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MTV1 Pull-down Assay in Arabidopsis
拟南芥MTV1 的pull-down实验   

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Abstract

This protocol is an example of how to analyse suspected interactions between proteins using a pull-down assay (Sauer et al., 2013). A bait protein of interest (in this case, MTV1 of Arabidopsis thaliana) is fused to a GST tag and expressed in bacteria. The protein is isolated and allowed to bind to a matrix of glutathione-conjugated agarose beads via the GST-tag. Unspecifically binding proteins from the bacterial lysate are removed from the matrix. A native plant protein extract is then passed over the matrix and binding between the bait GST-MTV1 and prey proteins can occur. Extensive washes remove unspecifically bound proteins and finally, bait and prey proteins are released from the beads. Immunoblot analysis is then used to identify the proteins that bound to GST-MTV1. Importantly, a negative control consisting of the GST-tag alone is analysed in parallel to exclude the possibility that prey protein binding to the GST-MTV1 bait was due to the GST-tag.

Keywords: Pull-down(拉下来), Interaction(相互作用), Clathrin(网格蛋白), MTV1(mtv1), EPSIN(epsin)

Materials and Reagents

  1. BL21 Escherichia coli (E. coli) cells containing a plasmid for expression of the recombinant GST-MTV1 fusion protein (the bait)
    Note: In this exemplary case, the MTV1 coding sequence was cloned into a modified pGEX-2T plasmid (General Electric Company, catalog number: 28-9546-53 ), in which the multiple cloning site had been replaced by a Gateway cloning cassette (Note 1).
  2. BL21 E. coli cells expressing the GST tag alone as negative control
  3. Murashige and Skoog medium mix with vitamins and MES buffer (Duchefa Biochemie BV, catalog number: M0255.0010 )
  4. Glutathione agarose (Sigma-Aldrich, catalog number: G4510 )
  5. Complete inhibitor (EDTA free) (Roche Diagnostics, catalog number: 11 873 580 001 )
  6. Phenylmethylsulfonyl fluoride PMSF (e.g. Sigma-Aldrich, catalog number: P7626 )
  7. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  8. Carbenicillin (e.g. Sigma-Aldrich, catalog number: C9231 ) (Note 2)
  9. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (e.g. Sigma-Aldrich, catalog number: I6758 )
    Note: Should be prepared as aqueous stock solution of 1 M and stored at -20 °C.
  10. Sodium dodecyl sulfate (SDS) (e.g. Sigma-Aldrich, catalog number: L3771 , or any other supplier)
    Note: An aqueous stock solution of 20% (weight/volume) can be prepared and sterilized by autoclaving at 121 °C for 15 min.
  11. Glycerol (e.g. Sigma-Aldrich, catalog number: G5516 , or any other supplier)
  12. Tris (hydroxymethyl) aminomethane (Tris) (e.g. Sigma-Aldrich, catalog number: 252859 , or any other supplier)
  13. Yeast extract (e.g. Sigma-Aldrich, catalog number: Y1625 , or any other supplier)
  14. Tryptone (e.g. Sigma-Aldrich, catalog number: 95039 , or any other supplier)
  15. Anti-GST polyclonal antibody (optional) (Carl Roth, catalog number: 3998 )
  16. Anti-CHC monoclonal antibody (optional) (BD Biosciences, catalog number: 610499 )
  17. Liquid nitrogen
  18. NaCl (any supplier)
  19. KCl (any supplier)
  20. Na2HPO4 (any supplier)
  21. KH2PO4 (any supplier)
  22. β-mercaptoethanol
  23. Liquid grown Arabidopsis seedlings of 6-8 days (see Recipes)
  24. MS medium (see Recipes)
  25. PBS (see Recipes)
  26. Wash buffer (see Recipes)
  27. Extraction buffer (see Recipes)
  28. Sample loading buffer (see Recipes)
  29. Liquid Lysogeny Broth (LB) growth medium (see Recipes)

Equipment

  1. Microcentrifuge for 1.5 and 2 ml standard reaction tubes (any manufacturer)
    Note: Either refrigerated or situated in a 4 °C cold-room, should be able to reach 16,000 x g.
  2. Refrigerated centrifuge for 50 ml conical “Falcon” type tubes (any manufacturer)
    Note: Should reach 4 °C and 3,000 x g.
  3. Sonicator device (micro tip sonotrode type)
    Note: We use the labsonic model of B. Braun, which is, however, no longer produced. But any tip style sonicator device that is suitable for small volumes (2-5 ml) will work, for example the UP100H device coupled to the MS3 sonotrode (Hielscher Ultrasound Technology).
  4. Poly-Prep Chromatography columns (Bio-Rad Laboratories, catalog number: 731-1550 )
  5. Erlenmeyer flasks
  6. Paper towels
  7. 1.5 ml microcentrifuge tubes capable of supporting 16,000 x g (any supplier)
  8. 50 ml polypropylene conical centrifuge tubes “Falcon” type (any supplier)
  9. 0.20 µm filter unit (e.g. Minisart®, Sartorious, catalog number: 17597 ) plus compatible 5 ml syringe
  10. Shaking incubator for bacteria 37 °C (any manufacturer)
  11. Shaking incubator 25 °C or orbital shaker at room temperature (any manufacturer)
  12. End-over-end (orbital) mixer (any manufacturer)
  13. Spectrophotometer capable of measuring optical density at 600 nm (any manufacturer)
  14. Mortar and pestle (about 10 cm diameter) (any manufacturer)

Procedure

  1. Planning ahead
    The pellets of bacterial culture expressing GST-MTV1 and GST can be prepared any time in advance, as this material can be stored at -80 °C for several weeks. The plant extract is preferentially prepared on the day of the actual pulldown experiment. To generate this plant material, calculate a total of 8-10 days (from seed sterilization to harvest). The pulldown experiment can be carried out in one day.

  2. Protein expression in bacteria
    1. From single bacterial colonies (or verified glycerol stocks) of GST-MTV1 (bait) and GST (negative control) expressing bacteria, grow overnight cultures in 10 ml LB with appropriate antibiotic selection (in this case, 100 μg/ml carbenicillin). Use 50 ml Falcon type tubes and grow cultures in a shaking incubator at 37 °C with agitation of at least 200 rpm. Tubes should not be fully closed to allow gas exchange.
    2. Next day, prepare two Erlenmeyer flasks (500 ml volume) each with 150 ml LB + 100 μg/ml carbenicilin and add the MTV1-GST and GST pre-culture, respectively. Grow at 25 °C on an orbital shaker at least at 200 rpm, until OD600 reaches 0.6. Growing at 25 °C helps to produce the proteins in soluble form.
    3. To induce the expression of GST-MTV1 and GST, add IPTG to a final concentration of 1 mM and continue to grow the cultures at 25 °C for another 3-4 h.
    4. Distribute each culture into three 50 ml tubes and centrifuge them at 4 °C at 3,000 x g for 5 min.
    5. Discard the supernatant and gently resuspend the pellet in ice cold PBS, then centrifuge again as above.
    6. Discard the supernatant and store the pellets at -80 °C. Pellets can be stored for several weeks. For one pulldown experiment, only one of the pellets (corresponding to 50 ml of bacterial culture) is used.

  3. Extract protein from bacteria
    1. Take one pellet of GST-MTV1 and GST (corresponding to 50 ml bacterial culture) and resuspend each in 2.5 ml ice cold extraction buffer.
    2. Use a sonicator to disrupt the bacterial cells until they are lysed, which is indicated by reduced opacity and increased viscosity. This has to be carried out on ice, and it is recommended to sonicate in intervals (e.g. 10 sec sonicating followed by 10 sec on ice and so on). Care has to be taken not to sonicate with too high intensity. If foaming or the formation of a white precipitate is detected, the intensity needs to be lowered.
    3. Transfer the lysed bacteria solution to 1.5 ml microcentrifuge tubes and centrifuge at 4 °C, 16,000 x g for 20 min.

  4. Reconstitute glutathione beads
    1. In the meantime, reconstitute the glutathione agarose beads. For each GST-MTV1 and GST, weigh 10 mg of the dry beads and add 2 ml washing buffer. Let the beads swell for 10 min while vortexing occasionally.
    2. Load the slurries onto two Poly-Prep columns, place them upright in an appropriate holder and let the buffer drain through the bottom opening by gravity.
      Note: Do not centrifuge or apply vacuum to increase flow rate! This applies to all subsequent washing steps.
    3. Then wash the column with 10 ml washing buffer, again letting the column drain by gravity.

  5. Protein binding to glutathione beads
    Of the centrifuged bacterial lysates, take 150 µl of the supernatant and mix with 75 µl sample loading buffer, boil for 5 min and store at -20 °C, this will serve later as a control to check protein expression and integrity after bacterial lysis, for instance by immunoblot analysis using anti-GST antibody. The rest of the supernatant is loaded onto the columns, one for GST-MTV1 and one for GST. If viscosity of the lysate seems high, 1-2 ml of extraction buffer can be added. Close column in- and outlets and place them onto a rotating wheel at 4 °C for 2 h. Let the lysate drain by gravity, then wash the columns 3 times with 10 ml washing buffer at 4 °C.

  6. Plant extract
    1. Blot the liquid grown Arabidopsis seedlings dry, using paper towels. Weigh 0.7 g and transfer to a mortar.
    2. Freeze with liquid nitrogen and grind to a fine powder with a pestle.
    3. Add 4 ml extraction buffer and let slowly thaw, grinding the sample even more.
    4. Transfer the sample to 1.5 ml microfuge tubes and centrifuge for 20 min at 4 °C at 16,000 x g.
    5. Pass supernatant through a 0.20 µm filter syringe and keep extract on ice. Per column, 1 ml extract will be needed.

  7. Pulldown
    1. Close column outlets.
    2. Per column, mix 1 ml extract with 1 ml extraction buffer and add it onto the washed column.
    3. Close column inlets and place them onto a rotating wheel at 4 °C for 1.5 h.
    4. Then open the column in- and outlets and collect the flowthrough.
    5. Mix 150 µl of the flowthrough of each column with 75 µl sample loading buffer and boil for 5 min, keep at -20 °C. These samples are the flowthrough, containing all the unbound plant proteins (and usually some bacterial proteins as well).
    6. Wash the columns 3 times with 10 ml washing buffer at 4 °C, again letting drain by gravity.
    7. The bead volume is now about 100 µl. Close the column outlet and re-suspend the beads in 200 µl washing buffer.
    8. Take 150 µl of the bead slurry and add 75 µl sample loading buffer. Boil the sample for 5 min, mix vigorously and centrifuge for 5 min at 16,000 x g.
    9. Pass the supernatant to a fresh tube and store at -20 °C. These samples are the eluates which contain the proteins that bound to GST-MTV1 or GST, respectively, plus the bait proteins themselves.
    10. Typically, the flowthrough and the eluate samples are loaded next to each other on a immunoblot for both GST and GST-MTV1 (see Figure 1). It is often advisable to correct for different protein amounts in flowthrough and eluate, a good starting point is to load 5 µl flowthrough and 20 µl eluate, but this has to be determined empirically.


      Figure 1. Typical result of an immunoblot usig anti-clathrin heavy chain (CHC) antibody (approx. 190 kDA). In the flowthrough (FT) of both GST and GST-MTV1 the protein is detected, indicating that CHC was present at equal levels during the incubation. After washing, only the eluate (EL) of GST-MTV1 produces a signal, indicating that CHC bound specifically to MTV1. In this blot, 5 µl of FT sample and 20 µl of EL sample were loaded to compensate for different protein levels.

    11. A protein which binds specifically to GST-MTV1 will be detected in both flowthrough samples, but only in the eluate sample of GST-MTV1. This is for example the case for clathrin heavy chain (CHC), which can be detected using a commercial anti-CHC antibody (see Figure 1).
    12. A protein which is detected in both eluate samples is binding unspecifically to either the GST tag or the glutathione-agarose matrix. In this case, it can help to increase duration and number of washing steps or use a different washing buffer with higher amounts of salts or detergents from step G 6 onwards.

Notes

  1. This protocol can in theory be used for any kind of protein fused to the GST tag. However, the conditions likely need to be optimized for each particular case. MTV1 is an example of a soluble protein that is easily expressed in E. coli and does not require any eukaryotic posttranslational modifications, such as glycosylation. Other proteins might require a different expression and buffer system.
  2. PMSF is highly toxic and very unstable in aqueous solutions, so the preparation has to be done with care. A common practice is to prepare a 200 mM stock solution in methanol, which can be stored at -20 °C for at least half a year.
  3. The considerably more expensive carbenicillin can be substituted with ampicillin using the same concentration. However, ampicillin is less stable, so carbenicillin is the preferred choice.
  4. Complete inhibitor comes in the form of small tablets, each good for a volume of 50 ml final buffer. If a smaller volume is desired, the tablet can be dissolved in 1 ml water to produce a 50x stock solution and any surplus can be stored at -20 °C for a couple of weeks without dramatic loss of activity.

Recipes

  1. Liquid grown Arabidopsis seedlings
    1. 200-300 seeds of Arabidopsis thaliana (We used Col-0 accession, but can be any accession, mutant or transgenic line.) are sterilized in a 1.5 ml microcentrifuge tube by a 15 min incubation in 70% ethanol with occasional vortexing.
    2. In a cleanbench, wash the seeds 5 times with 1 ml sterile water. After the last wash, keep the seeds in water, close the tube and keep it in darkness at 4 °C for 2-3 days for stratification.
    3. The stratified seeds are then passed to a 250 ml Erlenmeyer flask containing 100 ml MS medium and incubated in a plant growth chamber with slight agitation (such as an orbital shaker at 50 rpm). We use a 16 h light/8 h dark regime and 24 °C. Plants are grown for 6-8 days.
  2. MS medium (1 L)
    2.45 g Murashige and Skoog medium mix with vitamins and MES buffer
    10 g sucrose
    Add mili-Q water to 1 L
    Adjust pH to 5.8 using KOH
  3. PBS (1 L)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolve in 800 ml mili-Q water
    Adjust pH to 7.4 using HCl
    Then fill up to 1,000 ml
  4. Wash buffer
    PBS
    0.5% Triton X-100
    0.5 mM PMSF (made from a stock solution of 200 mM PMSF in methanol ) (Note 3)
  5. Extraction buffer
    Like wash buffer, but with the addition of 1x complete inhibitor (Note 4)
  6. Sample loading buffer (50 ml)
    15 ml 20% SDS aqueous stock solution
    15 ml glycerol
    15 ml Tris 0.5 M solution (pH 6.8)
    1.25 ml mili-Q water
    A small amount (spatula tip) of bromphenol blue, just enough to give a medium blue color.
    Prior to use, add 75 μl β-mercaptoethanol per 925 μl buffer.
  7. Liquid Lysogeny Broth (LB) growth medium (1 L)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Fill to 1 L with mili-Q water
    Autoclave at 121 °C for 15 min

Acknowledgments

This protocol was adapted from the previously published paper Sauer et al., 2013. Part of the work was funded by a Ramon y Cajal research stipend to M.S.

References

  1. Sauer, M., Delgadillo, M. O., Zouhar, J., Reynolds, G. D., Pennington, J. G., Jiang, L., Liljegren, S. J., Stierhof, Y. D., De Jaeger, G., Otegui, M. S., Bednarek, S. Y. and Rojo, E. (2013). MTV1 and MTV4 encode plant-specific ENTH and ARF GAP proteins that mediate clathrin-dependent trafficking of vacuolar cargo from the trans-Golgi network. Plant Cell 25(6): 2217-2235.

简介

该方案是如何使用下拉测定分析蛋白质之间可疑相互作用的实例(Sauer等人,2013)。 感兴趣的诱饵蛋白(在这种情况下,为拟南芥的MTV1)与GST标签融合并在细菌中表达。 分离蛋白质并使其通过GST标签结合到谷胱甘肽缀合的琼脂糖珠的基质上。 从基质中除去来自细菌裂解物的非特异性结合蛋白。 然后将天然植物蛋白提取物通过基质,并且可以发生诱饵GST-MTV1和猎物蛋白之间的结合。 大量洗涤去除非特异性结合的蛋白质,最后,诱饵和猎物蛋白质从珠子中释放。 然后使用免疫印迹分析来鉴定与GST-MTV1结合的蛋白质。 重要的是,平行地分析由GST标记单独组成的阴性对照,以排除猎物蛋白结合到GST-MTV1诱饵是由于GST标记的可能性。

关键字:拉下来, 相互作用, 网格蛋白, mtv1, epsin

材料和试剂

  1. BL21大肠杆菌(大肠杆菌)细胞(大肠杆菌),其含有用于表达重组GST-MTV1融合蛋白(诱饵)的质粒。
    注意:在该示例性情况下,将MTV1编码序列克隆到修饰的pGEX-2T质粒(通用电气公司,目录号:28-9546-53)中,其中多克隆位点已被Gateway 克隆盒(注1)。
  2. BL21 E。 表达单独表达GST标签的大肠杆菌细胞作为阴性对照
  3. Murashige和Skoog培养基与维生素和MES缓冲液混合(Duchefa Biochemie BV,目录号:M0255.0010)
  4. 谷胱甘肽琼脂糖(Sigma-Aldrich,目录号:G4510)
  5. 完全抑制剂(不含EDTA)(Roche Diagnostics,目录号:11 873 580 001)
  6. 苯基甲基磺酰氟PMSF(例如Sigma-Aldrich,目录号:P7626)
  7. Triton X-100(Sigma-Aldrich,目录号:T8787)
  8. 羧苄青霉素(例如,Sigma-Aldrich,目录号:C9231)(注2)
  9. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(例如Sigma-Aldrich,目录号:I6758)
    注意:应制备为1 M的储备水溶液,并储存在-20°C。
  10. 十二烷基硫酸钠(SDS)(例如Sigma-Aldrich,目录号:L3771或任何其他供应商)
    注意:可以制备20%(重量/体积)的水性储备溶液,并通过在121℃下高压灭菌15分钟灭菌。
  11. 甘油(例如Sigma-Aldrich,目录号:G5516或任何其他供应商)
  12. 三(羟甲基)氨基甲烷(Tris)(例如Sigma-Aldrich,目录号:252859或任何其他供应商)
  13. 酵母提取物(例如Sigma-Aldrich,目录号:Y1625或任何其他供应商)
  14. 胰蛋白胨(如 Sigma-Aldrich,目录号:95039或任何其他供应商)
  15. 抗GST多克隆抗体(可选)(Carl Roth,目录号:3998)
  16. 抗CHC单克隆抗体(可选)(BD Biosciences,目录号:610499)
  17. 液氮
  18. NaCl(任何供应商)
  19. KCl(任何供应商)
  20. (任何供应商)
  21. KH 2 PO 4 (任何供应商)
  22. β-巯基乙醇
  23. 液体培养6-8天的拟南芥幼苗(参见Recipes)
  24. MS介质(参见配方)
  25. PBS(请参阅配方)
  26. 洗涤缓冲液(见配方)
  27. 提取缓冲液(参见配方)
  28. 样品加载缓冲液(参见配方)
  29. 液体溶菌性肉汤(LB)生长培养基(参见配方)

设备

  1. 微量离心机用于1.5和2ml标准反应管(任何制造商) 注意:冷藏或位于4°C冷藏室,应该能够达到16,000 x g。
  2. 用于50ml锥形"Falcon"型管(任何制造商)的冷冻离心机
    注意:应达到4°C和3,000 x g。
  3. 超声波仪(微型超声波仪型)
    注意:我们使用B. Braun的labsonic模型,然而,它不再产生。 但是任何适用于小体积(2-5ml)的尖端型超声波仪器都可以工作,例如连接到MS3超声波探头(Hielscher Ultrasound Technology)的UP100H仪器。
  4. Poly-Prep色谱柱(Bio-Rad Laboratories,目录号:731-1550)
  5. 锥形瓶
  6. 纸毛巾
  7. 1.5 ml微量离心管,能够支持16,000 x g (任何供应商)
  8. 50ml聚丙烯锥形离心管"Falcon"型(任何供应商)
  9. 0.20μm过滤器单元(例如,Minisart ,Sartorious,目录号:17597)以及相容的5ml注射器
  10. 摇动细菌培养箱37°C(任何制造商)
  11. 在室温(任何制造商)摇动培养箱25°C或轨道摇床
  12. 端到端(轨道)混合器(任何制造商)
  13. 能够测量600nm(任何制造商)光密度的分光光度计
  14. 砂浆和杵(直径约10厘米)(任何制造商)

程序

  1. 提前规划
    表达GST-MTV1和GST的细菌培养物的沉淀可以在任何时间预先制备,因为这种材料可以在-80℃下储存数周。植物提取物优选在实际下拉实验的当天制备。为了生成这种植物材料,计算总共8-10天(从种子灭菌到收获)。下拉实验可在一天内进行。

  2. 细菌中的蛋白表达
    1. 从单个细菌菌落(或验证的甘油储备)的GST-MTV1  (诱饵)和GST(阴性对照)表达细菌,生长过夜 培养在含有适当抗生素选择的10ml LB中 病例,100μg/ml羧苄青霉素)。使用50ml Falcon型管并生长 培养物在振荡培养箱中在37℃下搅拌至少200 rpm。管不应完全关闭以允许气体交换
    2. 第二天,准备两个锥形烧瓶(500ml体积),每个具有150 ml LB +100μg/ml羧苄青霉素并加入MTV1-GST和GST预培养, 分别。 在定轨振荡器上在25℃下至少以200rpm生长, 直到OD 600达到0.6。 生长在25°C有助于生产蛋白质 以可溶形式
    3. 为了诱导GST-MTV1和GST的表达, 加入IPTG至终浓度为1mM并继续生长 在25℃下再培养3-4小时
    4. 将每种培养物分配到三个50ml管中,并在4℃下以3,000xg离心5分钟。
    5. 弃去上清液,轻轻地将沉淀物重悬于冰冷的PBS中,然后如上所述再次离心。
    6. 弃去上清液并将沉淀储存在-80℃。 颗粒可以 储存几个星期。 对于一个下拉实验,只有一个 使用颗粒(相当于50ml细菌培养物)
  3. 从细菌中提取蛋白质
    1. 取一粒GST-MTV1和GST(相当于50ml细菌 培养),并将其重悬于2.5ml冰冷的提取缓冲液中
    2. 使用超声波仪破碎细菌细胞,直到它们被裂解, 这通过降低的不透明性和增加的粘度来指示。 这有 在冰上进行,并且建议间隔声音   (例如 10秒超声处理,然后在冰上10秒,等等)。 护理 以不太高的强度进行声波处理。 如果发泡或 检测到白色沉淀物的形成,强度需要 降低。
    3. 将裂解的细菌溶液转移到1.5毫升微量离心管,并在4℃,16,000×g离心20分钟。

  4. 重组谷胱甘肽珠
    1. 同时,重组谷胱甘肽琼脂糖珠。 每个 GST-MTV1和GST,称重10mg干珠,并加入2ml洗涤 缓冲。 让珠子膨胀10分钟,同时偶尔涡旋。
    2. 将浆料装载到两个Poly-Prep柱上,将它们直立放入   适当的夹持器,让缓冲液通过底部开口排出 通过重力。
      注意:不要离心或施加真空以增加流速!这适用于所有后续的清洗步骤。
    3. 然后用10ml洗涤缓冲液洗涤柱,再次通过重力使柱排出。

  5. 蛋白结合谷胱甘肽珠
    在离心的细菌裂解物中,取150μl上清液并与75μl样品上样缓冲液混合,煮沸5分钟并储存在-20℃,这将用作对照以在细菌裂解后检查蛋白质表达和完整性,例如通过使用抗GST抗体的免疫印迹分析。将剩余的上清液加载到柱上,一个用于GST-MTV1,一个用于GST。如果裂解物的粘度似乎高,可以加入1-2ml提取缓冲液。关闭色谱柱入口和出口,并将其放置在4°C的旋转轮上2小时。让裂解液通过重力排出,然后在4℃下用10ml洗涤缓冲液洗涤柱子3次
  6. 植物提取物
    1. 使用纸巾对生长的拟南芥幼苗进行印迹。 称重0.7g并转移到研钵中。
    2. 用液氮冷冻并用研杵研磨成细粉末。
    3. 加入4毫升提取缓冲液,缓慢解冻,研磨样品更多。
    4. 将样品转移到1.5 ml微量离心管中,在4℃,16,000×g离心20分钟。
    5. 使上清液通过0.20微米过滤注射器,并保持在冰上的提取物。 每个柱,需要1ml提取物。

  7. 拉下
    1. 关闭色谱柱出口。
    2. 每个柱,用1ml提取缓冲液混合1ml提取物,并将其加到洗涤的柱上。
    3. 关闭柱入口,并将其放置在4℃的旋转轮上1.5小时。
    4. 然后打开色谱柱入口和出口,收集流出物。
    5. 混合150微升的每列的流通,75微升样品加载   缓冲液并煮沸5分钟,保持在-20°C。 这些样本是 流过,包含所有未结合的植物蛋白(和通常一些   细菌蛋白质)。
    6. 在4℃下用10ml洗涤缓冲液洗涤柱3次,再次通过重力排出
    7. 珠体积现在约100μl。 关闭柱出口,并重悬在200μl洗涤缓冲液中的珠子。
    8. 取150μl的珠粒浆液,加入75μl样品上样缓冲液。 煮沸样品5分钟,剧烈混合并在37℃离心5分钟 16,000 x g 。
    9. 通过上清液到一个新的管和存储 -20℃。 这些样品是含有蛋白质的洗脱液 结合到GST-MTV1或GST,加上诱饵蛋白
    10. 通常,流出液和洗脱液样品 在GST和GST-MTV1的免疫印迹上彼此相邻地加载 (参见图1)。 通常建议校正不同的蛋白质 在流过和洗脱液中的量,一个好的起点是加载5μl   流过和20μl洗脱液,但这必须确定 经验

      图1.免疫印迹应用的典型结果 抗网格蛋白重链(CHC)抗体(约190kDA)。 流式细胞术(FT)的GST和GST-MTV1检测蛋白, 表明CHC在孵育期间以相等的水平存在。 洗涤后,只有GST-MTV1的洗脱液(EL)产生信号, 表明CHC特异性结合MTV1。 在这个污点,5微升的FT   样品和20μlEL样品以补偿不同 蛋白质水平
    11. 特异性结合的蛋白质 GST-MTV1将在两个流穿样品中检测,但仅在 GST-MTV1的洗脱液样品。 这是例如网格蛋白的情况 重链(CHC),其可以使用商业抗CHC检测 抗体(见图1)
    12. 在两者中检测到的蛋白质 洗脱液样品非特异性地结合GST标签或 谷胱甘肽 - 琼脂糖基质。 在这种情况下,它可以帮助增加 持续时间和数量的洗涤步骤或使用不同的洗涤缓冲液 与从步骤G 6向前的较高量的盐或洗涤剂。

笔记

  1. 这个协议可以 理论用于与GST标签融合的任何种类的蛋白质。 然而, 可能需要针对每种特定情况优化条件。 MTV1是易于在大肠杆菌中表达的可溶性蛋白的实例,并且不需要任何真核翻译后修饰, 例如糖基化。 其他蛋白质可能需要不同的 表达和缓冲系统
  2. PMSF是高度毒性和非常   在水溶液中不稳定,因此必须进行制备 关心。 通常的做法是在中制备200mM储备溶液 甲醇,其可以在-20℃下储存至少半年
  3. 的   相当昂贵的羧苄青霉素可以被取代 氨苄青霉素使用相同浓度。 然而,氨苄青霉素较少 稳定,因此羧苄青霉素是首选
  4. 完成 抑制剂以小片剂的形式存在,每种都有利于体积 50ml最终缓冲液。 如果需要更小的体积,可以是片剂 溶解在1ml水中以产生50x储备溶液和任何剩余物 可以在-20℃下储存几个星期而没有显着损失 活动。

食谱

  1. 液体生长的拟南芥幼苗
    1. 拟南芥的200-300个种子(我们使用Col-0登录,但可以 是任何登记,突变体或转基因株系)   微量离心管在70%乙醇中孵育15分钟 偶尔涡旋。
    2. 在cleanbench,洗种子5次 用1ml无菌水。 最后一次洗涤后,保持种子在水中, 关闭管,并保持在黑暗中在4°C 2-3天 分层。
    3. 然后将分层的种子传递至250 ml Erlenmeyer烧瓶中,并在植物中温育   轻微搅拌的生长室(例如50℃的轨道摇床) rpm)。 我们使用16小时光/8小时黑暗区域和24℃。 植物生长 持续6-8天。
  2. MS培养基(1L)
    2.45g具有维生素和MES缓冲液的Murashige和Skoog培养基混合物 10g蔗糖 将mili-Q水加入1 L
    使用KOH将pH调节至5.8
  3. PBS(1 L)
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
    使用HCl
    调节pH至7.4 然后填充到1000毫升
  4. 洗涤缓冲液
    PBS
    0.5%Triton X-100 0.5mM PMSF(由200mM PMSF在甲醇中的储备溶液制备)(注3)
  5. 提取缓冲区
    像洗涤缓冲液,但加入1x完全抑制剂(注4)
  6. 加样缓冲液(50ml)
    15 ml 20%SDS水溶液 15ml甘油 15ml Tris 0.5M溶液(pH6.8) 1.25 ml mili-Q水
    少量(小铲尖)的溴酚蓝,刚好足以产生中等蓝色的颜色 使用前,每925μl缓冲液加入75μlβ-巯基乙醇
  7. 液体溶菌液(LB)生长培养基(1L) 10g胰蛋白胨
    5g酵母提取物
    10克NaCl
    用mili-Q水填充至1 L / 121℃高压灭菌15分钟

致谢

该方案改编自以前发表的论文Sauer等人,2013年。部分工作由Ramon y Cajal研究津贴资助。

参考文献

  1. Sauer,M.,Delgadillo,MO,Zouhar,J.,Reynolds,GD,Pennington,JG,Jiang,L.,Liljegren,SJ,Stierhof,YD,De Jaeger,G.,Otegui,MS,Bednarek, ,E。(2013)。 MTV1和MTV4编码植物特异性ENTH和ARF GAP蛋白,介导网格蛋白依赖性运输从跨高尔基网络液泡货物。 植物细胞 25(6):2217-2235。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sauer, M. (2014). MTV1 Pull-down Assay in Arabidopsis. Bio-protocol 4(12): e1152. DOI: 10.21769/BioProtoc.1152.
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