DNA "snot" in cell lysis/protein extraction - (Oct/06/2008 )
There is a sticky gooey snot-like substance in my cell lysate that I am told is DNA. It does not dissolve when boiled in SDS, It won't spin down and I don't know how to get rid of it.
Some have suggested using more extraction buffer (RIPA) but this dilutes the protein too much for my use. Any ideas?
Thanks!
Anything that you can do to shear the DNA will help with the viscosity. Sonication works well, but you can also just pass the sample through a syringe& with a needle >22G.
Bear in mind too, that some cell/tissue samples can also produce a thick 'snot' that is not DNA, but ECM collagen/proteoglycans that can be difficult to solubilize with out very harsh conditions (e.g. >6M Urea or >4M Guanidine).
Some have suggested using more extraction buffer (RIPA) but this dilutes the protein too much for my use. Any ideas?
Thanks!
yes one way is to use more RIPA but your concentration would drop....the best way is to keep on ice all the time, that is why in all protocls say even use cold PBS to wash your cells prior to lysis.....your problem is that you didn't do it on ice.....keep your cell pellet on ice....and use cold RIPA and immediately put your tube on ice.....this should eliminate the sticky clump.
we use DNase to get rid of that clump. just nearly 10ul would do.....but why use DNase when you can simply work on ice? don't forget ice....I've seen many people who are not ice friendly....they just don't like keeping their tubes on ice and that's a big mistake.
Bear in mind too, that some cell/tissue samples can also produce a thick 'snot' that is not DNA, but ECM collagen/proteoglycans that can be difficult to solubilize with out very harsh conditions (e.g. >6M Urea or >4M Guanidine).
Thank you for your advice. I tried both sonication (this is the low power, cleaning type) and passing the sample through a #27 needle and this did decrease the viscosity quite a bit but there was still goo that stuck to the pipette tip. I then passed the sample through a needle with a 5um filter installed and this caught all the goo but I am afraid of loosing DNA-interacting proteins that might be bound up in the goo.
There was additional advice offered that suggested keeping the sample cold which I will try today, then the urea/guanidine as a last resort.
http://www.protocol-online.org/forums/styl...lt/rolleyes.gif
I've had samples that I needed so concentrated that I had similar problems. Re-Boil the sample for a minute or two and load while it's still hot. A hot sample is not nearly as gooey as a room temp sample. The only major issue with samples this concentrated is that some proteins may not be able to enter the gel properly. You may want (if you can) to use lower percentage gels.
The problem with leaving the DNA in the sample is the likelihood of its creating variability between samples e.g. one pipette pulls out a wad of goo and the next does not. It turns out that the keeping everything cold idea did work and allowed me to pellet the DNA. My sample protein concentration is around 6.5 mg/ml which is fine for loading a gel etc. Since this is the simplest of all ideas it must be the best. I might try loading the "DNA" pellet on a gel just to rule out the possibility of loosing DNA-binding proteins but otherwise I will stick with this protocol.
Thanks to all who offered their advice!
Naive question, possibly, but is there any reason you can't just add some DNase I?