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Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
可视免疫沉淀(VIP)实验:一种简单通用的蛋白质 - 蛋白质相互作用可视化检测方法   

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参见作者原研究论文

本实验方案简略版
Journal of Cell Science
Jun 2015

Abstract

The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can be visualized by directly observing the beads bearing immunoprecipitates under a fluorescence microscope. This assay can examine a large number of protein combinations at one time, without requiring time-consuming procedures, including SDS-PAGE and immunoblotting. Furthermore, the VIP assay can examine complicated one-to-many and many-to-many protein interactions. Another important point of the VIP assay is the use of nanobodies for immunoprecipitation. A Nanobody is a single-domain antibody derived from Camelidae (camels and relatives). Because of its small size, high-affinity, high-specificity, and stability, anti-GFP Nanobody expressed in E. coli can be purified on a large scale, and used virtually inexhaustibly for immunoprecipitation experiments. Here we describe protocols for preparation of GST-tagged anti-GFP Nanobody and the VIP assay.

Keywords: Visible immunoprecipitation (VIP) (可视免疫沉淀(VIP)), Protein-protein interaction (蛋白质-蛋白质相互作用), Immunoprecipitation (免疫沉淀), Fluorescent protein (荧光蛋白), Nanobody (纳米抗体)

Background

Almost all proteins in cells function by interacting with other proteins. Revealing the protein-protein interaction network is the key to understand the functions of the proteins. Various methods such as yeast two-hybrid system, GST pull-down, and co-immunoprecipitation have been developed to analyze protein-protein interactions. Recently, we have developed a new method for protein-protein interaction analysis called visible immunoprecipitation (VIP) assay (Katoh et al., 2015). The most important advantage of VIP assay is that it is handy and convenient. This assay can examine a large number of protein combinations in a short time, without requiring time-consuming procedures, including SDS-PAGE and immunoblotting. Furthermore, the VIP assay can determine interactions between more than two proteins at a time. This powerful tool can be used to reveal the intricate architectures of multi-protein complexes, which cannot be determined by conventional protein-protein interaction assays. By taking advantage of the VIP assay, we have elucidated architectures of multi-subunit complexes, the BBSome (composed of 8 subunits) (Katoh et al., 2015), IFT-B (16 subunits) (Katoh et al., 2016), and IFT-A (6 subunits) (Hirano et al., 2017), all of which are involved in protein trafficking within the cilia.

Materials and Reagents

  1. 10 ml test tubes (ASIA KIZAI, catalog numbers: 2221C010B-10 )
  2. 50 ml tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  3. 15 ml tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  4. 6-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  5. 1.5 ml tubes (BIO-BIK, catalog number: RC-0150 )
  6. 0.2 ml 8-Tube Strips (Greiner Bio One International, catalog numbers: 673210 and 373270 )
  7. 96-well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 167008 )
  8. 0.22 µm PVDF filter (Millex-GV 0.22 µm PVDF 33 mm Gamma Sterilized) (Merck, catalog number: SLGV033RS )
  9. pGEX6P1-GFP-Nanobody (Addgene, catalog number: 61838 )
  10. Escherichia coli BL21-CodonPlus(DE3)-RIPL strain (Agilent Technologies, catalog number: 230280 )
  11. HEK293T cells (e.g., ATCC, catalog number: CRL-3216 )
  12. Fluorescent protein expression vectors (e.g., mEGFP-C1/N1 [Addgene, catalog numbers: 54759 and 54767 ], pmCherry-C1/N1 [Takara Bio, Clontech, catalog numbers: 632524 and 632523 ], or pTagRFP-C/N [Evrogen, catalog numbers: FP141 and FP142 ])
  13. Luria-Bertani (LB) medium (NACALAI TESQUE, catalog number: 20068-75 )
  14. LB agar plates (NACALAI TESQUE, catalog number: 20069-65 )
  15. Ampicillin (NACALAI TESQUE, catalog number: 02739-32 )
  16. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (NACALAI TESQUE, catalog number: 19742-94 )
  17. Polyethylene Glycol Mono-p-isooctylphenyl Ether (Triton X-100) (NACALAI TESQUE, catalog number: 12967-45 )
  18. Glutathione-Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  19. Phosphate buffered saline without Ca2+ and Mg2+ [PBS(-)] (NACALAI TESQUE, catalog number: 1148215 )
  20. CBB Stain One Super (NACALAI TESQUE, catalog number: 11642-31 )
  21. Dulbecco’s modified Eagle medium (DMEM), high glucose (NACALAI TESQUE, catalog number: 08458-16 )
  22. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  23. Opti-MEM (Thermo Fisher Scientific, catalog number: 31985070 )
  24. Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14112-52 )
  25. Polyethyleneimine (PEI) Max (Mw 40,000) (Polysciences, catalog number: 24765-2 )
  26. HEPES (NACALAI TESQUE, catalog number: 17546-05 )
  27. Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31320-05 )
  28. Glycerol (NACALAI TESQUE, catalog number: 17018-25 )
  29. Protease Inhibitor Cocktail for general use (100x) (NACALAI TESQUE, catalog number: 04080-11 )
    Note: This protease inhibitor cocktail contains 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, E-64, leupeptin hemisulfate monohydrate, and disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA).
  30. Bovine serum albumin (BSA) (NACALAI TESQUE, catalog number: 08587-84 )
  31. Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts (100x) (NACALAI TESQUE, catalog number: 25955-11 )
    Note: This protease inhibitor cocktail contains 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, E-64,leupeptin hemisulfate monohydrate, bestatin, and pepstatin A.
  32. Binding buffer (see Recipes)
  33. Washing buffer (see Recipes)
  34. 2 mg/ml PEI Max reagent (see Recipes)
  35. HEPES, NaCl, Triton X-100, and glycerol (HNTG) buffer (see Recipes)

Equipment

  1. Multichannel pipette (20-200 µl) (Greiner Bio One International, catalog number: 89008200 )
  2. Aspirator
  3. Large centrifuge (Hitachi Koki, model: CR22G )
  4. Cooling centrifuge that can be set at 4 °C (TOMY SEIKO, model: EX-136 )
  5. Desktop centrifuge (Eppendorf, model: 5415 R )
  6. Centrifuge for PCR tube (BM Equipment, model: ForceMini )
  7. Shaking incubator (TAITEC, model: BR-23FP )
  8. Rotator (TAITEC, model: RT-50 )
  9. Sonicator (Misonix, model: S-4000 )
  10. Fluorescence microscope (Keyence, model: BZ-8000 )
  11. Objective lens (Nikon, PlanApo 20x NA0.75 WD1.00)
  12. 500 ml flask (AGC Techno Glass, catalog number: 4551FK500R )
  13. Spectrophotometer (GE healthcare, model: UltrospecTM 2100 pro )
  14. Cuvette (GE Healthcare, catalog number: 80-2076-38 )

Procedure

  1. Preparation of GST-tagged anti-GFP Nanobody
    Here we introduce the method for preparing GST-tagged anti-GFP Nanobody. Under our conditions, we usually purify approximately 1 mg of the protein from 200 ml of E. coli culture.
    1. Pick up a single colony of E. coli carrying pGEX6P1-GFP-Nanobody, and inoculate a starter culture of 3 ml of LB medium plus 50 µg/ml ampicillin in a test tube with the colony, and incubate it overnight at 37 °C with vigorous shaking at 150-200 rpm.
    2. Inoculate a flask containing 200 ml of LB medium plus 50 µg/ml ampicillin with 2 ml of the overnight culture, and incubate it at 37 °C with shaking at 130 rpm until the culture reaches OD600 = 0.5. (It takes 2-4 h.)
    3. To induce expression of GST-tagged anti-GFP Nanobody, add 200 µl of 100 mM IPTG (to a final concentration 0.1 mM) to the flask, and incubate the flask for 4 h at 30 °C with shaking at 130 rpm.
    4. Transfer the culture to an appropriate centrifuge tube, and harvest the cells by centrifugation at 5,550 x g (e.g., 6,000 rpm in a Himac CR22G centrifuge) for 15 min at 4 °C.
    5. Pour away the supernatant and suspend the cells in 10 ml of binding buffer (see Recipes) by vortexing.
    6. After transferring the cell suspension to a 50 ml tube, disrupt the cells by sonication. Sonication is performed on ice for 5 rounds (15 sec ON and 15 sec OFF) at 30% amplitude using Astrason S4000.
    7. Add 0.5 ml of 20% Triton X-100 diluted in H2O to the tube to a final concentration of 1%, and incubate it for 20 min at 4 °C (or on ice) to help cell lysis.
    8. Centrifuge the tube at 20,700 x g (e.g., 12,000 rpm in a Himac CR22G centrifuge) for 20 min at 4 °C, and transfer the supernatant to a 15 ml tube.
      Note: We usually divide the supernatant into 2 x 5 ml. Hereafter we use 2 tubes.
    9. Add 2.5 ml (bed volume) of glutathione-Sepharose 4B beads equilibrated in PBS(-) to 5 ml of the supernatant, and allow binding of the Nanobody to the beads by rotating the tube overnight at 4 °C (12-24 h).
    10. Centrifuge the tube at 780 x g (e.g., 2,000 rpm in an EX-136 centrifuge) for 2 min at 4 °C, and remove the supernatant by aspiration.
    11. Add 10 ml of ice-cold washing buffer (see Recipes) to the precipitate.
    12. Repeat Steps A10 and A11 eight times.
    13. Keep the beads as a 1:1 slurry in the washing buffer (namely, 2.5 ml of the beads and 2.5 ml of the washing buffer). The beads can be stored for several months at 4 °C.
      Note: We usually divide the beads slurry into 1 ml (bed volume 500 µl) aliquots.
    14. Check the quality and estimate the quantity of purified GST-tagged anti-GFP Nanobody by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. Under our preparation conditions, approximately 1 µg/5 µl (bed volume of beads) of the Nanobody protein is purified. A typical result is shown in Figure 1.


      Figure 1. A typical result of purified GST-tagged anti-GFP Nanobody. Purified GST-tagged anti-GFP Nanobody and BSA were subjected to SDS-PAGE followed by CBB staining. By comparing the band intensity of the purified protein with that of BSA, the concentration of purified GST-tagged anti-GFP Nanobody was estimated to be approximately 1 μg/5 μl (bed vol.).

  2. VIP assay
    Here we introduce how to perform the VIP assay (Figure 2 and Video 1).


    Figure 2. Overview of VIP assay. Expression vectors for GFP-fusion and RFP-fusion proteins are co-transfected into HEK293T cells. After 24 h, expression of fluorescent proteins is confirmed by fluorescence microscopy. Next, lysates of the transfected cells are prepared, and processed for immunoprecipitation using GST-tagged anti-GFP Nanobody beads. Finally, the beads bearing immunoprecipitates are observed under a fluorescence microscope. When two proteins interact with each other, both GFP and RFP signals are detectable on the beads. On the other hand, when the two proteins do not interact, only GFP signals are observed.

    Video 1. Demonstration of VIP assay. The video shows how to perform the VIP assay.

    1. Seed HEK293T cells (approximately 8 x 105 cells) into a 6-well plate and incubate them for 24 h in 2 ml of DMEM high glucose supplemented with 5% FBS.
    2. Co-transfect the cells with expression vectors for GFP-fusion and RFP-fusion proteins using a PEI Max reagent (see Recipes).
      1. Mix 250 µl of Opti-MEM with 4 µg of DNA in a 1.5 ml tube (e.g., 2 μg of a GFP expression vector and 2 μg of an RFP expression vector).
      2. Mix 250 µl of Opti-MEM with 10 µl of 2 mg/ml PEI Max reagent in another 1.5 ml tube, and incubate for 5 min at room temperature.
      3. Mix the DNA solution with the PEI Max solution by vortexing, and incubate it for 20 min at room temperature.
      4. Add 500 µl of the DNA/PEI mixture to each well of the 6-well plate.
    3. After 24 h, check the expression of GFP-fusion and RFP-fusion proteins by observing the cells under a fluorescence microscope.
    4. Remove completely the medium from the plate by aspiration, add 250 µl of ice-cold HNTG buffer (see Recipes) to each well, and incubate the plate for 5-10 min on ice to detach adherent cells.
    5. Suspend the cells by pipetting, transfer the cell suspension to a 1.5 ml tube, and incubate the tube for 15 min on ice to lyse the cells.
    6. Centrifuge the tube at 16,100 x g (e.g., 13,200 rpm in a 5415 R centrifuge) at 4 °C for 15 min.
    7. Transfer 200 µl of the supernatant to 0.2 ml 8-Tube Strips containing GST-tagged anti-GFP Nanobody prebound to glutathione-Sepharose beads (approximately 5 µl (bed volume) of the beads), and incubate the 8-Tube Strips for 1 h at 4 °C with constant rotation using a tube rotator.
    8. Centrifuge the 8-Tube Strips for 30 sec, and remove the supernatant by aspiration.
    9. Add 180 µl of ice-cold HNTG buffer, and mix the content by tapping the 8-Tube Strips.
    10. Repeat Steps B8 and B9 three times.
    11. Finally, suspend the beads in 180 µl of ice-cold PBS(-), and transfer the beads suspension to a 96-well plate.
    12. Observe green and red fluorescence on the beads under a fluorescence microscope, and acquire images. To compare the fluorescence intensity, images are acquired under fixed conditions (exposure time and ISO sensitivity of the camera).
    13. Collect the beads if immunoblotting is required.

Data analysis

  1. Examples of VIP assay
    The intraflagellar transport (IFT)-B complex, which is responsible for ciliary protein trafficking is a huge protein complex consisting of 16 subunits. We have delineated the architecture of the IFT-B complex by determining not only binary interactions but also one-to-many and many-to-many protein interactions of IFT-B subunits by the VIP assay (Katoh et al., 2016). Here we show examples of interaction analyses of some IFT-B subunits.
    1. Experimental example 1: binary protein interaction
      The VIP assay was performed on GFP-tagged and mCherry(mChe)-tagged IFT74 and IFT81 (Figure 3A). The red signals in the fourth and fifth panels from the left indicate that IFT74 and IFT81 interact with each other.
    2. Experimental example 2: one-to-two protein interaction
      The VIP assay was performed on GFP-tagged IFT22 and mChe-tagged IFT74, IFT81, or IFT74 + IFT81 (Figure 3B). GFP-IFT22 did not interact with either mChe-IFT74 or mChe-IFT81 alone. However, GFP-IFT22 interacted with mChe-tagged IFT74 + IFT81 heterodimer (rightmost panel in Figure 3B). To confirm that both IFT74 and IFT81 are bound to IFT22, immunoblotting was performed after the VIP assay (Figure 3C). The results of the VIP assay and immunoblotting show that IFT22 interact with only the IFT74-IFT81 heterodimer (Figure 3D).
      As described above, the VIP assay is a versatile and flexible method that can comprehensively examine binary protein interactions and examine more complicated one-to-many and many-to-many protein interactions.
      Finally, we describe notes about the VIP assay. In this method, it is very important to use appropriate positive and negative controls. As a positive control, proteins already known to interact should be used. As a negative control, only fluorescent proteins are used. Using appropriate experimental controls facilitates determining whether protein-protein interactions are positive or negative.


      Figure 3. Interactions among IFT subunits revealed by VIP assay. A. Binary protein interaction. The VIP assay was performed on GFP- or mChe-tagged IFT74 and IFT81. B. One-to-two protein interaction. The VIP assay was performed on GFP-tagged IFT22 and mChe-tagged IFT74, IFT81, or IFT74 + IFT81. C. Immunoblotting was performed after the VIP assay shown in B. D. The interaction mode of IFT22, IFT74, and IFT88. (From Katoh and Nakayama, 2017; adapted with permission from YODOSHA Co., Ltd.)

Recipes

  1. Binding buffer
    PBS(-)
    5 mM DTT
    Protease Inhibitor Cocktail for general use (0.5 mM AEBSF, 0.15 µM aprotinin, 1 µM E-64, 1 µM leupeptin hemisulfate monohydrate, and 0.5 mM EDTA)
  2. Washing buffer
    PBS(-)
    5 mM DTT
    0.1% Triton X-100
  3. 2 mg/ml PEI Max reagent
    20 mg of PEI Max is dissolved in 10 ml of Milli-Q grade water sterilized by filtration
    The solution is divided into 1 ml aliquots, and can be stored at 4 °C for several months
    For more long-term, it can be stored at -20 °C
  4. HEPES, NaCl, Triton X-100, and glycerol (HNTG) buffer
    Note: This is an example of lysis buffer. Other lysis buffers can also be used for VIP assay.
    20 mM HEPES (pH7.4)
    150 mM NaCl
    0.1% (w/v) Triton X-100
    10% (w/v) glycerol
    Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts (1 mM AEBSF, 0.8 µM aprotinin, 15 µM E-64, 20 µM leupeptin hemisulfate monohydrate, 50 µM bestatin, and 10 µM pepstatin A)

Acknowledgments

This protocol was adapted from and used in Katoh et al. (2015), Katoh et al. (2016), and Katoh and Nakayama (2017). This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas ‘Cilia and Centrosome’ from the Ministry of Education, Culture, Sports, Science and Technology, Japan (grant number 15H01211 to Ka.N.); grants from the Japan Society for the Promotion of Science (grant numbers 15H04370 to Ka.N., and 15K07929 to Y.K.); and from the Takeda Science Foundation and the Uehara Memorial Foundation to Y.K. The authors declare no conflicts of interest.

References

  1. Hirano, T., Katoh, Y. and Nakayama, K. (2017). Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein-coupled receptors. Mol Biol Cell 28(3): 429-439.
  2. Katoh, Y. and Nakayama K. (2017). Visible immunoprecipitation assay: a convenient and versatile method for studying protein‒protein interactions using fluorescent fusion proteins. Experimental Medicine 35: 85-95 (written in Japanese).
  3. Katoh, Y., Nozaki, S., Hartanto, D., Miyano, R. and Nakayama, K. (2015). Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J Cell Sci 128(12): 2351-2362.
  4. Katoh, Y., Terada, M., Nishijima, Y., Takei, R., Nozaki, S., Hamada, H. and Nakayama, K. (2016). Overall architecture of the Intraflagellar Transport (IFT)-B complex containing Cluap1/IFT38 as an essential component of the IFT-B peripheral subcomplex. J Biol Chem 291: 10962-10975.

简介

可见的免疫沉淀(VIP)测定是常规免疫共沉淀的方便的替代方法(Katoh等人,2015)。通过处理来自共表达GFP融合蛋白和RFP融合蛋白的细胞的裂解物以用GST标记的抗GFP纳米抗体和谷胱甘肽琼脂糖珠粒进行免疫沉淀,可以通过在荧光显微镜下直接观察带有免疫沉淀物的珠来显现蛋白质 - 蛋白质相互作用。该检测方法可以一次检测大量的蛋白质组合,无需耗时的操作,包括SDS-PAGE和免疫印迹。此外,VIP测定可以检查复杂的一对多和多对多的蛋白质相互作用。 VIP测定的另一个重要的点是使用纳米抗体进行免疫沉淀。纳米抗体是来自骆驼科(骆驼和亲戚)的单域抗体。由于其体积小,高亲和力,高特异性和稳定性,因此在E中表达的抗GFP纳米抗体。大肠杆菌可以大规模纯化,并且实际上用于免疫沉淀实验。在这里,我们描述了制备GST标记的抗GFP纳米抗体和VIP测定的方案。


【背景】细胞中的几乎所有蛋白质都通过与其他蛋白质相互作用起作用。揭示蛋白质 - 蛋白质相互作用网络是了解蛋白质功能的关键。已经开发了多种方法,例如酵母双杂交系统,GST pull-down和免疫共沉淀来分析蛋白质 - 蛋白质相互作用。最近,我们开发了一种称为可见免疫沉淀(VIP)测定的蛋白质 - 蛋白质相互作用分析的新方法(Katoh等人,2015)。 VIP分析最重要的优点是方便,方便。该测定可以在短时间内检查大量的蛋白质组合,而不需要耗费时间的程序,包括SDS-PAGE和免疫印迹。此外,VIP测定可以一次确定两种以上蛋白质之间的相互作用。这个强大的工具可以用来揭示多蛋白复合物的复杂结构,这是传统的蛋白质 - 蛋白质相互作用测定所不能确定的。通过利用VIP测定,我们阐明了多亚基复合物的结构,BBSome(由8个亚基组成)(Katoh等人,2015),IFT-B(16亚基)( Katoh等人,2016)和IFT-A(6个亚基)(Hirano等人,2017),所有这些都涉及纤毛内的蛋白质运输。

关键字:可视免疫沉淀(VIP), 蛋白质-蛋白质相互作用, 免疫沉淀, 荧光蛋白, 纳米抗体

材料和试剂

  1. 10毫升试管(ASIA KIZAI,产品目录号:2221C010B-10)
  2. 50ml管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339652)
  3. 15ml试管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339650)。
  4. 6孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
  5. 1.5毫升管(BIO-BIK,目录号:RC-0150)

  6. 0.2毫升8管带(Greiner Bio One International,目录号:673210和373270)
  7. 96孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:167008)
  8. 0.22μmPVDF过滤器(Millex-GV0.22μmPVDF 33mm Gamma灭菌)(Merck,目录号:SLGV033RS)
  9. pGEX6P1-GFP-纳米抗体(Addgene,目录号:61838)
  10. 大肠杆菌BL21-CodonPlus(DE3)-RIPL菌株(Agilent Technologies,目录号:230280)
  11. HEK293T细胞(例如,ATCC,目录号:CRL-3216)
  12. 荧光蛋白表达载体(例如mEGFP-C1 / N1 [Addgene,目录号:54759和54767],pmCherry-C1 / N1 [Takara Bio,Clontech,目录号:632524和632523]或pTagRFP-C / N [Evrogen,目录号:FP141和FP142])
  13. Luria-Bertani(LB)培养基(NACALAI TESQUE,目录号:20068-75)
  14. LB琼脂平板(NACALAI TESQUE,目录号:20069-65)
  15. 氨苄青霉素(NACALAI TESQUE,目录号:02739-32)
  16. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(NACALAI TESQUE,目录号:19742-94)
  17. 聚乙二醇单辛基苯基醚(Triton X-100)(NACALAI TESQUE,目录号:12967-45)
  18. 谷胱甘肽-Sepharose 4B(GE Healthcare,目录号:17075601)
  19. 不含Ca 2+和Mg 2+的磷酸盐缓冲液[PBS( - )](NACALAI TESQUE,目录号:1148215)
  20. CBB Stain One Super(NACALAI TESQUE,目录号:11642-31)
  21. 达尔伯克改良伊格尔培养基(DMEM),高葡萄糖(NACALAI TESQUE,目录号:08458-16)
  22. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  23. Opti-MEM(Thermo Fisher Scientific,目录号:31985070)
  24. 二硫苏糖醇(DTT)(NACALAI TESQUE,目录号:14112-52)
  25. 聚乙烯亚胺(PEI)Max(Mw40,000)(Polysciences,目录号:24765-2)
  26. HEPES(NACALAI TESQUE,目录号:17546-05)
  27. 氯化钠(NaCl)(NACALAI TESQUE,目录号:31320-05)
  28. 甘油(NACALAI TESQUE,目录号:17018-25)
  29. 通用的蛋白酶抑制剂鸡尾酒(100x)(NACALAI TESQUE,目录号:04080-11)
    注意:这种蛋白酶抑制剂混合物含有4-(2-氨基乙基)苯磺酰氟盐酸盐(AEBSF),抑肽酶,E-64,亮氨酸素硫酸盐一水合物和二氢乙二胺四乙酸二钠二水合物(EDTA)。 >
  30. 牛血清白蛋白(BSA)(NACALAI TESQUE,目录号:08587-84)
  31. 用于哺乳动物细胞和组织提取物的蛋白酶抑制剂混合物(100x)(NACALAI TESQUE,目录号:25955-11)
    注意:该蛋白酶抑制剂混合物含有4-(2-氨基乙基)苯磺酰氟盐酸盐(AEBSF),抑肽酶,E-64,亮氨酸素硫酸盐半水合物,贝他汀和胃蛋白酶抑制剂A.
  32. 绑定缓冲区(请参阅食谱)
  33. 洗涤缓冲液(见食谱)
  34. 2毫克/毫升PEI最大试剂(见食谱)
  35. HEPES,NaCl,Triton X-100和甘油(HNTG)缓冲液(见食谱)

设备

  1. 多道移液器(20-200μl)(Greiner Bio One International,目录号:89008200)
  2. 吸气器
  3. 大型离心机(日立机械,型号:CR22G)
  4. 冷却离心机可以设置在4°C(TOMY SEIKO,型号:EX-136)
  5. 台式离心机(Eppendorf,型号:5415 R)
  6. 用于PCR管的离心机(BM Equipment,型号:ForceMini)
  7. 摇摇培养箱(TAITEC,型号:BR-23FP)
  8. 旋转器(TAITEC,型号:RT-50)
  9. 超声波仪(Misonix,型号:S-4000)
  10. 荧光显微镜(Keyence,型号:BZ-8000)
  11. 物镜(尼康,PlanApo 20x NA0.75 WD1.00)
  12. 500ml烧瓶(AGC Techno Glass,目录号:4551FK500R)
  13. 分光光度计(GE healthcare,型号:Ultrospec TM 2100 pro)
  14. 比色杯(GE Healthcare,目录号:80-2076-38)

程序

  1. GST-标记的抗GFP纳米抗体的制备
    这里我们介绍制备GST标记的抗GFP纳米抗体的方法。在我们的条件下,我们通常从200ml的E纯化约1mg的蛋白质。大肠杆菌培养。
    1. 拿起一个E的单个殖民地。携带pGEX6P1-GFP-纳米抗体的大肠杆菌接种3ml含有50μg/ ml氨苄青霉素的LB培养基的发酵剂培养物,在菌落的试管中接种,在37℃下剧烈振荡培养过夜-200转。
    2. 用2ml过夜培养物接种含有200ml LB培养基加50μg/ ml氨苄青霉素的烧瓶,并在37℃以130rpm振荡孵育,直到培养物达到OD 600 = 0.5 。 (需要2-4小时)
    3. 为了诱导GST-标记的抗GFP纳米抗体的表达,将200μl的100mM IPTG(终浓度0.1mM)加入到烧瓶中,并且将烧瓶在30℃下以130rpm振荡孵育4小时。 />
    4. 将培养物转移至合适的离心管中,并通过在4℃下以5,550xg(例如6,000rpm,在Himac CR22G离心机中)离心15分钟来收获细胞15分钟。
    5. 倒掉上清液,通过涡旋将细胞悬浮在10ml结合缓冲液(参见食谱)中。
    6. 将细胞悬浮液转移到50ml管后,通过超声破碎细胞。超声在冰上进行5轮(15秒ON和15秒OFF),使用Astrason S4000,幅度为30%。
    7. 加入0.5ml在H 2 O中稀释的20%Triton X-100至终浓度为1%,并在4℃(或在冰上)孵育20分钟以帮助细胞裂解。
    8. 在4℃下,将管子在20,700×g(例如,12,000rpm,在Himac CR22G离心机中)离心20分钟,并将上清液转移到15ml管中。
      注意:我们通常将上清液分成2 x 5 ml。此后,我们使用2管。
    9. 加入2.5ml(床体积)在PBS( - )中平衡的谷胱甘肽 - 琼脂糖4B珠粒至5ml上清液中,并通过在4℃(12-24小时)下旋转管子使纳米抗体与珠子结合, 。
    10. 在4℃下以780×g(例如2000rpm,在EX-136离心机中)离心试管2分钟,并通过抽吸去除上清液。 >

    11. 加入10毫升冰冷清洗缓冲液(见食谱)
    12. 重复步骤A10和A11八次。
    13. 将珠子保持在洗涤缓冲液中(即,2.5ml珠子和2.5ml洗涤缓冲液)为1:1浆液。珠子可以在4°C下储存数月。
      注意:我们通常将珠浆分成1毫升(床体积500微升)等份。
    14. 通过SDS-PAGE和考马斯亮蓝(CBB)染色检查纯化的GST-标记的抗GFP纳米抗体的质量和数量。在我们的制备条件下,纯化纳米抗体蛋白质的约1μg/5μl(床体积的珠)。一个典型的结果如图1所示。


      图1.纯化的GST-标记的抗GFP纳米抗体的典型结果。纯化的GST标记的抗GFP纳米抗体和BSA进行SDS-PAGE,然后进行CBB染色。通过比较纯化的蛋白质与BSA的条带强度,纯化的GST标记的抗GFP纳米抗体的浓度估计为约1μg/5μl(床体积)。

  2. VIP分析
    这里我们介绍如何进行VIP测定(图2和视频1)。


    图2. VIP测定概述。将GFP融合蛋白和RFP融合蛋白的表达载体共转染到HEK293T细胞中。 24小时后,通过荧光显微镜确认荧光蛋白的表达。接下来,制备转染的细胞的裂解物,并使用GST-标记的抗GFP纳米抗体珠粒进行处理以用于免疫沉淀。最后,在荧光显微镜下观察带有免疫沉淀物的珠子。当两种蛋白质相互作用时,GFP和RFP信号都可以在珠上检测到。另一方面,当两种蛋白质不相互作用时,只能观察到GFP信号。

    视频1

    1. 将HEK293T细胞(大约8×10 5个细胞)种入6孔板,并在2ml补充有5%FBS的DMEM高葡萄糖中孵育24小时。
    2. 使用PEI Max试剂与GFP融合蛋白和RFP融合蛋白的表达载体共转染细胞(见食谱)。
      1. 在1.5ml试管(例如,2μgGFP表达载体和2μgRFP表达载体)中混合250μlOpti-MEM与4μgDNA。
      2. 在另一个1.5 ml试管中混合250μlOpti-MEM和10μl2 mg / ml PEI Max试剂,室温孵育5分钟。
      3. 将DNA溶液与PEI Max溶液混合,涡旋混匀,在室温下孵育20分钟。

      4. 加入500μl的DNA / PEI混合物到6孔板的每个孔中
    3. 24小时后,在荧光显微镜下观察细胞,检查GFP融合蛋白和RFP融合蛋白的表达。
    4. 通过抽吸将培养基从培养板中完全除去,向每个孔中加入250μl冰冷的HNTG缓冲液(参见食谱),将培养板在冰上孵育5-10分钟以分离贴壁细胞。
    5. 通过移液悬浮细胞,将细胞悬液转移至1.5ml管中,并将管在冰上孵育15分钟以裂解细胞。
    6. 在4℃,以16,100×g(例如,在1350rpm,在5415R离心机中)离心试管15分钟。
    7. 将200μl上清液转移到0.2ml含有GST-标记的抗GFP纳米抗体的预先结合谷胱甘肽 - 琼脂糖珠(约5μl(床体积)的珠)的8-管带上,并将8管带孵育1小时在4°C的恒定旋转使用管旋转器。
    8. 离心8管带30秒,并吸取上清液。
    9. 加入180微升冰冷的HNTG缓冲液,并通过点击8管带混合内容。

    10. 重复步骤B8和B9三次
    11. 最后,将珠子悬浮在180μl冰冷的PBS( - )中,并将珠子悬浮液转移到96孔板上。
    12. 在荧光显微镜下观察珠子上的绿色和红色荧光,并获取图像。为了比较荧光强度,在固定条件下(相机的曝光时间和ISO感光度)获取图像。
    13. 收集珠子,如果需要免疫印迹。

数据分析

  1. VIP分析的例子
    负责睫状蛋白运输的眶内运输(IFT)-B复合物是由16个亚基组成的巨大蛋白质复合物。我们通过VIP测定不仅确定二元相互作用而且确定IFT-B亚基的一对多和多对多蛋白质相互作用(Katoh等, / em>,2016)。在这里,我们展示了一些IFT-B亚基的相互作用分析的例子。
    1. 实验例1:二元蛋白质相互作用
      对GFP标记的和mCherry(mChe)标记的IFT74和IFT81(图3A)进行VIP测定。在左边的第四和第五个面板中的红色信号表明IFT74和IFT81相互作用。
    2. 实验例2:一对二的蛋白质相互作用
      VIP测定在GFP标记的IFT22和mChe标记的IFT74,IFT81或IFT74 + IFT81(图3B)上进行。 GFP-IFT22不与单独的mChe-IFT74或mChe-IFT81相互作用。然而,GFP-IFT22与mChe标记的IFT74 + IFT81异二聚体相互作用(图3B中最右侧的面板)。为了证实IFT74和IFT81都与IFT22结合,在VIP测定后进行免疫印迹(图3C)。 VIP测定和免疫印迹的结果显示IFT22仅与IFT74-IFT81异二聚体相互作用(图3D)。
      如上所述,VIP测定法是一种灵活多变的方法,可以全面检查二元蛋白质相互作用并检查更复杂的一对多和多对多蛋白质相互作用。
      最后,我们描述关于VIP测定的笔记。在这种方法中,使用适当的正面和负面控制是非常重要的。作为阳性对照,应该使用已知相互作用的蛋白质。作为阴性对照,仅使用荧光蛋白。使用适当的实验对照便于确定蛋白质 - 蛋白质相互作用是正面的还是负面的。


      图3. VIP测定揭示的IFT亚基之间的相互作用。 :一种。二元蛋白质相互作用。 VIP测定在GFP-或mChe-标记的IFT74和IFT81上进行。 B.一对二的蛋白质相互作用。对GFP标记的IFT22和mChe标记的IFT74,IFT81或IFT74 + IFT81进行VIP测定。 C.在B中显示的VIP测定后进行免疫印迹。D.IFT22,IFT74和IFT88的相互作用模式。 (来自加藤和中山,2017;经YODOSHA Co.,Ltd.许可)

食谱

  1. 绑定缓冲区
    PBS( - )
    5 mM DTT
    通用的蛋白酶抑制剂混合物(0.5mM AEBSF,0.15μM抑肽酶,1μME-64,1μM亮抑酶肽半硫酸盐一水合物和0.5mM EDTA)。
  2. 清洗缓冲液
    PBS( - )
    5 mM DTT
    0.1%Triton X-100
  3. 2毫克/毫升PEI Max试剂
    将20mg PEI Max溶于10ml经过滤灭菌的Milli-Q级水中 该溶液分成1毫升等分试样,可以在4°C储存几个月
    对于更长期的,它可以被存放在-20°C
  4. HEPES,NaCl,Triton X-100和甘油(HNTG)缓冲液 注意:这是一个裂解缓冲区的例子。其他裂解缓冲液也可以用于VIP分析。
    20毫米HEPES(pH7.4)
    150 mM NaCl
    0.1%(w / v)Triton X-100
    10%(w / v)甘油
    与哺乳动物细胞和组织提取物一起使用的蛋白酶抑制剂混合物(1mM AEBSF,0.8μM抑肽酶,15μME-64,20μM亮肽素半硫酸盐一水合物,50μM苯丁抑菌素和10μM胃酶抑素A)

致谢

该协议改编自加藤等人(2015年),加藤等人并在其中使用。 (2016),Katoh和Nakayama(2017)。这项工作得到了日本文部科学省创新领域纤毛和中心体科学研究资助(部分15H01211至Ka.N.)的支持。日本科学促进会资助(授予号码15H04370至Ka.N.,授权号码15K07929至Y.K.);从武田科学基金会和上原纪念基金会到Y.K.作者宣称没有利益冲突。

参考

  1. Hirano,T.,Katoh,Y。和Nakayama,K.(2017)。 Intraflagellar运输 - 一种复合物介导睫状体进入和睫状G蛋白偶联受体的逆行运输。 Mol Biol Cell 28(3):429-439。
  2. Katoh,Y.和Nakayama K.(2017)。可见免疫沉淀试验:一种方便和多功能的方法研究蛋白质相互作用使用荧光融合蛋白。实验医学35:85-95(日文)。
  3. Katoh,Y.,Nozaki,S.,Hartanto,D.,Miyano,R.和Nakayama,K.(2015)。 通过使用荧光融合蛋白的可见免疫沉淀测定揭示的多亚基复合物的体系结构 J Cell Sci 128(12):2351-2362。
  4. Katoh,Y.,Terada,M.,Nishijima,Y.,Takei,R.,Nozaki,S.,Hamada,H。和Nakayama,K.(2016)。 含有Cluap1 / IFT38的Intraflagellar Transport(IFT)-B复合体的总体结构是IFT-B外周亚复合物 J Biol Chem 291:10962-10975。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Katoh, Y., Nakamura, K. and Nakayama, K. (2018). Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions. Bio-protocol 8(1): e2687. DOI: 10.21769/BioProtoc.2687.
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