Hello you all,
I am trying to isolate nuclear extract to study the transcription factors. I am mainly working with cell lines, I have found two kits for this.
https://www.biocat.com/bc/pdf/AY2002.pdf ( Nuclear Extraction Kit For Use with Transcription Factor Assays)
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011398_NEPER_Nuc_Cytoplasmic_Extract_Reag_UG.pdf (NE-PER Nuclear and Cytoplasmic Extraction Reagents).
The problem is that I have no experience working with these kits, I don't know how good they are. In addition, I need around 2-3 mg of NE of each biological replicate. So, using kit to get this amount of NE would be quite expensive if only it worth it.
So, I am just wondering what would be the best way to do this?
Thanks
Hi Trung,
there is no need to buy "Nuclear Extraction" Kits, since you can prepare the required buffers quite easily by your own. Here are some protocols:
http://www.abcam.com/ps/pdf/protocols/nuclear%20fractionation%20protocol.pdf
http://www.lamondlab.com/newwebsite/Protocols%20for%20Website/Cellular%20Fractionation%20Protocol.pdf
https://www.researchgate.net/post/Cell_fractionation_for_Western_Blot-protocol
I used previously a protocol similar to that from abcam and it worked quite well for me, also for transcription factor studies. If you don't have a Dounce homogenizer, you can also use a sonicator.
Good luck !
Thank you so much Eugen.
This is great news for me. I will try this and let you know the results. Since I don't have a Dounce homogenizer, can you please share with me the protocol that you used before. Thanks.
Best,
Trung
Here an easy protocols (without kits, without Dounce) for nuclear extraction adapted from :
Nucleic Acids Res. 1989 Aug 11;17(15):6419.
Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells.
Schreiber E, Matthias P, Müller MM, Schaffner W.
I used it a lot of times for several transcription factors.
PROTOCOL:
-wash cells with PBS
-add 1ml (100 mm dish) TEN
-collect the cells
- Centrifuge 2 min at 2000 rpm
- Aspirate supernatant
-wash pellet with PBS
- Centrifuge 2 min at 2000 rpm
- resuspend the cells in 400 ul cold Buffer A
- mix gently
- incubate 15 min in ice
- Vortex 10 seconds
- centrifuge 30 seconds at 13000 rpm at 4°
-resuspend the pellet in about 50 ul ( 3 volumes of cell pellet) of Buffer C
- mix (MixMate eppendorf or similar)15 minute 4°
- centrifuge 5 min at 13000 rpm at 4°
- recover the supernatant
- aliquot supernatant, quickly frozen it in liquid nitrogen and store at -80°.
Buffer A
10 mM hepes ph 7.9
10mM KCl
0.1 mM EGTA
0.1 mM EDTA
1 mM DTT
add mix of antiproteases just prior to use
antiphosphatase if necessary
Buffer C
20 mM Hepes ph 7.9
400 mM NaCl
1 mM EDTA
1 mM EGTA
1 mM DTT
add mix of antiproteases just prior to use
antiphosphatase if necessary
10% v/v glycerol ( my adaptation for storage of nuclear extracts in freezer)
Do not sonicate! You will have a high background of nucleic acids, cytoplasmic proteins and histones.
There is no need to use a homogenizer. Simple hypotonic extractions work well.
Try the preparation described in http://mcb.asm.org/content/30/20/4922.full#sec-1
With cell lines, its quite easy to make concentrated extracts. Depending on the cell line you are using, you may want to go down to as little as 10 mM KCl in Buffer A. You can also adjust the salt in Buffer C, from 0.3-0.5M, but if you go above 0.45 for longer extraction times, you risk salting off histones and generating a sludgy extract. You can also consider adding glycerol to your buffers (up to ~10%) for stabilizing some proteins, depending on the type of downstream use of the protein.
Best,
Mark
For later experiments with nuclear proteins obtained with this hypo/hypertonic extraction, keep in mind that your extract are in 300 mM NaCl.
Thank you so much Federio and Mark,.
I have combine all the info to see which one suits me the best, then I will let you all know the results and the final protocol. Thanks again.
Best,
Trung
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