to by syringed a couple of times to get the sample pipetable for Western analysis. Am I inactivating the DNAse with the phosphatase inhibitors? Any suggestions for preserving phosphorylation without syringing 30 samples prior to loading?
for protein extraction i do not use nucleases.
i think you get the appropriate cocktail
for the problem of your solution, i think you make your nuclease precipitating.
The activity of DNase I is dependent on the presence of divalent cations [principally magnesium and manganese], so if you have anything in your lysis buffer that chelates/precipitates with them the DNase I will not function. So the gooey-ness of your samples is probably because the cellular DNA is not getting chopped up.
I blot for phosphoproteins a lot, and I always used to include NaF and Na3Vo4 in the lysis buffer, until I tried using micrococcal nuclease. The activity of micrococcal nuclease depends on the presence of divalent cations [mostly Ca2+], but adding Ca2+ to a lysis buffer containing NaF and Na3Ov4 gives a white precipitate immediately and the micrococcal nuclease doesn't function. So my guess is that the Na3Ov4 is removing cations from your lysis buffer and preventing the DNase I from working.
On the plus side, since moving to the micrococcal lysis buffer I haven't seen any change in the detectability of phosphoproteins. I harvest cells, then wash them in ice-cold PBS before lysing them in Tris-buffered saline with 0.5% NP-40 containing 2 mM CaCl2 and 5 U per ml of micrococcal nuclease, plus an EDTA-free protease inhibitor cocktail. Once you've resuspended them in lysis buffer, you only need to incubate at room temperature for 5 minutes, then you can add SDS-PAGE sample buffer and boil them briefly before freezing - they won't be gooey, and the micrococcal nuclease in the lysates doesn't screw up Westerns.