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Purification of His-ubiquitin Proteins from Mammalian Cells
从哺乳动物细胞中纯化带His标签泛素化蛋白   

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Abstract

This protocol is used to purify His-tag ubiquitin conjugated protein. In this particular case, cells were transfected with His-tag ubiquitin and p53 which allowed us to purify using His-tag and reveal the WB using antibodies against p53 to see just the p53-ubiquitinate. The present protocol can be used in general for His-tag proteins expressed in mammalian cells.

Materials and Reagents

  1. EDTA free protease inhibitors (F. Hoffmann-La Roche, catalog number: 05892988001 )
  2. Proteasome inhibitor MG-132 (Calbiochem®, catalog number: 474790-5MG )
  3. Phosphate buffered saline (PBS)
  4. Tris
  5. Ni-NTA-Agarose beads (QIAGEN, catalog number: 30210 )
  6. Urea
  7. Guanidinium-HCl ( Sigma-Aldrich, catalog number: G4505-25G )
  8. Imidazole (Sigma-Aldrich, catalog number: 12399 )
  9. Triton X-100
  10. Glycerol
  11. β-mercaptoethanol
  12. SDS
  13. Bromophenol blue
  14. Lysis buffer (see Recipes)
  15. Wash buffer (see Recipes)
  16. Elution buffer (see Recipes)
  17. 4x laemmeli buffer (see Recipes)

Equipment

  1. Centrifuges
  2. Sonicator
  3. Western blotting equipment
  4. Tissue culture plates
  5. 1.5 ml eppendorf tube
  6. 15 ml falcon tube

Procedure

  1. Seed 1 x 106 cells on 10 cm tissue culture plates.
  2. After 8-12 h (around 40% of confluency) cells were transfect with 0.8 μg of plasmid for the His tag protein.
  3. 4 h before stop the culture add proteasome inhibitor MG-132 to a final concentration of 25 μM.
  4. 48 h after transfection aspirate the medium and wash the cells twice with pre-chilled PBS (cell can be keeping freezing on the plates at -80 °C until the day of the purification).
  5. Scrape cell with 1 ml of lysis buffer and transfer it in 1.5 ml eppendorf tube.
  6. Sonicate cells on ice twice for 10 sec with 1 min break (the sonicator probe should go under the lysate surface to avoid foaming but taking care do not touch the walls of the eppendorf tube).
  7. Spin cells 10 min at 11,000 rpm at 4 °C.
  8. Transfer the supernant in to 15 ml falcon tube and add 4 ml more of lysis buffer.
  9. Add 75 μl of Ni2+-NTA-agarose beads pre-equilibrate with lysis buffer (take 150 μl of Ni2+-NTA-agarose bead and spin 2 min at 4,000 rpm, discard the supernant and wash with 150 μl of lysis buffer four times, last time add 75 μl of lysis buffer and use).
  10. Incubate for 4 h at RT with gently agitation.
  11. Wash the beads at RT incubating 5 min with the following buffers:
    1. Once in lysis buffer.
    2. Once in wash buffer.
    3. Twice in wash buffer plus 0.1% Triton X-100 (spin 2 min at 4,000 rpm each time to recover the beads).
  12. Elute products by incubating beads in 75 μl of elution buffer for 20 min at RT with gentle agitation.
  13. Spin the beads and transfer supernant in to an eppendorf.
  14. Add 4x laemmeli buffer and boil for western blot analysis.

Recipes

  1. Lysis buffer
    6 M guanidinium-HCl
    0.1 M Na2HPO4/NaH2PO4
    10 mM Tris-HCl (pH 8)
    0.005 M imidazol
    0.01 M β-mercaptoethanol
  2. Wash Buffer
    8 M urea
    0.1 M Na2HPO4/NaH2PO4
    10 mM Tris-HCl (pH 6.8)
    0.005 M imidazol
    0.01 M β-mercaptoethanol
  3. Elution buffer
    0.2 M imidazol
    0.15 M Tris-HCl (pH 6.8)
    30% glycerol
    0.72 M β-mercaptoethanol
    5% SDS
  4. 4x laemmeli buffer
    2 ml 1 M Tris-HCl (pH 6.8)
    4.0 ml 20% (w/v) SDS
    4.0 ml glycerol
    4 mg bromophenol blue
    0.2 ml 14.3 M β-mercaptoethanol

Acknowledgments

This protocol was adapted from Gajjar et al. (2012). This work was supported by INSERM, La Ligue Contre le Cancer, and RECAMO CZ.1.05/2.1.00/03.0101. M.M.C. was supported by JSPS, AXA Research Fund, and Fundacao para a Ciencia e Tecnologia of Portugal. M.G. was supported by a research fellowship from Indo-French Centre for the Promotion of Advanced Research (IFCPAR) and from the ARC.

References

  1. Gajjar, M., Candeias, M. M., Malbert-Colas, L., Mazars, A., Fujita, J., Olivares-Illana, V. and Fahraeus, R. (2012). The p53 mRNA-Mdm2 interaction controls Mdm2 nuclear trafficking and is required for p53 activation following DNA damage. Cancer Cell 21(1): 25-35.
  2. Xirodimas, D., Saville, M. K., Edling, C., Lane, D. P. and Lain, S. (2001). Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo. Oncogene 20(36): 4972-4983.

简介

该方案用于纯化His标签泛素缀合的蛋白。 在这种特殊情况下,细胞用His标签泛素和p53转染,这允许我们使用His标签进行纯化,并使用针对p53的抗体显示WB以仅观察p53泛素化。 本方案通常可用于在哺乳动物细胞中表达的His标签蛋白。

材料和试剂

  1. EDTA游离蛋白酶抑制剂(F.Hoffmann-La Roche,目录号:05892988001)
  2. 蛋白酶体抑制剂MG-132(Calbiochem ,目录号:474790-5MG)
  3. 磷酸盐缓冲盐水(PBS)
  4. Tris
  5. Ni-NTA-琼脂糖珠(QIAGEN,目录号:30210)
  6. 尿素
  7. 胍盐酸盐(Sigma-Aldrich,目录号:G4505-25G)
  8. 咪唑(Sigma-Aldrich,目录号:12399)
  9. Triton X-100
  10. 甘油
  11. β-巯基乙醇
  12. SDS
  13. 溴酚蓝
  14. 裂解缓冲液(见配方)
  15. 洗涤缓冲液(见配方)
  16. 洗脱缓冲液(见配方)
  17. 4x laemmeli缓冲区(参见配方)

设备

  1. 离心机
  2. 超声波仪
  3. Western印迹设备
  4. 组织培养板
  5. 1.5 ml eppendorf管
  6. 15 ml falcon管

程序

  1. 在10cm组织培养板上种1×10 6个细胞
  2. 8-12小时后(约40%的汇合)细胞用0.8μgHis标签蛋白的质粒转染。
  3. 在停止培养前4小时,加入蛋白酶体抑制剂MG-132至终浓度为25μM。
  4. 转染后48小时,吸出培养基,用预冷的PBS洗涤细胞两次(细胞可以保持在-80℃的板上冷冻,直到纯化的天为止)。
  5. 用1ml裂解缓冲液刮取细胞,并将其转移到1.5ml eppendorf管中。
  6. 在冰上超声处理细胞两次,每次1分钟(超声波探针应在裂解液表面下方,以避免起泡,但注意不要接触埃彭道夫管壁)。
  7. 在4℃下以11,000rpm旋转细胞10分钟。
  8. 将上清液转移到15 ml falcon管中,加入4 ml更多的裂解缓冲液
  9. 加入75μL用裂解缓冲液预平衡的125μLNi 2+ 2+ -NTA-琼脂糖珠(取150μLNi 2+ + -NTA-琼脂糖珠,并旋转2分钟, 4,000rpm,弃去上清液,用150μl裂解缓冲液洗涤4次,最后加入75μl裂解缓冲液并使用)。
  10. 在室温下轻轻摇动孵育4小时
  11. 在室温下孵育珠子用以下缓冲液洗涤珠子5分钟:
    1. 一次在裂解缓冲液中。
    2. 一次在洗涤缓冲液中。
    3. 两次在洗涤缓冲液加0.1%Triton X-100(每次4,000 rpm旋转2分钟,以回收珠子)。
  12. 洗脱产物通过将珠子在75μl洗脱缓冲液中孵育20分钟,在RT轻轻搅拌。
  13. 旋转珠子,将上清液转移到eppendorf
  14. 加入4x laemmeli缓冲液,煮沸进行western印迹分析

食谱

  1. 裂解缓冲液
    6M盐酸胍
    0.1M Na 2 HPO 4钠/NaH 2 PO 4 4/1/2 10mM Tris-HCl(pH8)
    0.005 M咪唑 0.01 Mβ-巯基乙醇
  2. 洗涤缓冲液
    8 M尿素
    0.1M Na 2 HPO 4钠/NaH 2 PO 4 4/1/2 10mM Tris-HCl(pH6.8) 0.005 M咪唑 0.01 Mβ-巯基乙醇
  3. 洗脱缓冲液
    0.2 M咪唑 0.15 M Tris-HCl(pH 6.8)
    30%甘油 0.72μMβ-巯基乙醇 5%SDS
  4. 4x laemmeli缓冲区
    2ml 1M Tris-HCl(pH 6.8)
    4.0ml 20%(w/v)SDS
    4.0ml甘油 4mg溴酚蓝
    0.2ml14.3μMβ-巯基乙醇

致谢

该方案改编自Gajjar等人(2012)。 这项工作得到INSERM,La Ligue Contrele Cancer和RECAMO CZ.1.05/2.1.00/03.0101的支持。 M.M.C. 由JSPS,AXA研究基金和葡萄牙基金会赞助。 M.G. 得到了印度 - 法国促进高级研究中心(IFCPAR)和ARC的研究奖学金的支持。

参考文献

  1. Gajjar,M.,Candeias,M.M.,Malbert-Colas,L.,Mazars,A.,Fujita,J.,Olivares-Illana,V.and Fahraeus,R。(2012)。 p53 mRNA-Mdm2相互作用 控制Mdm2核转运并且是DNA损伤后p53活化所需的。 Cancer Cell 21(1):25-35。
  2. Xirodimas,D.,Saville,M.K.,Edling,C.,Lane,D.P.and Lain,S。(2001)。 p14ARF对体内泛素化p53和Mdm2的水平的不同影响 。 Oncogene 20(36):4972-4983
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Illana, V. O. and Farhaeus, R. (2012). Purification of His-ubiquitin Proteins from Mammalian Cells. Bio-protocol 2(17): e258. DOI: 10.21769/BioProtoc.258.
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Hailong Zhang
Department of Pediatrics, Emory University, USA
Sorry, I have a question, when you preparing your Lysis buffer and Wash buffer, what's the pH value for the 0.1M Na2HPO4/NaH2PO4? because different pH value needs different ratio between these two reagents.
8/11/2014 1:49:22 PM Reply
Vanesa Olivares-Illana
Interacciones Biomoleculares y Cancer, Instituto de Fisica, Mexico

Hello, the pH should be 8

8/11/2014 2:10:38 PM


Hailong Zhang
Department of Pediatrics, Emory University, USA

Thanks Vanesa,Let me make sure for the Lysis buffer: the pH should be the same as Tris-Hcl? so the 0.1M Na2HPO4/NaH2PO4 pH should be 8 in Lysis buffer and 6.8 in Wash buffer? or both are the same pH:7.0? Please confirm.Thank you again.

8/11/2014 2:26:00 PM


Vanesa Olivares-Illana
Interacciones Biomoleculares y Cancer, Instituto de Fisica, Mexico

yes, indeed for lysis buffer pH should be 8 and for wash buffer should be 6.8. Hope you have a nice experiment!!

8/12/2014 10:05:06 AM


Hailong Zhang
Department of Pediatrics, Emory University, USA
In your step "9", pre-equilibrate beads with lysis buffer, the last volume should be 75ul but not 75ml.
8/11/2014 11:44:35 AM Reply
Vanesa Olivares-Illana
Interacciones Biomoleculares y Cancer, Instituto de Fisica, Mexico

yes! indeed is 75ul

8/11/2014 2:09:12 PM


Hailong Zhang
Department of Pediatrics, Emory University, USA

Thanks for your reply

8/11/2014 2:26:29 PM


simone bosso
sapienza
Hi,I've read the reference-papers and in both of them TrisHCL concentration in Lysis and Wash Buffers is 10mM not 100mM as written above: which is the actual TrisHCL concentration?

I've another question: why is the volume of lysis buffer requested for NiNTA beads incubation (5ml) so large?

Many Many Thanks, Simone.
10/6/2013 11:37:57 AM Reply
Vanesa Olivares-Illana
Interacciones Biomoleculares y Cancer, Instituto de Fisica, Mexico

yes you are completely right! the good concentration is 10mM, im sorry for this mistake.
On the other hand 5ml is the volume that for my proteins gave the best yield

10/7/2013 6:56:00 AM


Bio-protocol Editorial Team
bio-protocol.org

Per author's request, the concentration of Tris-HCl has been corrected to 10mM in the protocol.

Sincerely,
Bio-protocol editing team

10/24/2013 11:14:49 PM


Giuseppe Bosso
University la Sapienza Rome

I have this question: in order to obtain the input (starting material/whole cell extract) can I save 15-30ul of lysate before diluting it with 4ml more of lysis buffer and beads incubation? Many Thanks
Giuseppe.
9/21/2013 8:20:11 AM Reply
Vanesa Olivares-Illana
Interacciones Biomoleculares y Cancer, Instituto de Fisica, Mexico

Hello and sorry for the delay in the answer!!. Yes you can keep 15-30ul of the lysate.
cheers
Vanesa

9/26/2013 7:02:36 AM


Why is the pH actually changing between lysis and washing buffer?
1/22/2013 7:59:03 AM Reply
Lin Fang
Department of Pediatrics, School of Medicine, Stanford University, USA


When pH > pI, a protein has a net negative charge. When pH < pI, a protein has a net positive charge.

In the higher pH of lysis buffer(8.0), your His-tagged protein binds to the Nickle beads strongly, but at the same time some nonspecific proteins are negatively charged and bind to the Nickle beads.

In the lower pH of washing buffer (6.8), your His-tagged protein still remain on the beads with strong affinity due to its His tag. The nonspecific proteins bound to the beads have less (if not zero) negative charges and dissociate from Nickle beads. So after washing step, the nickle beads should only bind to the His-tagged protein.

1/27/2013 7:09:13 AM


This may be a silly question, but is it the final pH of the solution that varies between buffers, or only the pH of the TRIS used?

Thanks much!
12/14/2012 11:15:28 AM Reply
Lin Fang
Department of Pediatrics, School of Medicine, Stanford University, USA

I am not the author, but let me try to answer your question: I would think it is commonly accepted that the pH is the Tris pH. As a result, the buffer ends up in the same (or similar) pH as Tris. But it is a good lab practice to always check pH of the buffer especially when it is your first time to make such buffer. Another caution is that samples you will add in the buffer does not maximize its buffer capacity, which is also done through sensibly checking pH.

12/22/2012 9:44:20 PM


Vanesa Olivares-Illana
Interacciones Biomoleculares y Cancer, Instituto de Fisica, Mexico

Hello and sorry for the delay in the answer.
Im agree with Lin, is commonly accepted that the pH is the Tris pH. Just one point, in the case of wash buffer you have also the component of phosphate buffer around 8, but you slowly will go down the pH (because of the explanation of Lin) of each buffer to improve the purification.

1/28/2013 8:20:27 AM