Nuclear extraction - (May/26/2010 )
Hi,
I've reviewed the forum archives which were helpful, but still have a problem:
I'm working on a DNA binding, nuclear protein, and am interested in separating nuclear and cytoplasmic fractions, both for cleaner results, as well as for recognizing nuclear-cytoplasmic translocation.
I found a protocol in which cells are swollen using hypotonic buffer, then burst with 0.5% IGPAL added to buffer for 15'. Nuclei are then extracted with a high salt buffer.
I was somewhat concerned that 15 minutes with a 0.5% IGPAL buffer would also partially lyse nuclei, and indeed, my cytoplasmic fraction is contaminated with nuclear proteins.
I've noticed that following swelling, all protocols use either mechanical (homogenization or syringe) or detergent methods for releasing the cytoplsamic fraction. Does anyone have good experience with a similar protocol? I'd prefer not to use homogenization, since I would like to be able to work with small volumes.
Regards, Benny.
beenyg on May 26 2010, 12:37 PM said:
I've reviewed the forum archives which were helpful, but still have a problem:
I'm working on a DNA binding, nuclear protein, and am interested in separating nuclear and cytoplasmic fractions, both for cleaner results, as well as for recognizing nuclear-cytoplasmic translocation.
I found a protocol in which cells are swollen using hypotonic buffer, then burst with 0.5% IGPAL added to buffer for 15'. Nuclei are then extracted with a high salt buffer.
I was somewhat concerned that 15 minutes with a 0.5% IGPAL buffer would also partially lyse nuclei, and indeed, my cytoplasmic fraction is contaminated with nuclear proteins.
I've noticed that following swelling, all protocols use either mechanical (homogenization or syringe) or detergent methods for releasing the cytoplsamic fraction. Does anyone have good experience with a similar protocol? I'd prefer not to use homogenization, since I would like to be able to work with small volumes.
Regards, Benny.
Hi there,
I just incubate the cells with 400ul buffer A (10mM hepes 7.9, 10mM KCl, EDTA, EGTA, Betamercapto. plus inhibitors) 10 min in ice. Then I add 10ul of 10% NP40 and lysed by gently vortexing. Centrifugation and recolection of the citosolic fraction. Normally I dont have any contamination. (Then you have to wash several times the pellet and you continue with the nuclear extraction)
A colleague has a good protocol that uses homogenisation, he is able to process 500 ul as a minimum volume with 2.0 ml maximum for the homogeniser he uses. We did have to buy in homogenisers for this (I think from Kimble-Kontes). It needs to be a ball ended pestle rather than a cylinder one if you go this route. This proceedure is followed by a sucrose cushion centrifugation to clean the fractions up.
If you are planning on looking at both cytoplasm and nucleus, you should check that neither fraction is contaminated with the other (i.e. nucleus with no cytoplasm, as well as cytoplasm with no nucleus).