I would like to measure enzymatic activity of some nuclear proteins, but I have troubles with the extraction and keeping its native form. Sonication seems to be a must in my cells to release nuclear proteins.
If you directly lyze or homogenize the cells in the presence of liquid nitrogen, the lysate can be directly used for assay of nuclear proteins. in liquid nitrogen, the cell slurry/ pellet immediately becomes dehydrated and using glass or zircon beads cells can be effectively lyzed!
Please look at the following below links which may help you in your analysis:
https://www.activemotif.com/documents/41.pdf
https://www.abcam.co.jp/ps/products/219/ab219177/documents/ab219177%20Nuclear%20Extract%20Kit%20(website).pdf
https://www.cellbiolabs.com/sites/default/files/AKR-172-nuclear-cytosolic-fractionation-kit.pdf
https://www.promocell.com/app/uploads/product-information/manual/PK-CA577-K266-100.pdf
Thanks
Shamsher S. Kanwar
So the cells are not sufficiently crushed though nitrogen liquid grinding? and further steps must be done on the slurry (powder) to release nuclear proteins. Am I right ?
If you mix cell slurry and zircon or glass beads together, add liquid nitrogen and grind, most of the cells shall lyse. Moreover, if you take cell slurry + glass beads + SDS-PAG sample buffer, and grind; most of the cells will rupture! Centrifuge this preparation after 1: 1 dilution with upper Tris buffer (pH 6.8), take supernatant, heat above boiling point for 3-4 minutes and load on SDS-PAGE directly. Perform electrophoresis and result shall be before you.
Good luck!
Shamsher S. Kanwar
What I meant is that grinding the cells by nitrogen liquid in mortar&pestle does not release nuclear proteins. Nitrogen liquid releases only cytoplasmic molecules and may causes damage to organelles. So, ground cells (powder obtained upon nitrogen liquid grinding) must furhter be subjected to lysis by SDS, glass beads..to efficiently release nuclear stuffs or organelle content. Right?
Hi,
TO avoid sonication and loss of its native structure, the nuclear proteins you desire could be extracted as suggested by Prof. Shamsher, in the presence of liquid nitrogen.
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