Hello, Recently I'm doing Nuclear fractionation experiment with THP1 cell line.
But everytime i try it, I get jelly like DNA pellet in nuclear extract.
It's so sticky that I can't do pipetting on it.
I think maybe because of it, I can't extract nuclear protein properly (when I measured concentration by Bradford assay, its concentration was not detectable)
I have already tried sonication to breakdown genomic DNA. but jelly like DNA pellet has not be dissolved
Is there anyone that experienced same problem?
Please let me know how you have solved this problem
Hi Seungheon Yoo
Could you share which nuclear fractionation protocol you’re following, and what buffers or detergents are present in your extract? Additionally, do you primarily require nuclear extract for WB or downstream functional assays?
Hi Lucian Miles.
I have used Nuclear Extraction Kit by Chemicon
I don't know about exact composition of its buffer (Cell lysis buffer, Nuclear extraction buffer)
but when I tried manual method,
I followed protocol of this paper.
https://doi.org/10.1002/cpz1.1042
buffer composition
Hypotonic lysis buffer
10 mM Tris pH 7.5
10 mM NaCl
3 mM MgCL2
0.3% NP-40
10% glycerol
Nuclear lysis buffer
20 mM Tris pH 7.5
150 mM KCl
3 mM MgCl2
0.3% NP-40
10% glycerol
I want to use each fraction extract in WB and IP experiment
Thank you for your answering.
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