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Nuclear extracts from myeloid cells? - (Jun/12/2008 )

Hi all,

I've attempted several times to isolate cytoplasmic and nuclear extracts from 32D myeloid cells (also Ba/f3 and K562 cells). The protocol I'm using first lyses cells in buffer containing NP-40 (0.1%), then washes them, and lyses nuclei in nuclear extract buffer (high salt concentration). Every time I've done this (15+ times!), my cytoplasmic and nuclear extracts are both contaminated with the other fraction, but the nuclear extracts are almost always REALLY contaminated with cytoplasmic extract. I've tried letting the cells sit in the cytoplasmic extract buffer for longer, but that seems to increase the number of nuclei that lyse prematurely.

I'm wondering if anyone has had success in isolating clean nuclear extracts from these cells, and if so, how?

Thanks!

-montana-

QUOTE (montana @ Jun 12 2008, 08:12 AM)
Hi all,

I've attempted several times to isolate cytoplasmic and nuclear extracts from 32D myeloid cells (also Ba/f3 and K562 cells). The protocol I'm using first lyses cells in buffer containing NP-40 (0.1%), then washes them, and lyses nuclei in nuclear extract buffer (high salt concentration). Every time I've done this (15+ times!), my cytoplasmic and nuclear extracts are both contaminated with the other fraction, but the nuclear extracts are almost always REALLY contaminated with cytoplasmic extract. I've tried letting the cells sit in the cytoplasmic extract buffer for longer, but that seems to increase the number of nuclei that lyse prematurely.

I'm wondering if anyone has had success in isolating clean nuclear extracts from these cells, and if so, how?

Thanks!

I have personally not done these cells, but it seems if you are not already doing it, you need to find a protocol for suspension cell nuclear extract (if you can't find one for 32D).

Some things to consider:

1. Do people do hypotonic treatment (swell cells) first?
2. You don't have to isolate nuclear and cytoplasmic fractions from the same bunch of cells. You can first divide your cells in two, use one for purely nuclear and another for purely cytoplasmic isolation, that way you can be rigorous with each. (of course, only you can decide whether doing this is appropriate depending upon your exact experiment)

If you have time and energy, you can also wade through these links:

http://search.vadlo.com/b/q?sn=158621799&a...ract+&rel=0

..

-cellcounter-

What markers are you using to assess the purity
of your cytoplasmic and nuclear fractions? Are you sure these proteins
are expressed exclusively in the nucleus?
One of the problems obtaining pure cytoplasmic extracts
from myeloid cells is that the cells are almost entirely nuclear.

-mikew-

QUOTE (mikew @ Jun 12 2008, 05:58 PM)
What markers are you using to assess the purity
of your cytoplasmic and nuclear fractions? Are you sure these proteins
are expressed exclusively in the nucleus?
One of the problems obtaining pure cytoplasmic extracts
from myeloid cells is that the cells are almost entirely nuclear.



I'm using H3 as a nuclear marker and tubulin as a cytoplasmic marker. Cytoplasmic contaminates in my nuclear extracts are more of a problem for what I'm trying to do, although clean extracts from both the cytoplasm and the nucleus would be ideal. I have had zero luck finding a protocol for suspension cells.

Would increasing the concentration of NP-40 in my lysis buffer work, or do you think that would cause more nuclei to lyse early?

-montana-

Hi again,

Cell counter made a good point. Do you swell your cells first in a
hypotonic buffer (often 10 mM NACl or KCl)? This is impt.
Also, if you are already doing that, I wouldn't suggest increasing the NP-40 concentration, BUT you could extend the lysis time with NP-40. I have seen this vary from 30 seconds to 5 minutes. I add NP-40 to my swollen cells, vortex a little, and leave for about 1 minute. But the lysis time could be extended.

-mikew-

Thanks for all of your help so far.

I do swell the cells. The cytoplasmic extract buffer that I use has 10 mM KCl in it. The cells aren't swelled prior to lysis by NP-40 in this protocol, we just resuspend them in the cytoplasmic extract buffer (Hepes, KCl, EDTA, DTT, NP-40). Would swelling them first and then adding the NP-40 later help? Can I swell the cells, spin them down, dump the sup, and then resuspend in buffer with NP-40?

QUOTE (mikew @ Jun 13 2008, 02:49 PM)
Hi again,

Cell counter made a good point. Do you swell your cells first in a
hypotonic buffer (often 10 mM NACl or KCl)? This is impt.
Also, if you are already doing that, I wouldn't suggest increasing the NP-40 concentration, BUT you could extend the lysis time with NP-40. I have seen this vary from 30 seconds to 5 minutes. I add NP-40 to my swollen cells, vortex a little, and leave for about 1 minute. But the lysis time could be extended.

-montana-

Hi Montana,

Swelling the cells prior to NP-40 addition is really important for isolating
pure fractions (in my experience).
I swell the cells for 15 minutes in buffer with 10 mM KCl. After this I simply
add NP-40 to a final concentration of 0.125%. I spin down the lysed cells to recover the nuclei.
The supernatant from this step is your cytoplasmic fraction. It can be respun after collection to get rid of any debris.
Wash nuclei and then proceed to nuclear protein isolation.

-mikew-

Sweet, I will try this sometime this week and let you all know how it goes.

Thanks!

QUOTE (mikew @ Jun 15 2008, 03:32 PM)
Hi Montana,

Swelling the cells prior to NP-40 addition is really important for isolating
pure fractions (in my experience).
I swell the cells for 15 minutes in buffer with 10 mM KCl. After this I simply
add NP-40 to a final concentration of 0.125%. I spin down the lysed cells to recover the nuclei.
The supernatant from this step is your cytoplasmic fraction. It can be respun after collection to get rid of any debris.
Wash nuclei and then proceed to nuclear protein isolation.

-montana-

Success! Thanks for your help everyone!

QUOTE (montana @ Jun 16 2008, 11:44 AM)
Sweet, I will try this sometime this week and let you all know how it goes.

Thanks!

QUOTE (mikew @ Jun 15 2008, 03:32 PM)
Hi Montana,

Swelling the cells prior to NP-40 addition is really important for isolating
pure fractions (in my experience).
I swell the cells for 15 minutes in buffer with 10 mM KCl. After this I simply
add NP-40 to a final concentration of 0.125%. I spin down the lysed cells to recover the nuclei.
The supernatant from this step is your cytoplasmic fraction. It can be respun after collection to get rid of any debris.
Wash nuclei and then proceed to nuclear protein isolation.

-montana-

If you want real fractionation, you need to be doing ultracentrifugation - excellent protocol here

-bob1-