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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.

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Nuclear Extraction from Arabidopsis thaliana

Plant Science > Plant cell biology > Organelle isolation
Authors: Fang Xu
Fang XuAffiliation: Department of Botany and Michael Smith Laboratories, University of British Columbia, Vancouver, Canada
For correspondence: xufang@mail.ubc.ca
Bio-protocol author page: a188
 and Charles Copeland
Charles CopelandAffiliation: Department of Botany and Michael Smith Laboratories, University of British Columbia, Vancouver, Canada
Bio-protocol author page: a189
Vol 2, Iss 24, 12/20/2012, 9660 views, 5 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.306

[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.


How to cite this protocol: Xu, F. and Copeland, C. (2012). Nuclear Extraction from Arabidopsis thaliana. Bio-protocol 2(24): e306. DOI: 10.21769/BioProtoc.306; Full Text



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4/28/2015 12:57:08 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 8:06:36 AM  

Fang Xu (Author)
Department of Botany and Michael Smith Laboratories,University of British Columbia

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.

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3/30/2015 1:36:32 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 10:35:49 AM  

Fang Xu (Author)
Department of Botany and Michael Smith Laboratories,University of British Columbia

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 11:33:31 AM  

Ahmet B
Universität Bonn

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

3/30/2015 5:14:29 PM  

Fang Xu (Author)
Department of Botany and Michael Smith Laboratories,University of British Columbia

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.

4/19/2016 3:09:35 AM  

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

Reply

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11/16/2014 6:44:56 PM  

oji plant
psc

Hi,
please you can tell me the catalog number of Phenylmethanesulfonylfluoride (PMSF), I need to order it.
thanks

Reply

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8/27/2014 6:33:45 AM  

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 10:36:02 AM  

Fang Xu (Author)
Department of Botany and Michael Smith Laboratories,University of British Columbia

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.

8/28/2014 2:25:03 AM  

charukesi Rajulu
Rothamsted Research

Thanks a lot.

Reply

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3/24/2014 9:28:41 AM  

Adrianne Brown
Delaware State University

Can you reuse the lysis buffer after you add DTT and PMSF?

3/26/2014 4:59:05 PM  

Fang Xu (Author)
Department of Botany and Michael Smith Laboratories,University of British Columbia

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 5:20:54 PM  

Adrianne Brown
Delaware State University

Thank you very much!!!

Reply

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