欢迎您, 登录 | 注册

首页 | English

X
加载中

Protein microarray is probably the only technique currently available for systematic investigation of protein-protein interactions. This protocol describes an optimized method to probe yeast protein microarrays for protein-protein interactions using purified V5-epitope tagged fusion protein. It should also apply to any other proteins with appropriate modifications.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Probing Yeast Protein Microarrays for Protein-protein Interactions Using V5-epitope Tagged Fusion Protein Probes
通过V5-抗原表位标签的融合蛋白标记酵母蛋白芯片探究蛋白之间的相互作用

系统生物学 > 蛋白质组学 > 蛋白质芯片/微阵列
作者: Joseph Fasolo
Joseph FasoloAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a22
 and Michael Snyder
Michael SnyderAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: mpsnyder@stanford.edu
Bio-protocol author page: a1789
Vol 2, Iss 5, 3/5/2012, 5913 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.123

[Abstract] Protein microarray is probably the only technique currently available for systematic investigation of protein-protein interactions. This protocol describes an optimized method to probe yeast protein microarrays for protein-protein interactions using purified V5-epitope tagged fusion protein. It should also apply to any other proteins with appropriate modifications.
Keywords: Kinase(激酶), Protein-protein interaction(蛋白质-蛋白质相互作用), Phosphorylation(磷酸化), Protein microarray(蛋白芯片), MAP Kinase(MAP激酶)

[Abstract] 目前,蛋白芯片可能是唯一一种验证蛋白之间相互作用的有效的工具。这种方法是通过用V5-抗原表位标签的融合蛋白标记酵母蛋白芯片,探究蛋白之间的相互作用。同时,这种方法也可以应用于其它任何一个修饰的蛋白。

Materials and Reagents

  1. Zirconia beads (Biospec Products, catalog number: 11079110zx 1.0mm dia)
  2. His-tag affinity resin (His Spintrap, GE Life sciences, catalog number:
    28-4013-53 )
  3. Protein microarray (Life Technologies, catalog number: PAH0525101 )
  4. V5-AlexaFluor 647 antibody (Life Technologies, catalog number: 451098 )
  5. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  6. Potassium chloride
  7. Sodium chloride
  8. Triton X-100
  9. Dithiothreitol (DTT) (Promega, catalog number: P1170 )
  10. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-250MG )
  11. Imidazole (Sigma-Aldrich, catalog number: I5513-5G )
  12. ATP (for kinases)
  13. BSA
  14. Tween-20
  15. Yeast extract
  16. Peptone
  17. Galactose
  18. Yeast nitrogen base
  19. (NH4)2SO4
  20. Raffinose (Sigma-Aldrich, catalog number: R0514-100G )
  21. Agar
  22. Lysis buffer (see Recipes)
  23. Wash buffer 1 (see Recipes)
  24. Wash buffer 2 (see Recipes)
  25. Wash buffer 3 (see Recipes)
  26. Elution buffer (see Recipes)
  27. Probe buffer (see Recipes)
  28. Blocking buffer (see Recipes)
  29. 3x YEP-GAL (see Recipes)
  30. Synthetic complete minus uracil media (Sc-ura) (see Recipes)

Equipment

  1. FastPrep Cell Lyser (MP Bio, catalog number: 116004500 - 1 each )
  2. High speed centrifuges
  3. JA-10 (or comparable) rotor
  4. Axon 4200 AL microarray reader
  5. 50 ml conical tube
  6. Wheel
  7. Humidified chamber

Procedure

  1. Probe protein purification
    1. Construct a V5-His6-protein fusion construct (i.e. pYES-DEST52, Life Technologies, Invitrogen™) of the yeast protein you wish to profile by using the methods outlined by Fasolo and Snyder (2009).
    2. Yeast (Y258 strain optimized for protein purification (Zhu et al., 2001)) that have been transformed with a pYES-DEST52 fusion construct under control of the GAL1 inducible promoter are grown in Sc-Ura/Dex overnight in 5 ml starter cultures.
    3. The next day OD600 are determined for the starter cultures and used to inoculate a 400 ml. Sc-Ura/Raffinose culture to an OD600 = 0.1. The culture is grown at 30 °C shaking to an OD600 = 0.55-0.6 and induced with 200 ml 3XYEP-GAL for 6 h at 30 °C shaking.
    4. Cell pellet is harvested by centrifugation using a JA-10 (or comparable) rotor by spinning 400 ml volumes of cell suspension at 4,000 rpm, 4 °C, 5 min.
    5. Wash cell pellet in ice cold PBS buffer by transferring the cell pellet to 50 ml conical tube and centrifuging in a tabletop centrifuge at 3,000 rpm, 4 °C, 5 min.
    6. Re-suspend cell pellet in ice cold PBS once more and aliquot evenly into 4 screw cap 2.0 ml. FASTPREP compatible tubes (pellet weight is ~0.5 g/tube).
    7. Spin cell suspension at 14,000 rpm, 4 °C, 1 min in a tabletop microfuge and aspirate supernatant. Snap freeze cell pellet immediately in liquid nitrogen and store at -80 °C or lyse immediately (you can stop here and proceed on the following day if not prepared for following steps).
    8. Add 0.5 mm zirconia beads, and lysis buffer at a 1:1:1 ratio to cell pellet and store on ice.
    9. Place tubes on FastPrep machine for 6 cycles of lysis at maximum setting for 1 min each, with 1 min on ice in between each interval.
    10. Spin suspension down in tabletop microcentrifuge at 14,000 rpm, 4 °C, 10 min, 10 min and collect supernatant.
    11. Repeat steps 9 and 10 then discard extracted pellet.
    12. Combine supernatant from both extractions into a single tube and incubate with Ni2+, or Co2+ affinity resin for 3 h/4 °C/rotating on a wheel.
    13. Centrifuge resin and save small aliquot of cleared supernatant for quality control.
    14. Wash resin 2x 10 min in wash buffer 1 /4 °C/rotating on a wheel.
    15. Wash resin 2x 10 min in wash buffer 2 /4 °C/rotating on a wheel.
    16. Wash resin 1x 10 min in wash buffer 3 /4 °C/rotating on a wheel.
    17. Collect washed resin in G25 or comparable column and elute with elution buffer and store at -80 °C until ready to use in probing assay (stop here is not ready to proceed).

  2. Protein microarray probing
    1. Dilute the purified protein probe over a concentration range of 5-500 μg/ml in the probe buffer (concentration of probe is empirically determined for each protein-protein interaction assay).
    2. Block the arrays in blocking buffer for 1 h by shaking at 50 rpm on a stage at 4 °C.
    3. After blocking, transfer the arrays to a humidified chamber, and add 90 μl of diluted probe directly to the array surface. Overlay the arrays with a raised lifter slip and incubate static (no shaking) in the humidor for 1.5 h.
    4. Wash the arrays 3 times for 1 min each in probe buffer in three 50-ml conical tubes.
    5. To detect interactions, dilute the V5-AlexaFluor 647 antibody to 260 ng/ml in probe buffer and mix thoroughly by shaking on a wheel for 30 min at 4 °C.
    6. After washing the arrays several times as indicated, add antibody solution directly to array and overlay with a raised lifter slip as before. Incubate the arrays for 30 min/static/4 °C.
    7. Finally, wash the arrays for 1 min (3x) in probe buffer, and spin in a 50-ml conical tube at 800 x g in a tabletop centrifuge for 5 min at RT. Air-dry the arrays in a slide holder in the dark for 30 min prior to scanning the array at 647 nm on an Axon 4200 AL microarray reader (or comparable).

Notes

  1. Protein microarrays used in this protocol are produced using full-length GST-fusion fusion proteins that were purified using glutathione sepharose beads. Protein arrays produced using proteins purified with antibody-conjugated matrices may result in high background when probed with anti-V5 antibody for detection due to the presence of residual IgG contamination. It is therefore important to test the background of each array with a negative control consisting of the detection antibody alone.
  2. Protein probe concentration must be high enough to dilute to working concentration in probe buffer without effecting solute concentration of probe buffer. This can be accomplished by scaling up culture to >10 L if necessary. Downstream applications like molecular weight cut-off columns may be used to exchange solutions and remove excess imidazole from final probe solution.
  3. Imidazole gradient in wash steps may be altered to optimize protein purification which can vary from protein to protein; protein probes must be at ~80-90% pure in order to which can be assessed by mass spectrometry and SDS-PAGE.

Recipes

  1. Lysis buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    10% glycerol
    0.1% Triton X-100
    0.5 mM DTT
    1 mM PMSF
    1x complete protease inhibitor tablet (F. Hoffmann-La Roche)
  2. Wash buffer 1
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    50 mM Imidazole
    10% glycerol
    0.1% Triton X-100
  3. Wash buffer 2
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    100 mM Imidazole
    10% glycerol
    0.1% Triton X-100
  4. Wash buffer 3
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    150 mM Imidazole
    10% glycerol
    0.1% Triton X-100
  5. Elution buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    0.1% Triton X-100
    500 mM NaCl
    0.5 mM DTT
    500 mM imidazole
    2 mM MgCl2
    25% glycerol (if freezing at -80 °C)
  6. Probe buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    1% glycerol
    2 mM MgCl2
    0.5 mM DTT
    0.05% Triton X-100
    50 mM NaCl
    500 μM ATP (for kinases)
    1% BSA
  7. Blocking buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    1% BSA
    0.1% Tween-20
  8. 3x YEP-GAL (yeast extract–peptone–galactose)
    30 g yeast extract
    60 g peptone
    Make up to 700 ml with dH2O
    Add 300 ml of sterile filtered 20% galactose to media after autoclaving.
  9. Synthetic complete minus uracil media (Sc-ura)
    1.5 g yeast nitrogen base
    5 g (NH4)2SO4
    2 g Sc-ura drop out mix (commercially available)
    20 g raffinose (or dextrose for starter culture media and SD-ura plates)
    20 g agar (for plates)

Acknowledgments

This protocol was developed in the Snyder Lab, Department of Genetics, Stanford University, Stanford, CA, USA. It was adapted from Fasolo et al. (2011), and Fasolo and Snyder, (2009). This work was supported by the NIH.

References

  1. Fasolo, J., Sboner, A., Sun, M. G., Yu, H., Chen, R., Sharon, D., Kim, P. M., Gerstein, M. and Snyder, M. (2011). Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes. Genes Dev 25(7): 767-778.
  2. Fasolo, J. and Snyder, M. (2009). Protein microarrays. Methods Mol Biol 548: 209-222.

材料与试剂

 

1.       氧化锆beads

2.       His-标签亲和树脂

3.       蛋白芯片

 

仪器

 

1.       FastPrep 细胞振荡器

2.       高速离心机

3.       Axon 4200 AL芯片信号输出器

 

步骤

 

1.       蛋白标记纯化

1)            根据Fasolo and Snyder (2009)的方法,构建V5-His6-蛋白。如,pYES-DEST52)。

2)            已经转入GAL1诱导启动子驱动pYES-DEST52融合载体的酵母,于加有DEX的缺尿嘧啶培养基上过夜培养。

3)            次日,将酵母转接到400ml Sc-Ura/Raffinose培养基中,OD600=0.1,震荡培养至OD600 = 0.55 - 0.6时,加入200 mL 3XYEP-GAL诱导培养6h

4)            4,000 rpm/4°C/5 min,集菌。

5)            将收集的细胞分装到50ml锥型管中,用预冷的PBS缓冲液洗涤细胞(3,000 rpm/4°C/5 min)。

6)            用预冷的PBS缓冲液重悬细胞。平均分装到2.0ml带有螺丝帽的管中。

7)            14000 rpm/4°C/1 min,离心。离心结束立即用液氮栋存于-80°C(如果你没有准备下一步,可以于此步)。

8)            加入0.5mm氧化锆beads和裂解buffer111),冰上贮存。

9)            按照震荡1分钟,冰上放置1分钟的方法,震荡裂解细胞。

10)        14000rpm/4°C/10 min /10 min,离心收集上清。

11)        重复910

12)        将上清集中到一个管中与Ni2+ , or Co2+亲和树脂于4°C旋转孵育3h

13)        用缓冲液14°C洗涤树脂两次,每次10分钟。

14)        用缓冲液24°C洗涤树脂两次,每次10分钟。

15)        用缓冲液34°C洗涤树脂两次,每次10分钟。

16)        用洗脱缓冲液洗涤树脂收集蛋白。贮存于-80°C直到使用。

2.       蛋白芯片标记

1)            用标记缓冲液稀释蛋白探针至浓度于5–500 μg/mL

2)            四度用封闭液封闭芯片(50 rpm1h)。

3)            封闭以后,将芯片转入湿润的容器内,直接向芯片表面加入90μL稀释的探针。随后,将芯片于湿润的升降机器上(不震动)孵育1.5h

4)            50ml管中用探针缓冲液洗涤芯片3次,每次1分钟。

5)            用探针缓冲液稀释V5-AlexaFluor 647 抗体至260 ng/mL,于4°C震荡孵育30分钟。

6)            5)中稀释的抗体与芯片静置孵育(30 min/ 4°C)。

7)            室温洗涤芯片(3X探针缓冲液,800个,5分钟),于黑暗中空气干燥芯片30分钟后,通过Axon 4200 AL microarray reader647nm扫描芯片。

 

试剂配方

 

1.       裂解缓冲液

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),150 mM NaCl 10%甘油, 0.1% Triton X-100 0.5 mM DTT1 mMPMSF 蛋白酶抑制剂。

2.       洗脱缓冲液1

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),150 mM NaCl50mM 咪唑(PH7.410% 甘油,0.1% Triton X-100

3.       洗脱缓冲液2

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),150 mM NaCl50mM 咪唑(PH7.410% 甘油,0.1% Triton X-100

4.       洗脱缓冲液3

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),150 mM NaCl50mM 咪唑(PH7.410% 甘油,0.1% Triton X-100

5.       洗脱缓冲液

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),0.1% Triton X-100500 mM NaCl0.5 mM DTT50mM 咪唑(PH7.4),2mM MgCl225% 甘油。

6.       探针缓冲液

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),0.1% Triton X-100500mM NaCl0.5 mM DTT50mM 咪唑(PH7.4),2mM MgCl21% 甘油,50mM ATP1% BSA

7.       封闭缓冲液

PBS0.01M磷酸缓冲液,0.0027M Kcl0137M Nacl PH7.4),1% BSA0.1% 吐温-20

8.       3× YEP–GAL

30g酵母提取物,60g蛋白胨,700ml水,300ml抽虑的20%半乳糖。

9.       缺尿嘧啶培养基

1.5g酵母无氨基氮源,5g(NH 4 ) 2 SO42 g Sc-ura20g棉籽糖,20g琼脂粉。

 

注意事项

 

1.       用这种方法使用的蛋白芯片使用的是全长GST融合蛋白。使用抗体检测蛋白可能会出现很强的背景,因为IgG的原因。所以,每一次检测负对照是很重要的。

2.       蛋白探针的浓度必须稀释至工作浓度。所使用的仪器必须洗去多余的咪唑。

3.       在洗涤蛋白的时候,要根据蛋白的差异,调节咪唑的浓度。为了达到质谱或者SDS-PAGE的需要,蛋白探针的纯度必须达到约80-90%

 

参考文献

 

1.        Fasolo J., Sboner A., Sun M.G., Yu H., Chen R., Sharon D., Kim P.M., Gerstein M., Snyder M. (2011). Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes. Genes and Development 25(7): 767-78. 

2.        Fasolo J., Snyder M. (2009). Protein microarrays. Methods in Molecular Biology 548: 209-22. 

 


English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Fasolo, J. and Snyder, M. (2012). Probing Yeast Protein Microarrays for Protein-protein Interactions Using V5-epitope Tagged Fusion Protein Probes. Bio-protocol 2(5): e123. DOI: 10.21769/BioProtoc.123; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook