I'm having a lot of problems performing nucleus/cyto fractionation in DAUDI cell line. The protein recovery is really low. Do you have any suggestions?
To be able to anwer the question you should provide some basic information how you go for nuclear isolation - kit? if yes, from which company? home made protocol? if yes, how does it look like? cell number? treatment of the cells? in which volume do you finally take your nuclei? ... just to mention some points that should be clearified.
For adherent cells, I am working with a kit from Active Motif, personally I am using the Nuclear Complex Co-IP kit but colleages of mine were also using the simple Nuclear Extract kit and never faced any problem.
I'm using an homemade protocol based on hypotonic buffered followed by an high salt buffer in order tio disreupt nuclei. Usually everything is working until i'm lysing 20 milions of cells but the scaling up always failed...
I cannot sonicate since I assessed precipitation...
The kit I am working with is also based on some hypotonic buffer, and it uses later on some enzymatic digest of nuclei.
Instead of sonication you can use a syringe with a thin needle and give 5-20 strokes with it. This is used either to disrupt cells or to reduce viscosity of proteins and to fragment DNA.
How about going for less than 20 million cells and then pooling the material in one of the last steps?
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