I've extracted fractioned protein lysates from adherent breast cancer cells. I've had good results with the cytosolic and mitochondrial fractions. However, the last fractioned obtained is a nuclear suspension and I've been obtaining subpar results in Western blotting. I'm positive this is due to the viscosity of the sample, due to the presence of DNA. I was wondering if anyone would have a good method for getting rid of DNA in the sample and, if by sonication, a suggestion for the best conditions to sonicate this kind of samples (to make optimisation easier) Thanks!
yes, you could try sonication. Keep your suspension on ice and give a couple of strong pulses. you will see that viscosity is reduced. Another option would be to add DNaseI
I use Benzonaze - it's a very potent nuclease (RNA and DNA). I add it to RIPA lysates (1-5U/50uL lysate) and then leave overnight at 4C. This aids in release of chromatin associated factors as well (like histones). Next day, centrifuge the samples at at max speed for 5-10 minutes to pellet any undigested large DNA and insoluble membranes, etc. If you centrifuge out the DNA prior to nuclease digestion, you may loose histone proteins in the pellet - this can or may not be useful.
The lysate should resolve nicely. Good Luck!
Nuclease treatment is a great approach. Another method that may work is shearing with a 27-28g needle (such as an insulin or tuberculin syringe). I usually add lysis buffer to the nuclear pellet and try to get it off the bottom of the tube, and then add an equal volume 2X SDS sample buffer. Next pipet the sample it into the syringe, insert the plunger, and expel into a tube. After this, you should be able to draw the sample back up through the needle and passage up and down three of four more times. If the sample is too viscous to pipet initially, I find that simply increasing the volume will solve the problem. I usually boil after shearing, but boiling first can also make the sample a bit easier to pipet. Good luck.
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