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Nuclear Extraction from Arabidopsis thaliana
拟南芥中细胞核的分离   

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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: 0 4693132001 )
  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.

简介

该方案是从拟南芥细胞中分离核。 它们可以进一步用于其他实验,如核蛋白检测,核蛋白免疫沉淀等。

材料和试剂

  1. Tris-HCl(pH 7.4)
  2. 甘油
  3. KCl
  4. EDTA(pH7.5)
  5. MgCl 2
  6. 蔗糖
  7. Triton X-100
  8. Murashige和Skoog基础培养基(Sigma-Aldrich,目录号:M0404-10L)
  9. 苯基甲磺酰氟(PMSF)
  10. 二硫苏糖醇(DTT)
  11. 蛋白酶抑制剂(PI)(完全无EDTA)(Roche Diagnostics,目录号:04693132001)
  12. Phytagel(Sigma-Aldrich,目录号:P8169-1KG)
  13. 液氮
  14. 裂解缓冲液(LB)(参见配方)
  15. 含有0.2%Triton X-100(NRBT)的核重悬浮缓冲液(参见配方)
  16. 核重悬缓冲液(NRB)(见配方)
  17. 核储存缓冲液(NSB)(参见配方)
  18. MS(参见配方)

设备

  1. 离心机(例如:Eppendorf离心机5810 R,可以冷藏并装50 ml试管)
  2. 砂浆和杵
  3. 100μm和40μm尼龙网(BD Biosciences,Falcon ,目录号:REF352360,REF352340)。
  4. 50ml锥形管

程序

在MS上种子生长拟南芥种子2周或在土壤中生长4周。 收集约1g拟南芥组织(约50个平板生长的植物的幼苗,约20个土壤生长的植物的叶子),在液氮中冷冻,然后按照下列步骤。 > 注意:始终将样品保存在冰上。

  1. 使用冷的研钵和研杵在液氮中将组织研磨成细粉末。 将粉末收集到50ml锥形管中
  2. 向粉末中加入2ml冷的裂解缓冲液,并通过温和摇动或吸移将混合物匀化。
    注意:如果样品从过量的液氮中冷冻,请等待,直到其解冻,以便可以均质化。
  3. 依次通过100μm和40μm尼龙网过滤匀浆。
  4. 在4℃下以1500×g离心过滤的匀浆10分钟以沉淀细胞核。
  5. 弃去上清液,向沉淀中加入3ml NRBT。 通过移液重新悬挂细胞核。
  6. 在4℃下将样品以1500×g离心10分钟。 重复步骤5和6两次。
  7. 弃去上清液,向沉淀中加入3ml NRB。 通过移液重新悬挂细胞核。
  8. 在4℃下以1,500×g离心10分钟以沉淀细胞核,并弃去上清液。 此步骤的目的是删除Triton X-100。 核可以用于任何目的。 例如,它们可以用于使用western印迹检测核蛋白。 如果核不能立即使用,它们应该重新悬浮在400μlNSB缓冲液中,在液氮中快速冷冻,并储存在-80℃。 核可持续至少半年。

食谱

  1. 裂解缓冲液(LB)
    20mM Tris-HCl(pH7.4)2ml(2M) 25%甘油25ml
    20mM KCl 1ml(2M) 2mM EDTA 0.4ml(0.5M) 2.5mM MgCl 2 0.25ml(1M) 250mM蔗糖12.5ml(2M) 将H <2> 添加到100ml ml / 在使用前立即加入DTT和PMSF至终浓度为1mM。
  2. 含有0.2%Triton X-100(NRBT)的核重悬浮缓冲液 20mM Tris-HCl(pH7.4)2ml(1M) 25%甘油25ml
    2.5mM MgCl 2 0.25ml(1M) 0.2%Triton X-100 0.2ml
    将H <2> 添加到100ml ml /
  3. 核重悬缓冲液(NRB)
    20mM Tris-HCl(pH7.4)2ml(1M) 25%甘油25ml
    2.5mM MgCl 2 0.25ml(1M) 将H <2> 添加到100ml ml /
  4. 核存储缓冲区(NSB)
    20mM Tris-HCl(pH7.4)2ml(1M) 25%甘油25ml
    2.5mM MgCl 2 0.25ml(1M) 蔗糖15.1克
    将H <2> 添加到100ml ml / 使用前加入PI(1/100稀释)
  5. MS
    蔗糖10g
    Murashige和Skoog基础培养基4.4 g
    Phytagel 3 g
    将H 2 O加至1L,使用KOH调节pH至5.6-5.8,并高压灭菌30分钟。

致谢

该协议改编自Xu等人的研究文章(2012)。 我们感谢加拿大自然科学和工程研究理事会(NSERC)发现计划和中国科学技术部973计划的资助,资助号为2011CB10070。

参考文献

  1. Cheng,Y.T.,Germain,H.,Wiermer,M.,Bi,D.,Xu,F.,Garcia, Wirthmueller,L.,Despres,C.,Parker,J.E.,Zhang,Y.and Li,X. (2009)。 核孔复合体组分MOS7/Nup88是先天免疫和防御调节剂核聚集所需的拟南芥。植物细胞 21(8):2503-2516。
  2. Xu,F.,Xu,S.,Wiermer,M.,Zhang,Y.and Li,X.(2012)。 细胞周期蛋白L同源物MOS12和MOS4相关复合物是正确剪接植物抗性基因所必需的 。 Plant J 70(6):916-928。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xu, F. and Copeland, C. (2012). Nuclear Extraction from Arabidopsis thaliana. Bio-protocol 2(24): e306. DOI: 10.21769/BioProtoc.306.
提问与回复

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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