I am trying to isolate a nuclear protein NF-kB from raw cells in order to do a western blot.
I have searched some protocol for nuclear isolation and finally came up with one. But in that protocol, apart from adding 420mM Nacl for NE buffer , they have added an extra 400mM using 5M NaCl directly onto nuclear pellet and again 1 pellet volume of NE buffer. Can anyone tell why extra NaCl should be added, and if we add double the volume of NE buffer, won't the protein get diluted? I'm attaching that protocol along with this.
The increase in salt concentration is to have better lysis of your samples.
When I have performed cytoplasmic vs nuclear extraction, I used the Thermo Pierce NE-PER extraction kit. This has given me pretty good results. I have tried to look online for the recipes for the different buffers, but they are probably protected, so you would have to buy their kit. I am including their entire protocol. Maybe this can help you.
http://www.piercenet.com/instructions/2160872.pdf
I think you have misunderstood the protocol. They say add 1 pellet volume of NE buffer and readjust the salt concentration to 400mM. I would assume that with such a non-tight pellet if u resuspend in 50µl NE buffer, the final volume will be more and then u will have to add more salt to bring the salt concentration to 400mM (approx present in NE buffer).
Its normal to use High salt buffers in nuclear extraction protocols to extract chromatin associated proteins without lysing/making a mess with sticky DNA (similar protocols like that given by Anne above is also available from other companies- all based on somewhat same basic principle: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/nxtractbul.pdf )
Good Luck
Anti-oxidants can be used in this purpose. For precipitation acid media can be used.
Thanks
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