I'm already extracting total protein with RIPA buffer. I now want to extract proteins only from the nucleus and only from the cytosol. The proteins shall be used for Western Blotting.
Hi Luis,
I'm attaching a paper that has a protocol for isolation of nuclei from human HeLa cells. If you follow this protocol, the first supernantant from the nuclear recovery step (1st low g centrifugation) would consistute the cytoplasmic subcellular fraction. Dialyze against an appropriate buffer and use this fraction for assessment of cytoplasmic proteins. Carry out the remainder of the nuclear extraction procedure to recover protein from the nuclear subcellular fraction. you can determine the efficiency of the fractionation by blotting for compartment specific proteins (i.e., a DNA polymerase of your choice vs. a glycolysis protein (but not glyceraldehyde-3-phosphate dehydrogenase as this 37kDa protein is also the nuclear human uracil-DNA glycosylase!)).
Hope this helps. Let me know if you want any more info and I'll see what I can do to help.
Good luck!
Tom
Article Determination of human DNA polymerase utilization for the re...
Hi Luis,
I'm attaching a paper that has a protocol for isolation of nuclei from human HeLa cells. If you follow this protocol, the first supernantant from the nuclear recovery step (1st low g centrifugation) would consistute the cytoplasmic subcellular fraction. Dialyze against an appropriate buffer and use this fraction for assessment of cytoplasmic proteins. Carry out the remainder of the nuclear extraction procedure to recover protein from the nuclear subcellular fraction. you can determine the efficiency of the fractionation by blotting for compartment specific proteins (i.e., a DNA polymerase of your choice vs. a glycolysis protein (but not glyceraldehyde-3-phosphate dehydrogenase as this 37kDa protein is also the nuclear human uracil-DNA glycosylase!)).
Hope this helps. Let me know if you want any more info and I'll see what I can do to help.
Good luck!
Tom
Article Determination of human DNA polymerase utilization for the re...
Just a follow-up. The protocol I mentioned is for a pretty large amount of cells, but it works well if you scale it down too.
Another reference; basically the same protocol but with a better suite of protease inhibitors added.
Mr. Thomas, thank you very much for your help! I didn't imagine I was gonna receive this ammount of information! Thank you again!
If I need more info even after this, I'll come back here for sure!
You're welcome Luis, and thanks for the upvote. Hope it works out! :)
Ms. Mónica, thank you for your help too! Unfortunately, I don't think my teacher is quite open to buy anything right now, so I must prepare my own extraction buffers at least for now...
Mr. Winters, I read your two articles and noticed that you used dounce homogenizer to lyse the cells (to get the nuclear fractions). Well, I don't have one here at the lab and really don't know anyone around the other labs near mine who may have one. Moreover, you didn't use any detergent. I was thinking about a protocol using a detergent with a different astringency for the cell lysis as we don't have here any homogenizers and any sonicators. Besides, we don't have all the proteinases inhibitors you used. We're using here roche proteinase inhibitor tablets, do you think it can substitute that proteinases inhibitors you used? Thank you very much.
Hi Luis. Yes, since you are primarily interested in recovering protein to western blot, rather than native/active protein, doing a differential partial extraction with detergent should work. I looked over the results for the kits Monica mentioned and they looked good, so you might want to look up the original papers that are cited in the kit literature to figure out which detergents to use and how exactly to make up the extraction buffers and run the protocol (concentrations, spins, etc.). As for the Roche protease inhibitor tablets, yes, I'm sure they'll probably work, but call me old-fashioned I guess, because I actually like to know what the composition of my buffers are (components and concentrations) and Roche never really reports what's in their cOmplete tablets and what the final concentration per unit volume is (i.e., per ml or L).
Best wishes Luis!
Tom
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