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Protein Immunoprecipitation Using Nicotiana benthamiana Transient Expression System
采用本氏烟草瞬时表达系统进行蛋白质免疫共沉淀实验   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Aug 2014

Abstract

Nicotiana benthamiana (N. benthamiana) is a useful model system to transiently express protein at high level. This protocol describes in detail how to transiently express protein in N. benthamiana and how to carry out protein immunoprecipitation in this expression system. This protocol can be broadly used for investigation on protein-protein interaction, protein purification and other related protein assay.

Materials and Reagents

  1. Nicotiana benthamiana seeds
  2. Agrobacterium strains harboring binary vector (e.g. pCambia1300, pBIN19) with the targeted gene fused with protein purification tag
    Note: In this protocol, we use 3x FLAG tag as an example.
  3. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  4. MES (pH 5.6) (Sigma-Aldrich, catalog number: M8250 )
  5. Protease Inhibitor (Roche, catalog number: 04693132001 )
  6. Nonidet P 40 substitute (Fluka, catalog number: 74385 )
  7. Induction medium (see Recipes)
  8. Infiltration solution (see Recipes)
  9. Extraction buffer (see Recipes)
  10. TBS (see Recipes)
  11. 4x SDS (see Recipes)

Equipment

  1. Anti-FLAG beads (Sigma-Aldrich, catalog number: A2220 )
  2. 3x FLAG peptides (Sigma-Aldrich, catalog number: F3290 )
  3. Protein G sepharose beads (GE healthcare, catalog number: 17-0618-01 )
  4. Test tubes, flask and shaker machine for culturing Agrobacterium
  5. Centrifuge
  6. 1 ml blunt syringe (BD, catalog number: 309659 )

Procedure

  1. Grow Nicotiana benthamiana at 22 °C (16 h day/ 8h night) to the age of 4 weeks. Grow Agrobacterium strains harboring binary vector with the targeted gene fused with 3x FLAG overnight at 28 °C at 200 rpm in 3 ml LB with proper antibiotic selection. With a sterilized pipette tip, inoculate a tiny amount of Agrobacterium glycerol stock into the test tube.
  2. Prepare induction medium and inoculate agrobacterium from the LB medium into the induction medium. (1/100 dilution for overnight culture or 1/25 for about 8 h culture). This can be done in the morning to prepare for an evening infiltration, or at night to prepare for infiltration the next morning.
  3. Incubate at 28 °C, with shaking at 200 rpm, for ~8-12 h, so that the Agrobacterium cell is in the log growth phase. The best OD value should be 0.6-0.8.
  4. Centrifuge Agrobacterium culture in 50 ml Falcon tube for 10 min at 3,200 x g to pellet the cells.
  5. Prepare infiltration solution.
  6. Resuspend Agrobacterium pellet in ~5-10 ml of infiltration solution.
  7. Measure the OD of Agrobacterium cell and add appropriate amounts of infiltration solution to dilute the Agrobacteria to the desired OD (usually 0.1-0.3 OD). Make at least 1 ml infiltration solution per leaf. If you want to co-express two or multiple proteins, mix different kinds of Agrobacteria, with the desired OD for each one. It’s recommended to carry out preliminary expression experiment to test the protein expression level for different constructs. You can adjust the OD value for each kind of Agrobacteria based on its protein expression level in N. benthamiana. For instance, you can use larger OD value for Agrobacteria expressing lower protein while use smaller OD value for Agrobacteria expressing abundant protein.
  8. Water the N. benthamiana plants about 30 min before the infiltration to make the infiltration easier.
  9. Choose the proper leaves for infiltration. Choose the healthiest looking plants, and avoid leaves that are torn or otherwise damaged. The ideal leaves are the young leaves, ~3-5 cm across, use 2-3 leaves per plant. One leaf is about 0.1 g of tissue, which is enough for measuring protein expression. For immunoprecipitation, 10 leaves are usually enough.
  10. Use a 1 ml blunt syringe to inject the solution into the underside of the leaf. Swirl the solution in the tube before drawing it up into the syringe, in order to evenly suspend the agrobacterium. Try to be as gentle as possible when injecting, to avoid damaging the leaf. The infiltrated area can easily be seen as a dark, water-soaked region. If the infiltrated region stops expanding without pushing too hard, infiltrate again in a different area of the leaf. There is no specific requirement for the infiltration regions as long as the infiltration can cover the leaf. Try to finish the infiltration using less than 5 different infiltrations per leaf. There is short video showing the Agrobacteria infiltration of N. benthamiana: https://www.youtube.com/watch?v=GHc7PU_jG2M. After infiltration, the plants can be grown at the same condition as before.
  11. Harvest the N. benthamiana leaves without the main vein at 36-48 h after agrobacterium infection. 1 g tissue usually is enough. Time point experiment might need to be carried out to decide at which time point the protein express the highest.
  12. Grind the tissues into a fine powder in liquid nitrogen using a cold mortar and pestle.
  13. Add extraction buffer [see Recipes, with 2% PVPP (polyvinylpolypyrolidone), 10 mM DTT, 1x protease inhibitor (PI) and 1 mM PMSF] into the mortar (2 ml extraction buffer per gram plant tissue), place it in the 4 °C fridge, wait until the powder start to thaw and homogenize the mixture by grinding.
  14. Transfer all the liquid into the 1.5 tubes and centrifuge at highest speed for 10 min at 4 °C.
  15. Transfer the supernatant into new tubes and centrifuge for another time.
  16. Transfer the supernatant into the tubes and add 20% NP40 to a final concentration of 0.15%.
  17. Add 20 μl protein G sepharose beads into the sample and incubate for 30 min in the cold room to pre-clear the sample (the beads were pre-equilibrated using 1 ml extraction buffer and the supernatant buffer was discarded after centrifugation at 12,000 x g for 30 sec).
  18. Pellet the beads by centrifugation at 12,000 x g for 30 sec and transfer the supernatant into new tubes. 60 μl of the supernatant is taken as the input sample, which stands for the sample before immunoprecipitation.
  19. Add 10 μl anti-FLAG beads into the supernatant. The beads also need to be equilibrated as shown in step 18. Incubate the beads at 4 °C for 3 h on an Eppendorf tube rotator.
  20. Pellet the beads by centrifugation and take 60ul of supernatant for flow-through sample.
  21. Wash the beads with 1 ml extraction buffer (with 0.15% NP40) for 3 times. For each washing step, pellet the beads by centrifuging at 12,000 x g for 1 min at 4 °C, discard the supernatant and add 1 ml extraction buffer.
  22. Add 100 μl diluted 3x FLAG peptide (dilute the 3x FLAG stock 1/10 in TBS containing 1 mM EDTA with 1x protease inhibitor. The 3x FLAG stock is 2.5 mg/ml in TBS which can stored at -20 °C for years) into the beads and incubate in the cold room for 1 h on an Eppendorf tube rotator.
  23. Pellet the beads by centrifugation. Transfer the supernatant into a new tube. Add 35 μl 4x SDS loading buffer into the supernatant. This fraction is the elution sample containing the purified immunoprecipitated protein. Add 60 μl 1x SDS loading buffer into the tube containing the beads. The beads sample might contain the residue protein that fails to elute from the beads.
  24. Add proper volume of 4x SDS loading buffer to the samples of input and flow-through. Boil all the samples at 95-100 °C for 5-10 min. And analyze the samples using Western blot.

Representative data

A representative experiment result was shown in the Figure 1. The figure was adapted from the Xu et al. (2014) as listed in the reference.


Figure 1. bHLH84-HA co-immunoprecipitates with SNC1-FLAG. Two samples were used in the Co-IP experiment including sample with coexpression of bHLH84 and SNC1-FLAG and sample with expression of only bHLH84, which serves as the negative control.

Notes

  1. Negative control samples are always recommended during the immunoprecipitation process. For immunoprecipitation of single protein, it is suggested to express the empty vector to serve as the negative control. For co-immunoprecipitation of two or more proteins, it is suggested to express the empty vector without the bait protein together with the relative vectors containing the prey proteins. Based on the experiment design, more negative controls can be included accordingly.
  2. As long as the protein can be pulled down, the immunoprecipitation experiment is highly reproducible although the IP efficiency might be vary slightly in different trials.

Recipes

  1. Induction medium
    Note: You also need to add proper antibiotics to the following media.
    Reagent
    Stock concentration
    Sterilization
    Volume /100 ml
    Final concentration
    K2HPO4
    10.5 g/L
    Mix all together with water and autoclave
    96.85 ml

    KH2PO4
    4.5 g/L

    (NH4)2SO4
    1.0 g/L

    NaCitrate:2H2O
    0.5 g/L

    Glycerol
    0.5%

    Glucose
    20%
    Filter
    1 ml
    0.2%
    MgSO4
    1 M
    Filter
    100 µl
    1 mM
    Acetosyringone
    100 mM
    Filter
    50 µl
    50 µl
    MES (pH 5.6)
    0.5 M
    Filter
    2 ml
    10 mM

  2. Infiltration solution
    Reagent
    Stock concentration
    Sterilization
    Amount per 20 ml
    Final concentration
    MS powder

    NA
    0.088 g
    4.4 mg/L
    MES
    0.5 M
    Filter
    0.4 ml
    10 mM
    Acetosyringone
    100 mM
    Filter
    30 µl
    150 µM

  3. Extraction buffer
    10% glycerol
    25 mM Tris-HCl (PH 7.5)
    1 mM EDTA
    150 mM NaCl
  4. TBS
    10 mM Tris-HCl
    150 mM NaCl (PH 7.4)
  5. 4x SDS
    200 mM Tris-HCl (PH 6.8)
    8% SDS
    40% glycerol
    0.04% bromophenol blue
    400 mM DTT

Acknowledgments

We are grateful for the financial supports from Natural Sciences and Engineering Research Council (NSERC) (Canada) Discovery grant program. The transient protein expression in N. benthamiana was adapted from Van den Ackerveken et al. (1996). The IP protocol was modified from Moffett et al. (2002).

References

  1. Moffett, P., Farnham, G., Peart, J. and Baulcombe, D. C. (2002). Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death. EMBO J 21(17): 4511-4519.
  2. Van den Ackerveken, G., Marois, E. and Bonas, U. (1996). Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87(7): 1307-1316.
  3. Xu, F., Kapos, P., Cheng, Y. T., Li, M., Zhang, Y. and Li, X. (2014). NLR-associating transcription factor bHLH84 and its paralogs function redundantly in plant immunity. PLoS Pathog 10(8): e1004312.

简介

本塞姆氏烟草 ( )是一种用于高水平瞬时表达蛋白质的有用的模型系统。 该协议详细描述了如何在N中瞬时表达蛋白质。 本bent和如何在此表达系统中进行蛋白质免疫沉淀。 这个协议可以广泛用于调查蛋白质 - 蛋白质相互作用,蛋白质纯化和其他相关的蛋白质测定。

材料和试剂

  1. 本尼特烟草种子
  2. 携带具有与蛋白质纯化标记融合的靶基因的二元载体(例如pCambia1300,pBIN19)的土壤杆菌菌株
    注意:在此协议中,我们使用3x FLAG标签作为示例。
  3. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  4. MES(pH 5.6)(Sigma-Aldrich,目录号:M8250)
  5. 蛋白酶抑制剂(Roche,目录号:04693132001)
  6. Nonidet P 40 substitute(Fluka,catalog number: 74385)
  7. 感应介质(见配方)
  8. 渗透解决方案(参见配方)
  9. 提取缓冲液(参见配方)
  10. TBS(参见配方)
  11. 4x SDS(见配方)

设备

  1. 抗FLAG珠(Sigma-Aldrich,目录号:A2220)
  2. 3x FLAG肽(Sigma-Aldrich,目录号:F3290)
  3. 蛋白G琼脂糖珠(GE healthcare,目录号:17-0618-01)
  4. 用于培养土壤杆菌的试管,烧瓶和振动机。
  5. 离心机
  6. 1ml钝的注射器(BD,目录号:309659)

程序

  1. 在22℃(16小时一天/8小时夜晚)至4周龄时生长本塞姆氏烟草。将具有与3x FLAG融合的靶基因的二元载体的土壤杆菌菌株在28℃,200rpm下在具有适当抗生素选择的3ml LB中过夜生长过夜。使用灭菌的移液管吸头,将少量的农杆菌甘油原液接种到试管中。
  2. 制备诱导培养基并将土壤杆菌从LB培养基接种到诱导培养基中。 (1/100稀释过夜培养或1/25稀释约8小时培养)。这可以在早上进行以准备晚上渗透,或在晚上准备第二天早上的渗透。
  3. 在28℃下以200rpm摇动孵育约8-12小时,使得细菌细胞处于对数生长期。最佳OD值应为0.6-0.8
  4. 在50ml Falcon管中以3,200×g离心10分钟,离心土壤杆菌培养物以沉淀细胞。
  5. 准备渗透溶液。
  6. 在〜5-10ml的浸润溶液中重悬土壤杆菌沉淀
  7. 测量细菌细胞的OD,并加入适量的浸润溶液以将土壤杆菌稀释至所需的OD(通常为0.1-0.3OD) 。每片至少1ml浸润溶液。如果你想共表达两个或多个蛋白质,混合不同种类的农杆菌,每个都有所需的OD。建议进行初步表达实验以测试不同构建体的蛋白表达水平。您可以根据其在 N中的蛋白质表达水平调整每种土壤杆菌的OD值。 benthami 。例如,您可以使用较大的OD值代表表达较低蛋白质的土壤杆菌,同时使用较小的OD值表达表达丰富蛋白质的农杆菌。
  8. 加水。本氏烟植物在浸润前约30分钟使渗透更容易。
  9. 选择适当的叶子渗透。选择最健康的植物,避免被撕裂或损坏的叶子。理想的叶子是年轻的叶子,〜3-5厘米跨,每株使用2-3片叶子。一片叶是约0.1g组织,这足以测量蛋白质表达。对于免疫沉淀,10片叶通常就足够了。
  10. 使用1毫升钝的注射器将溶液注入叶子的下侧。在将其拉入注射器之前,将溶液在管中旋转,以便均匀地悬浮农杆菌。尽量在注射时尽可能温柔,以免损伤叶子。渗透区域可以很容易地看作是一个黑色的,水浸泡的区域。如果渗透区域在不太推力的情况下停止膨胀,则再次在叶的不同区域中渗入。只要渗透可以覆盖叶片,对渗透区域没有特定要求。尝试完成渗透使用每叶少于5个不同的渗透。有短视频显示农杆菌 N的渗透。本bent:: https://www.youtube.com/watch?v=GHc7PU_jG2M。浸润后,植物可以在与以前相同的条件下生长
  11. 收获 N。本生烟草在土壤杆菌感染后36-48小时没有主静脉。 1g组织通常就足够了。可能需要进行时间点实验以决定蛋白质在哪个时间点表达最高。
  12. 使用冷的研钵和杵将组织在液氮中研磨成细粉
  13. 添加提取缓冲液[参见配方,用2%PVPP(聚乙烯聚吡咯烷酮),10mM DTT,1×蛋白酶抑制剂(PI)和1mM PMSF]到研钵中(每克植物组织2ml提取缓冲液),将其置于4° C冰箱中,等待粉末开始解冻并通过研磨使混合物均匀
  14. 将所有液体转移到1.5管中,并在4℃以最高速度离心10分钟
  15. 将上清液转移到新管中,再离心一次。
  16. 将上清液转移到管中,加入20%NP40至终浓度为0.15%
  17. 向样品中加入20μl蛋白G琼脂糖珠,并在冷室中孵育30分钟以预清除样品(使用1ml提取缓冲液预平衡珠,在12,000rpm离心后弃去上清液缓冲液, xg 30秒)。
  18. 通过在12,000×g离心30秒来沉淀小珠,并将上清液转移到新管中。取60μl上清液作为输入样品,代表免疫沉淀前的样品
  19. 加入10μl抗FLAG珠到上清液。珠子也需要如步骤18所示平衡。在Eppendorf管旋转器上在4℃下孵育珠子3小时。
  20. 通过离心沉淀小珠,并取60ul上清液用于流通样品
  21. 用1ml提取缓冲液(含0.15%NP40)洗涤珠子3次。对于每个洗涤步骤,通过在4℃下以12,000xg离心1分钟来沉淀珠粒,弃去上清液并加入1ml提取缓冲液。
  22. 加入100μl稀释的3×FLAG肽(用1×蛋白酶抑制剂将含有1mM EDTA的TBS中的3×FLAG储备液稀释1/10)3×FLAG储备液在TBS中为2.5mg/ml,可在-20℃下储存多年)珠子并在冷室中在Eppendorf管旋转器上孵育1小时。
  23. 通过离心沉淀珠粒。将上清液转移到新管中。加入35μl4x SDS加载缓冲液到上清液。该级分是含有纯化的免疫沉淀蛋白的洗脱样品。加入60μl1XSDS加载缓冲液到含有珠子的管中。珠子样品可能含有不能从珠子洗脱的残留蛋白质
  24. 向输入和流通样品中加入适当体积的4x SDS上样缓冲液。 将所有样品在95-100℃煮沸5-10分钟。 并使用Western blot分析样品

代表数据

在图1中示出了代表性的实验结果。该图根据在参考文献中列出的Xu等人(2014)修改。


图1.bHLH84-HA与SNC1-FLAG共免疫沉淀。在Co-IP实验中使用两个样品,包括具有bHLH84和SNC1-FLAG的共表达的样品和仅表达bHLH84的样品 用作阴性对照。

笔记

  1. 在免疫沉淀过程中总是推荐阴性对照样品。对于单个蛋白质的免疫沉淀,建议表达空载体作为阴性对照。对于两种或更多种蛋白质的共免疫沉淀,建议表达没有诱饵蛋白的空载体以及含有猎物蛋白的相关载体。基于实验设计,可以相应地包括更多的阴性对照。
  2. 只要蛋白质可以被下拉,免疫沉淀实验是高度可重现的,虽然IP效率可能在不同的试验中略有不同。

食谱

  1. 感应介质
    注意:您还需要在以下媒体中添加适当的抗生素。
    试剂
    库存集中
    灭菌
    体积/100 ml
    最终集中
    K 2 HPO 4
    10.5 g/L
    用水和高压釜将所有混合物混合
    96.85 ml

    KH 2 PO 4
    4.5 g/L

    (NH 4) 2 4
    1.0 g/L

    柠檬酸钠:2H 2 O·dm / 0.5 g/L

    甘油
    0.5%

    葡萄糖
    20%
    过滤
    1 ml
    0.2%
    MgSO 4 4 / 1 M
    过滤
    100微升
    1 mM
    Acetosyringone
    100 mM
    过滤
    50微升
    50微升
    MES(pH 5.6)
    0.5 M
    过滤
    2 ml
    10 mM

  2. 浸润溶液
    试剂
    库存集中
    灭菌
    每20 ml的金额
    最终集中
    MS粉末

    NA
    0.088克
    4.4 mg/L
    MES
    0.5 M
    过滤
    0.4 ml
    10 mM
    Acetosyringone
    100 mM
    过滤
    30微升
    150μM/

  3. 提取缓冲区
    10%甘油 25mM Tris-HCl(PH7.5)
    1mM EDTA
    150mM NaCl
  4. TBS
    10mM Tris-HCl
    150mM NaCl(PH7.4)
  5. 4x SDS
    200mM Tris-HCl(PH 6.8)
    8%SDS
    40%甘油 0.04%溴酚蓝
    400 mM DTT

致谢

我们感谢自然科学和工程研究委员会(NSERC)(加拿大)发现补助计划的财政支持。在N中的瞬时蛋白表达。本尼曼人改编自Van den Ackerveken等人(1996)。 IP协议修改自Moffett等人(2002)。

参考文献

  1. Moffett,P.,Farnham,G.,Peart,J.and Baulcombe,D.C。(2002)。 植物NBS-LRR蛋白结构域在疾病抗性相关细胞死亡中的相互作用。 21(17):4511-4519。
  2. Van den Ackerveken,G.,Marois,E.and Bonas,U.(1996)。 在宿主植物细胞内发生细菌无毒性蛋白AvrBs3的识别。单元 87(7):1307-1316。
  3. Xu,F.,Kapos,P.,Cheng,Y.T.,Li,M.,Zhang,Y.and Li,X.(2014)。 NLR相关转录因子bHLH84及其旁系同源基因在植物免疫中的功能冗余。 > PLoS Pathog 10(8):e1004312。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xu, F., Copeland, C. and Li, X. (2015). Protein Immunoprecipitation Using Nicotiana benthamiana Transient Expression System. Bio-protocol 5(13): e1520. DOI: 10.21769/BioProtoc.1520.
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