Dear Goutam,

I am giving this protocol below, it really worked well for me...

Preparation of nuclear extract

Nuclear extract can be prepared according to the method of Schreiber et al (Schreiber et al., 1990). Cells at a concentration of 1 x 106 cell/well are pre‐incubated with or without test compound. The cells are then washed with PBS, dislodged and pelleted by centrifugation and resuspended in the cell lysis buffer [10 mM HEPES; pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% Nonidet‐40 and 0.5 mM PMSF along with the protease inhibitor cocktail (Sigma)] and allowed to swell on ice for 15‐20 min with intermittent mixing. Tubes are vortexed to disrupt cell membranes and then centrifuged at 12,000 g at 4°C for 10 min.

The supernatant is stored at ‐70°C till further use as cytoplasmic extract.

The pelleted nuclei were washed thrice with the cell lysis buffer and resuspended in the nuclear extraction buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF with protease inhibitor cocktail and incubated in ice for 30 min.

Nuclear extract is collected by centrifugation at 12,000 g for 15 min at 4°C. Protein concentration of the nuclear extract is estimated using Bradford’s reagent (BioRad, USA). The extract is either immediately used or stored at ‐70°C till further use.

Dear Goutam,

I am giving this protocol below, it really worked well for me...

Preparation of nuclear extract

Nuclear extract can be prepared according to the method of Schreiber et al (Schreiber et al., 1990). Cells at a concentration of 1 x 106 cell/well are pre‐incubated with or without test compound. The cells are then washed with PBS, dislodged and pelleted by centrifugation and resuspended in the cell lysis buffer [10 mM HEPES; pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% Nonidet‐40 and 0.5 mM PMSF along with the protease inhibitor cocktail (Sigma)] and allowed to swell on ice for 15‐20 min with intermittent mixing. Tubes are vortexed to disrupt cell membranes and then centrifuged at 12,000 g at 4°C for 10 min.

The supernatant is stored at ‐70°C till further use as cytoplasmic extract.

The pelleted nuclei were washed thrice with the cell lysis buffer and resuspended in the nuclear extraction buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF with protease inhibitor cocktail and incubated in ice for 30 min.

Nuclear extract is collected by centrifugation at 12,000 g for 15 min at 4°C. Protein concentration of the nuclear extract is estimated using Bradford’s reagent (BioRad, USA). The extract is either immediately used or stored at ‐70°C till further use.

Very good protocol above! Thank you

There is a kit we use from Sigma: Nuclei EZ Prep. Extremely easy to use and reliable. Essentially consists of a lysis buffer and nuclei storage buffer. Works with HEK293- don't know about NIHT3/Neuro2A.

Hi everybody thank you so much!!..,Debasis I was using a similar protocol but there was lots of debris in the nucleus I like to ask whether your protocol isolate pure nuclei...Many thanks

This protocol needs repeated washes of the nuclear pellet.. before that it has to be ensured that your cells have been disrupted properly by timely observing the sample under the microscope. You can see two different sizes of spherical structures but after thorough treatment with the cell lysis buffer the smaller spherical structures (nuclei) will be more and larger (intact cells) will be negligible.

Remember, do not stick to the time limit of 15-20 min but adjust it as per your observation. Also, keep the sample strictly in ice.

Yes, my nuclear fraction was very pure because I had put EMSA for a nuclear protein with both the fractions, cytosolic and nuclear, there was a clear difference rather contrast among the two. I also detected beta actin in the nuclear fraction, which was completely absent in the cytosolic fraction.

Debasis, that is a great help..thanku so much :)

Hi Debasis,

Do you have any idea about the protocol for isolation of nucleaus from brain tissues?

Thanking you,

Best

How I can demonstrate that I obtained nuclear fraction? 

If the nuclear fraction is used for western blotting then any loading control specific to the nucleus should be used such as lamin B1, TATA binding protein TBP, PCNA, histone H1, histone H3 which are exclusively present in nucleus and not cytoplasm. 

 As Debasis said.. I preferred Lamin B1 and Histone H3 :) 

Is this protocol suitable for a downstream experiment as a DNA Affinity binding assay?

Yes, I have used this protocol for EMSA - NFkB binding to DNA... so I can say that it is indeed an experiment of choice...

Hi Goutam and Debasis, 

Did you actually detect LaminB in the nuclear extract (using the protocol you provided) on western blot? I used the similar protocol for brain tissues and I am looking for a loading control to use for the extracts. 

Thank you! 

See protocol in

Article: Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine

Is that protocol useful for nuclear protein extraction from renal tissue? 

 I use NE-PER™ Nuclear and Cytoplasmic Extraction Reagents from fisher scientific.. It works well with my cells. 

Hi community. I want to ask you if this protocol works with tissue (brain tissue) o do you have another protocl for tissues? Thanks in advance

Hi Octavio, The above protocol by Debashish worked well with my cells, but its not very good for the tissues, I use the following protocol for tissue fractionation. and it works good with me. Good luck.

Hi Debasis Sahu - Could you kindly share your downstream EMSA protocol? Thank you!

Many thanks for you all ! How much cell lysis buffer should I use for 1x106 cell/well? What is the pellet/lysis buffer ratio?

Hello guys, '

Is it a must to add PMSF ? I only have protease/phosphates inhibitor cocktail ?

Hi Doaa

PMSF is a phosphatase inhibitor and because you already added phosphatase inhibitor cocktail, I don't think you must add it.

Good luck

Thank you Ahmed

Hi Mohamed Meghil

Would you please share the composition of  NE-PER™ Nuclear and Cytoplasmic Extraction Reagents from fisher scientific.....

i am facing difficulty in fractionation .....

Aditi Arora

Fisher Scientific doesn't release the composition of each reagent in that kit. They named them as Cytoplasmic extraction reagent I and II and nuclear extraction reagent (as a secret)

Here is the link for the kit if you need more info https://www.thermofisher.com/order/catalog/product/78833

Hi Debasis and Goutam,

The above discussed protocol for nuclear extraction looks great. I want to know what happens after nuclear lysis.

Does the supernatant also contain chromatin and its associated proteins.?

Goutam Kumar Tanti Debasis Sahu

I tried using the thermo kit NEPR . but its too expensive to use but if you wants good nuclear protein then i would recommend that

Hi everyone

Below I am attaching the link of full paper which I found this protocol worked efficiently with H9c2 cells. No kit is needed to buy.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853285/

Good luck

Thank you

Hi Goutam Kumar Tanti ,

I am now trying to get the nuclear protein from mice brain tissue but I didn't have a good protocol for that. Did you already find out the protocol for nuclear protein extraction in mice tissue?