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Nuclear Extraction from Arabidopsis thaliana    

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Original research article

A brief version of this protocol appeared in:
The Plant Journal
Jun 2012

Abstract

This protocol is to isolate nuclei from Arabidopsis cells. They can be further used for other experiments, such as nuclear protein detection, nuclear protein immunoprecipitation and so on.

Materials and Reagents

  1. Tris-HCl (pH 7.4)
  2. Glycerol
  3. KCl
  4. EDTA (pH 7.5)
  5. MgCl2
  6. Sucrose
  7. Triton X-100
  8. Murashige and Skoog basal medium (Sigma-Aldrich, catalog number: M0404-10L )
  9. Phenylmethanesulfonylfluoride (PMSF)
  10. Dithiothreitol (DTT)
  11. Proteinase inhibitor (PI) (complete EDTA-free) (Roche Diagnostics, catalog number: 04693132001 )
  12. Phytagel (Sigma-Aldrich, catalog number: P8169-1KG )
  13. Liquid nitrogen
  14. Lysis buffer (LB) (see Recipes)
  15. Nuclei resuspension buffer with 0.2% Triton X-100 (NRBT) (see Recipes)
  16. Nuclei resuspension buffer (NRB) (see Recipes)
  17. Nuclei storage buffer (NSB) (see Recipes)
  18. MS (see Recipes)

Equipment

  1. Centrifuges (e.g. Eppendorf centrifuge 5810 R that can be refrigerated and will hold 50 ml tubes)
  2. Mortar and pestle
  3. 100 μm and 40 μm nylon mesh (BD Biosciences, Falcon®, catalog number: REF352360 , REF352340 )
  4. 50 ml conical tube

Procedure

Grow Arabidopsis seeds on MS for 2 weeks or on soil for 4 weeks. Collect approximately 1 g of Arabidopsis tissue (seedlings of about 50 plate-grown plants, leaves of approximately 20 soil-grown plants), freeze in liquid nitrogen, and then follow the steps listed below.
Note: Always keep the sample on ice.

  1. Grind the tissue to a fine powder in liquid nitrogen using a cold mortar and pestle. Collect the powder into a 50 ml conical tube.
  2. Add 2 ml cold Lysis buffer into the powder and homogenize the mixture by gentle shaking or pipetting.
    Note: If the sample is frozen from excess liquid nitrogen, wait until it is thawed enough that it can be homogenized.
  3. Filter the homogenate through a 100 μm and 40 μm nylon mesh sequentially.
  4. Centrifuge the filtered homogenate at 1,500 x g at 4 °C for 10 min to pellet the nuclei.
  5. Discard the supernatant and add 3 ml NRBT to the pellet. Re-suspend the nuclei by pipetting.
  6. Centrifuge the sample at 1,500 x g at 4 °C for 10 min. Repeat step 5 and 6 twice more.
  7. Discard the supernatant and add 3 ml NRB to the pellet. Re-suspend the nuclei by pipetting.
  8. Centrifuge at 1,500 x g at 4 °C for 10 min to pellet the nuclei, and discard the supernatant. The purpose of this step is to remove the Triton X-100. The nuclei can now be used for any purpose. For example, they can be used to detect nuclear protein using a western blot. If the nuclei cannot used immediately, they should be re-suspended in 400 μl NSB buffer, quickly frozen in liquid N2, and stored at -80 °C. The nuclei can last for at least half a year.

Recipes

  1. Lysis buffer (LB)
    20 mM Tris-HCl (pH 7.4) 2 ml (2 M)
    25% glycerol 25 ml
    20 mM KCl 1 ml (2 M)
    2 mM EDTA 0.4 ml (0.5 M)
    2.5 mM MgCl2 0.25 ml (1 M)
    250 mM sucrose 12.5 ml (2 M)
    Add H2O to 100 ml
    Add DTT and PMSF to a final concentration of 1 mM respectively, immediately before use.
  2. Nuclei resuspension buffer with 0.2% Triton X-100 (NRBT)
    20 mM Tris-HCl (pH 7.4) 2 ml (1 M)
    25% glycerol 25 ml
    2.5 mM MgCl2 0.25 ml (1 M)
    0.2% Triton X-100 0.2 ml
    Add H2O to 100 ml
  3. Nuclei resuspension buffer (NRB)
    20 mM Tris-HCl (pH 7.4) 2 ml (1 M)
    25% Glycerol 25 ml
    2.5 mM MgCl2 0.25 ml (1 M)
    Add H2O to 100 ml
  4. Nuclei storage buffer (NSB)
    20 mM Tris-HCl (pH 7.4) 2 ml (1 M)
    25% Glycerol 25 ml
    2.5 mM MgCl2 0.25 ml (1 M)
    Sucrose 15.1 g
    Add H2O to 100 ml
    Add PI before use (1/100 dilution)
  5. MS
    Sucrose 10 g
    Murashige and Skoog basal medium 4.4 g
    Phytagel 3 g
    Add H2O to 1 L, adjust pH to 5.6-5.8 using KOH and autoclave for 30 min.

Acknowledgments

This protocol was adapted from the research article Xu et al. (2012). We are grateful for financial support from Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery program and the 973 program of the Chinese Ministry of Science and Technology, grant number 2011CB10070.

References

  1. Cheng, Y. T., Germain, H., Wiermer, M., Bi, D., Xu, F., Garcia, A. V., Wirthmueller, L., Despres, C., Parker, J. E., Zhang, Y. and Li, X. (2009). Nuclear pore complex component MOS7/Nup88 is required for innate immunity and nuclear accumulation of defense regulators in Arabidopsis. Plant Cell 21(8): 2503-2516.
  2. Xu, F., Xu, S., Wiermer, M., Zhang, Y. and Li, X. (2012). The cyclin L homolog MOS12 and the MOS4-associated complex are required for the proper splicing of plant resistance genes. Plant J 70(6): 916-928.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Xu, F. and Copeland, C. (2012). Nuclear Extraction from Arabidopsis thaliana. Bio-protocol 2(24): e306. DOI: 10.21769/BioProtoc.306.
Q&A

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Md Mostafa Kamal
UGAS, Iwate University, Japan
Hi Fang:
Do I have to lysis the nuclei for mass spec study? or use the intact nuclei for tryptic digestion?

Best,
Kamal
10/9/2018 12:52:57 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

Hi Kamal, I think it depends on your purpose. You might also want to check with the MS facility

10/9/2018 7:23:25 AM


Gabriel Angelo Saraiva Raimundo
Universidade Federal de Viçosa
Hi, Fang I stored the cytoplasm Fraction after step 4 and did steps 5 to 8 as your protocol says to. to obtain the nuclear fraction, but my western blot showed a little of contamination from nucleus to cytoplasm and vice versa. I used anti UGPase to evaluation of cytoplasm and anti Histone 3 for the nuclear fraction. What could I do to improve my nucleus-cytoplams fractioning? Thank you, Gabriel
2/22/2018 6:30:57 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

Hi Gabriel, You can wash more times with NRBT to avoid the contamination from cytosol. To minimize contamination from nuclear fraction, try to do the whole process quickly and gently to avoid the damage of the nuclear envelope.

2/22/2018 1:21:10 PM


HUAN CHEN
University of South Carolina
Hi Fang, I am curious about that why the Triton was removed in Step 7 using NRB? Thanks,
2/21/2018 8:03:29 PM Reply
Fang Xu
Cold Spring Harbor Laboratory

Hi Huan, If you want to stock the nucleus, it's better to remove the triton to avoid any potential damage to the nuclear envelope.

2/22/2018 1:11:22 PM


HUAN CHEN
University of South Carolina

Thank you!

4/11/2018 7:29:50 PM


Anjil Kumar Srivastava
Durham University, Durham
Hi Fang, I want to purify the membrane protein and nuclear protein to see the difference between the interaction of two proteins. What changes I can made to get the membrane proteins. Thanks Anjil
2/16/2018 12:53:19 PM Reply
Fang Xu
Cold Spring Harbor Laboratory

Hi Anjil, You just save the cytosolic potion and do another centrifugation at16000g to get rid of other pellet. The supernatant is then subjected to ultra-centrifugation(100000g for 1hr), by which the membrane will be pellet down. All the best! Fang.

2/16/2018 12:59:36 PM


Jennifer Deke
Universität Hamburg
Dear Fang,

when I want to proceed with nuclei stored in Nuclei Storage Buffer, how do I get them out of it? Centrifuging did not seem to have worked well. Do you have a suggestion?

Best
8/11/2017 6:51:53 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

Hi Jennifer, thanks for your question. I usually centrifuge for 10min at 1500g to pellet the nuclei. And it works well for me, did you try it?.

8/23/2017 4:56:41 AM


Jennifer Deke
Universität Hamburg

Hi Fang,

thank you for your answer.
Yes we did, and in the other buffers it worked, but not in the storage buffer. We resuspended the nuclei pellet in storage buffer, froze it in liquid nitrogen and stored it a few days in -80°C. After thawing it on ice we tried to centrifuge it, but we only obtained a rather smeary pellet after centrifugation at 20,000xg for 10 min. At less speed no pellet could be seen.

Best

8/23/2017 6:49:40 AM


Fang Xu
Cold Spring Harbor Laboratory

It's really odd, it never happens to me.you can try to spin longer time at1500g. But if you use higher speed it will break the nuclei. I don't know whether there's something differ for your reagent. You can try to add the washing buffer without triton to dilute it and then spin it. I never did it as I never have problems to get the nuclei. But I think this method can be tried.

8/23/2017 6:57:29 AM


Jun Li
The University of Adelaide
Dear Fang,
I want to extract nuclear from Arabidopsis using the protocol which is highly similar with yours. I want to make Lysis buffer without DTT and PMSF, and what temperature can store the Lysis buffer (without DTT and PMSF) ? How can I sterilize the Lysis buffer without DTT and PMSF? Additionally, if my EDTA is PH=8, can I directly use this EDTA into lysis buffer?
4/27/2017 11:34:59 PM Reply
Fang Xu
Cold Spring Harbor Laboratory

Hi Jun, sorry for missing your questions,you can autoclave your lysis buffer without adding DTT, PMSF and PI. Also I would think it's not a big problem if you directly use the EDTA ph=8. Hope this is still helpful for you. Thanks.

8/23/2017 5:01:35 AM


Ahmet B
Universität Bonn
Dear Fang,

I tested the protocol and had my nuclei extraction run through mass spectrometry after my nuclear protein IP. but when I checked results , I realized the chloroplast contamination. there were high abundance of chloroplast proteins.
Do you have any suggestions to prevent this ? For example adding a slow centrifugation step before/after filtering, etc..?
4/28/2015 12:57:08 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

Yes, you can add a centrifugation step after filtering (100g for 10 min). You can also wash the nuclear with one more time and make sure that the nuclear pellet has no green color. When you wash the the pellet,try to pipette enough to complete suspend the pellet. I hope those suggestions will help.

4/28/2015 8:06:36 AM


Ahmet B
Universität Bonn
If I want to use nuclei immediately for a co-IP, again should I resuspend with 400 μl NSB buffer after I discard the supernatant at the end of Step 8 ?
3/30/2015 1:36:32 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

NSB buffer is used for store the nuclei as you can tell that it contains high concentration of Glycerol and Sucrose. For Co-IP experiment, you need to break the nuclear envelope and solubilize the protein for Co-IP. I use different buffer instead of NSB to suspend the nuclei for further step. Here is the recipe for your reference:20 mM HEPES-KOH pH 7.9, 2.5 mM MgCl2, 100 mM KCl, 20% (v/v) Glycerol, 0.2 mM EDTA, 0.2% Triton X-100, 1 mM DTT (add before use), Protease inhibitors (add before use), optional: Phosphatase inhibitors (add before use).

3/30/2015 10:35:49 AM


Ahmet B
Universität Bonn

but how much volume, do you suggest to resuspend the pellet from last step?

3/30/2015 11:33:31 AM


Fang Xu
Cold Spring Harbor Laboratory

Yes,suspend the pellet using the new buffer instead the NSB. How much volume depends on how you want the nuclear diluted. I usually suspend nuclear of 10g Arabidopsis in 1-2 mL buffer.

3/30/2015 5:14:29 PM


Emilio Gutierrez
SLU

Hi Fang,

I am using this protocol for Co-Ip experiment.. I would like to know how your break the nuclear envelope.. I have seen that you use different buffer to NSB, adding this buffer is enough to break the nuclei or I need some additional step?

Thanks

4/19/2016 3:09:35 AM


Fang Xu
Cold Spring Harbor Laboratory

Hi Emilio, NSB is used to store the nuclei which won't break the nuclear membrane. I use either sonicaction or sonication combined with high salt to break the membrane. I have worked with different proteins. It seems that some protein is easy to be released from Nuclei, while for some protein, it's hard. You can play with the condition. Sorry for missing your questions earlier and hope this is helpful for you.

8/23/2017 5:09:17 AM


oji plant
psc
Hi,
please you can tell me the catalog number of Phenylmethanesulfonylfluoride (PMSF), I need to order it.
thanks
11/16/2014 6:44:56 PM Reply
Charukesi Rajulu
Rothamsted Research
Hai,
Thanks for this protocol.
If I need to take the cytoplasmic fraction, at which step I should take the supernatent?
Thanks
8/27/2014 6:33:45 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

The supernatant after centrifugation in step 4 is the cytoplasmic fraction. You can transfer the supernatant carefully into a new container if you need all of them. Otherwise, you can take a small portion for analysis and dump the rest.

8/27/2014 10:36:02 AM


Charukesi Rajulu
Rothamsted Research

Thanks a lot.

8/28/2014 2:25:03 AM


Adrianne Brown
Delaware State University
Can you reuse the lysis buffer after you add DTT and PMSF?
3/24/2014 9:28:41 AM Reply
Fang Xu
Cold Spring Harbor Laboratory

I highly recommend you to use lysis buffer with freshly added DTT and PMSF. DTT is a relatively unstable compound due to air oxidation. PMSF is also very unstable in aqueous solutions (110 min at pH 7, 55 min at pH 7.5, and 35 min at pH 8, all at 25°C). I usually make lysis buffer stock without DTT and PMSF. I aslo make 1M DTT and 100mM PMSF (in ethanol or isopropanol). When I start the experiment, I add DTT and PMSF freshly into the lysis buffer.

3/26/2014 4:59:05 PM


Adrianne Brown
Delaware State University

Thank you very much!!!

3/26/2014 5:20:54 PM