Protocol for nuclei extraction or isolation using IGEPAL - (Feb/28/2006 )

Hello everyone,

I was wondering if someone had a good protocol for nuclei isolation using Igepal.

The protocol I have does not seem to work that well.

Thanks for any help

LeserattePD

I like this one; what type of cells are you using?

NUCLEAR EXTRACTION OF TISSUE CELLS

This procedure was developed for HNEK cells grown in KGM, from Clonetics. All steps are performed on ice, with ice-cold reagents, in pre-chilled centrifuge tubes; this is to retard the activity of any proteases present as much as possible.

aspirate medium
wash with ice-cold PBS
add 3 mL ice-cold PBS and scrape cells gently into 15mL tube
centrifuge 5 minutes at 500 rpm, 4OC
carefully aspirate supernatant with pipet
resuspend pellet in 1 mL ice cold BUFFER I
incubate 15 minutes on ice to allow cells to swell
add Igepal-CA630 to 1% (100 ul of a 10% stock solution)
vortex 10 seconds
centrifuge 2-3 minutes at maximum speed (~15K RPM)
carefully aspirate supernatant with pipet; this is the cytoplasmic fraction
resuspend the pellet in 175 ul ice cold BUFFER II
vortex 30 seconds; rotate vigorously at 4OC 30 minutes
centrifuge 15 minutes at maximum speed; remove the supernatant to a fresh, chilled tube. leave on ice if using immediately, or store aliquots at –80OC until use. DO NOT freeze/thaw.
assay for protein concentration (Pierce’s BCA kit).
You will need:

PBS, tissue-culture grade
10 % Igepal-CA630 (w/v in dH2O)
tissue culture scrapers

A. 100 mL Buffer I – membrane lysis
1 mL 1 M Hepes, pH 8.0
150 μl 1 M MgCl2
1 mL 1 M KCl
100 μl 1 M DTT
dH2O to 100 mL
*filter sterilize and store at 4OC.
*aliquot enough for one use and add protease inhibitors (1000:1)

B. 100 mL Buffer II – nuclear envelope lysis
2 mL 1 M Hepes pH 8.0
150 μl 1 M MgCl2
25 mL glycerol
42 mL 1 M NaCl
40 μl 0.5 M EDTA
100 μl 1 M DTT
dH2O to 100 mL
*filter sterilize and store at 4OC.
*aliquot enough for one use and add protease inhibitors (1000:1)

Thanks a lot. It's for CHO cells (maybe HepG2s later).

oh, hey, this is probably obvious but let's be sure....all those centrifugations are at 4 degrees C, not 40 degrees C...the font transferred strangely

and please note...I get the best nuclear protein yield from a 6-well dish of mostly-confluent cells using those volumes of reagent

good luck, and please do let me know if it works for you!

Hi aimikins,

I'm not actually interested in the nuclei protein content, I just need intact nuclei for FACS to see how many of the nuclei have the labelled oligo I transfected in them. Therefore I've adapted your protocol up to the point before you add buffer II, so hopefully that will work. Should know tomorrow.

Thanks for your help.

LeserattePD

Hi aimikins,

I've tried the protocol now and we do get good nuclei isolation without cell debris, however the nuclei tend to stick together. I'd like to use them in FACS, so I need to single nuclei and I was wondering if you got any recommendations as to what to do.

I'm thinking about trying to squeeze them through a needle repeatedly or maybe adding some SDS. DNAse is out (I want to look at a labelled oligo in the nucleus), RNAse possibly?

Thanks again for your help

LeserattePD

I don't know enough to help you; I'm sorry! I just go straight through the protocol to get my nuclear proteins; I have recovered the cytoplasmic fraction but I have never tried to isolate intact nuclei

anyone else?

(ps I'm glad the protocol worked for you

Is the oligo fluorescently labelled? If so, you shouldn't need to isolate the nuclei. Just do the FACS directly on the cells. If not, so you have to indirectly detect the oligo, couldn't you just permeabilse the cells with Triton X-100? Cells tend to stick together before FACS but you could probably gate clumps out.

If you want to confirm the oligo is in the nucleus, I would have though microscopy of some kind was your best bet with a nuclear co-stain (DAPI/ Hoeschst).

All the best,
Ceri

wait a minute...why does it have to be FACS? can't you lyse the nuclei and do a southern? if you want to quantify, there is densitometry

Dear Ceri,

The oligo is fluorescently labelled. Unfortunately I do know that I need the oligo in the nucleus to be functional in my experiments (targeted gene therapy). I also know that I get it into most cells without problems, however only in a certain percentage of these cells is the oligo really in the nucleus.
As far as I know there is no way that FACS can tell you if the labelled oligo is colocalized spatially inside a cell with a nucleus stain. You can only say that the cell has the oligo not exactly where it is. Please correct me if I'm wrong about that.

I've already done confocal microscopy to show that only in a certain percentage of cells is the oligo really in the nucleus. I want to use that FACS to get good quantitative data, basically to compare different transfection methods and their efficiency for the nucleus localisation of oligo.