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Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo
通过双分子荧光互补(BiFC)试验进行体内蛋白质之间的相互作用的可视化分析   

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Abstract

Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1-158 amino acid residues, called Venus-N, and its C-terminal 159-239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is proportional to the strength of protein-protein interaction (Morell et al., 2008); and fourth, the BiFC signals are derived from intrinsic protein-protein interaction, rather than from extrinsic fluorophores that may not reflect true protein-protein interaction due to their nonspecific association with cellular macromolecules or subcellular compartments. However, some limitations of BiFC include slow maturation (T1/2 ~ 1 hour) of an eventually stable BiFC complex (Hu et al., 2002), making it unsuitable for real-time observation of transient interaction that disappears prior to BiFC detection, and enhanced BiFC background at high expression levels due to fusion-independent association between two non-fluorescent fragments association. BiFC signals generated by in vivo protein-protein interaction can be validated by amino acid mutation introduced at the protein-protein contact surfaces. This imaging technique has been widely used in different cell types and organisms (Kerppola, 2006).

Keywords: BiFC(互补试验), Venus(金星), BRD4(BRD4), P53(p53), HCT116(HCT116)



Figure 1. Protein fragments of Venus (super enhanced YFP) constructs. A. Venus protein (amino acids 1-239; accession number: CAO79509) was dissected into two fragments at residue 158 to generate Venus N-terminus (top) and Venus C-terminus (bottom). B. Schematic drawing of Venus-N-p53 and Venus-C-PDID fusion fragments. Venus-N-p53 and Venus-C-PDID contain Venus-N-terminus and Venus-C-terminus fused respectively to p53 (amino acids 1-393; Gully et al., 2012) and the phosphorylation-dependent interaction domain (PDID, amino acids 287-530) of human Brd4 (Wu et al., 2013), in which a flexible linker containing two copies of Gly4Ser peptide is introduced to allow optimal space contacts between Venus-N-terminus and Venus-C-terminus and also to prevent steric hindrance between the Venus fragment and its fused protein of interest. AscI and XbaI indicate the positions of restriction enzyme-cutting sites used for generating fusions from PCR-amplified DNA fragments. An initiation codon for methionine (M) was added to allow translation of Venus-C-PDID. It should be noted that, although linker peptides ranging from 5 to 17 amino acids are often used (Remy and Michnick, 2007), the exact length and the sequence nature of the linkers have not been systematically analyzed (Kerppola, 2013). C. BiFC fluorescence signal is produced when Venus-N and Venus-C are in close proximity brought together via p53-PDID interaction in the cell.

Materials and Reagents

  1. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
  2. Antibiotics (Penicillin/Streptomycin) (Sigma-Aldrich, catalog number: P0781 )
  3. Cell culture medium (Complete: With 10% FBS and antibiotics; Antibiotic-free: with 10% FBS only)
  4. Formaldehyde (Thermo Fisher Scientific, catalog number: F79-500 )
  5. Triton X-100 (Sigma-Aldrich, catalog number: 79284 )
  6. BSA (Sigma-Aldrich, catalog number: A3059 )
  7. Lipofectamine 2000 (Life Technologies, Invitrogen™, catalog number: 11668-019 )
  8. Venus-N-p53 (Gully et al., 2012) (Santa Cruz, catalog number: sc8334 ) and Venus-C-PDID (Wu et al., 2013) plasmids (Santa Cruz, catalog number: sc5384 ) (see Figure 1B)
  9. Primary antibodies against Venus
    e.g. Anti-full-length-GFP antibody (Santa Cruz, catalog number: sc8334 or sc9996 )
    Anti-C-terminal GFP antibody (Santa Cruz, catalog number: sc5384)
    or β-actin (Sigma-Aldrich, catalog number: A5441 )
  10. Secondary antibody conjugated with a fluorescence dye emitting wavelength other than that of Venus (excitation 488 nm, emission 515 ± 15 nm) or Hoechst 33258 (excitation 350 nm, emission 461 nm), for example, Alexa Fluor® from Life Technologies
  11. Sodium chloride (Thermo Fisher Scientific, catalog number: 7647-14-5 )
  12. Potassium chloride (Thermo Fisher Scientific, catalog number: 7447-40-7 )
  13. Sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O) (Thermo Fisher Scientific, catalog number: S373500 )
  14. Potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: 7778-77-0 )
  15. Aluminum foil (grocery store)
  16. Permanent mounting medium (Vector Laboratories, catalog number: H-5000 )
  17. Microscope slides (Thermo Fisher Scientific, catalog number: 12-544-7 )
  18. Nail polish (grocery store)
  19. 10x Phosphate Buffered Saline (PBS) (see Recipes)
  20. 3.7% Formaldehyde (freshly prepared) (see Recipes)
  21. Phosphate Buffered Saline with Triton X-100 and BSA (PBSTB) (see Recipes)
  22. Hoechst 33258 (Sigma-Aldrich, catalog number: 861405 ) (see Recipes)

Equipment

  1. Glass bottom culture dish (35-mm glass bottom plate containing a 14-mm center microwell, poly-D-lysine coated) (MatTek, catalog number: P35GC-1.5-14-C )
  2. Tissue culture hood (NuAire, model: Class II, Type A2 )
  3. 37 °C cell culture incubator (Thermo Fisher Scientific, Forma®, model: Series II , water-jacketed and HEPA filtered)
  4. Confocal fluorescence microscope (Nikon, model: Eclipse TE-2000E/C1 )
  5. Rocker (Labnet International, model: Rocker 25 )

Software

  1. NIS Elements Basic Research (version 2.2)
  2. Nikon EZ-C1 Free Viewer (version 3.90)

Procedure

     Note: Steps 1 to 7 performed in a tissue culture hood; steps 8 to 10 done on regular bench.

  1. The day before transfection: Seed log-phase growing cells of interest (2 x 105 cells in 2 ml) in a 35-mm glass bottom culture dish and allow overnight incubation for proper cell attachment and expansion in a 37 °C cell culture incubator.
    Note: Optimum cell cultures are 30% to 40% confluent with a low percentage of overlapping cells on the day of transfection.
    Note: Pre-warm culture medium and 1x PBS to 37 °C.
  2. Rinse cells twice with 2 ml of 37 °C 1x PBS.
    Note: Avoid center glass area when pipetting solutions at all steps.
  3. Replace with 1 ml antibiotic-free medium.
  4. Co-transfect Venus-N-p53 and Venus-C-PDID constructs with Lipofectamine 2000 according to manufacture's instructions.
    Note: 0.5 μg of each construct (total 1 μg DNA) plus 2.5 μl of Lipofectamine 2000 in 25 μl of Opti-MEM works well with HCT116 cells.
    Note: Negative controls, such as Venus-N-p53 with Venus-C linked to a non-interacting protein (or domain, e.g., Brd4 amino acids 149-284 described in Wu et al., 2013), Venus-N-p53 with Venus-C, Venus-N with Venus-C-PDID, or Venus-N with Venus-C, should be included in parallel for comparison.
  5. Leave cells at 37 °C in a cell culture incubator.
  6. Replace transfection medium with complete medium 6 hours post-transfection.
  7. Incubate cells for 24 h at 37 °C in a cell culture incubator.
    Note: Incubation time after transfection can vary by the level of protein expression. Pilot experiments to test the optimum expression time and the levels of protein expression for Venus-N-p53 and Venus-C-PDID are beneficial (see Figure 2).
    Note: The amounts of transiently expressed proteins should be titrated to the levels of the endogenous proteins, reflecting endogenous protein interaction in vivo.


    Figure 2. Venus-N-p53 and Venus-C-PDID protein expression. Western blot analysis of Venus protein expression in p53-null (p53-/-) HCT116 cells, 24 hours post-transfection. Antibodies: Venus-N-p53, Venus-C-PDID, and β-Actin.

  8. Wash cells for 5 min with 2 ml of 1x PBS on a rocker at a speed of 10-20 rpm, total three times.
    Note: This step is important to reduce the background signals of Hoechst 33258 due to non-specific adherence of transfection DNA deposits to the plate surfaces.
  9. Prepare for cell imaging:

    For live-cell imaging:
    Note: Perform steps a to c (see below) on a rocker at the speed of 10-20 rpm.
    Note: Avoid center glass area when pipetting solutions at all steps.
    1. Stain DNA in the nucleus with Hoechst 33258, 2 ml (5 μg/ml), for 30 min at room temperature.
      Note: For multiple dishes, prepare one dish at a time before next Hoechst 33258 staining so there is enough time for fluorescence microscope visualization.
    2. Remove staining solution and wash cells for 5 min with 2 ml of 1x PBS, total three times.
    3. Add 1 ml of 1x PBS for fluorescence detection.
    4. Visualize fluorescence signals under a fluorescence microscope.
    5. Acquire lower magnification images and then higher magnification images of bright field, Venus, and Hoechst 33258.
      Note: This step may take up to 30 to 60 min depending on adjusting position/focus and higher resolution of images desired.
    6. Typical results are shown in Figure 3.


      Figure 3. BiFC results. Direct visualization of p53-PDID interaction in vivo by BiFC live-cell imaging performed with p53-null (p53-/-) HCT116 cells transiently expressing Venus-N-p53 and Venus-C-PDID. A. Two different magnification images were obtained by using the 10x ocular lens in combination with a 20x or 60x objective lens. A 20x objective lens is typically used for observing a large number of cells and providing a general glimpse of cellular localization, whereas a 60x objective lens allows more detailed localization within subcellular compartments (Kerppola, 2006). Merge: combined Venus (pseudo-colored green), Hoechst 33258 (pseudo-colored blue), and bright field signals. B. Magnified images of p53-PDID interaction shown in A (i and ii) from 600x images. The BiFC signals co-localize with nuclear DNA staining (Hoechst 33258) as presented in pseudo-colored cyan. Images were obtained by Nikon Eclipse TE-2000E/C1 confocal fluorescence microscopy using NIS Element Basic Research software and further processed by Nikon EZ-C1 software.
      Note: Fixed cell images are virtually the same as live-cell imaging (data not shown).

    For fixed-cell imaging:
    Note: Perform steps a to k (see below) on a rocker at a speed of 10-20 rpm.
    Note: Avoid center glass area when pipetting solutions at all steps.
    1. Fix cell with 1 ml 3.7% formaldehyde in PBS for 15 min at room temperature.
      Note: 3.7% formaldehyde must be freshly prepared.
    2. Remove formaldehyde solution and wash fixed cells 5 min with 2 ml of 1x PBS, total three times.
    3. Remove wash solution and incubate fixed cells with 2 ml of 1x PBS containing 0.25% Triton X-100 to permeabilize cells for 30 min at room temperature.
    4. Wash cells for 5 min with 2 ml of 1x PBS, total three times.
      Note: Skip antibody incubation procedures (steps e to i) if only signals from Venus and Hoechst 33258 (i.e., without fluorophore-conjugated antibody amplification) are needed.  However these steps are helpful for verifying protein expression in transfected cells.
    5. Incubate permeabilized cells with 2 ml of PBSTB for 30 min to block non-specific antibody binding.
    6. Replace PBSTB with primary antibody (against protein of interest or GFP) in 1 ml of PBSTB for 1 h at room temperature or overnight at 4 °C.
      Note: Start with 1:500 antibody dilution to determine the best condition. Two primary antibodies can be used at the same time.
    7. Wash cells for 5 min with 2 ml of 1x PBS, total three times.
    8. Incubate with secondary antibody (conjugated with fluorophores) in 1 ml of PBSTB for 1 hour at room temperature in the dark (wrap with aluminum foil).
      Note: Start with 1:1,000 antibody dilution to determine best condition.
    9. Wash cells for 5 min with 2 ml of 1x PBS, total three times.
    10. Stain cell nucleus DNA with Hoechst 33258, 2 ml (0.5 μg/ml), for 10 min.
    11. Wash cells for 5 min with 2 ml of 1x PBS, total three times.
    12. Add 1 ml of 1x PBS for fluorescence detection.
    13. Visualize reconstituted fluorescence signal under a fluorescence microscope.
    14. Acquire lower magnification images and then higher magnification images of bright field, Venus, Hoechst 33258, or other signals from fluorophore-conjugated secondary antibody.
  10. When needed, permanent preservation of samples can be done by the following steps:
    1. Remove PBS and use razor blade to separate the bottom glass (i.e., coverslip) from the petri dish (see Figure 4A).
      Note: Do not touch the cell-attached side in the center circle of coverslip.
    2. Drop 50 μl of mounting medium on a microscope slide (see Figure 4B).
    3. Gently tilt the coverslip (see Figure 4C), with the cell-attached side facing down, to mount with mounting medium on a microscope slide (see Figure 4D).
    4. Gently press coverslip in the center with a pipet tip to remove air bubbles (see Figure 4E) and remove excess mounting medium around the edges with a paper towel (see Figure 4F).
    5. Mark microscope slides and seal coverslip with nail polish around the edges for 15 min, or until dry, to prevent sample movement and drying (see Figure 4G).
    6. Store at -20 °C or 4 °C in the dark.


      Figure 4.  Illustration of permanent preservation of BiFC samples

Recipes

  1. 10x PBS (1 L)
    80 g NaCl
    2 g KCl
    21.7 g Na2HPO4.7H2O
    2 g KH2PO4
    Add ddH2O to 1 L
    Autoclave and stored at room temperature
    Dilute with ddH2O to make 1x PBS (autoclave required, then store at room temperature)
  2. 3.7% Formaldehyde (freshly prepared for 10 ml)
    1 ml 37% Formaldehyde
    1 ml 10x PBS
    8 ml sterilized ddH2O
  3. PBSTB (freshly prepared for 100 ml)
    10 ml 10x PBS
    250 μl Triton X-100
    1 g BSA
    Add sterilized ddH2O to 100 ml
  4. Hoechst 33258
    Dissolve in 1x PBS to make final stock concentration 10 mg/ml
    Dilute to desired concentration with 1x PBS
    Stored at 4 °C

Acknowledgments

We thank Dr. Shwu-Yuan Wu for technical help and discussions during the development and writing of this protocol. The protocol detailed here was extended primarily from the procedures described in Wu et al. (2013). This work was supported in part by NIH grants (CA103867 and CA124760), CPRIT grants (RP110471 and RP120340), and a Welch Foundation grant (I-1805).

References

    1. Ding, Z., Liang, J., Lu, Y., Yu, Q., Songyang, Z., Lin, S. Y. and Mills, G. B. (2006). A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. Proc Natl Acad Sci U S A 103(41): 15014-15019.
    2. Gully, C. P., Velazquez-Torres, G., Shin, J. H., Fuentes-Mattei, E., Wang, E., Carlock, C., Chen, J., Rothenberg, D., Adams, H. P., Choi, H. H., Guma, S., Phan, L., Chou, P. C., Su, C. H., Zhang, F., Chen, J. S., Yang, T. Y., Yeung, S. C. and Lee, M. H. (2012). Aurora B kinase phosphorylates and instigates degradation of p53. Proc Natl Acad Sci U S A 109(24): E1513-1522. 
    3. Hu, C. D., Chinenov, Y. and Kerppola, T. K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9(4): 789-798. 
    4. Kerppola, T.K. (2006). Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protocols 1(3): 1278-1286.
    5. Kerppola, T. K. (2013). Design of fusion proteins for bimolecular fluorescence complementation (BiFC). Cold Spring Harb Protoc 2013(8): 714-718.
    6. Morell, M., Espargaro, A., Aviles, F.X., and Ventura, S. (2008). Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry. Nat Protocols 3(1): 22-33.
    7. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. and Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1): 87-90. 
    8. Remy, I. and Michnick, S.W. (2007). Application of protein-fragment complementation assays in cell biology. BioTechniques 42(2): 137-145.
    9. Wu, S. Y., Lee, A. Y., Lai, H. T., Zhang, H. and Chiang, C. M. (2013). Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting. Mol Cell 49(5): 843-857.

简介

双分子荧光互补(BiFC)测定是用于使用活细胞成像或固定细胞直接观察蛋白质 - 蛋白质相互作用的方法。这里描述的协议是基于我们最近的论文描述人类染色质衔接子和转录辅因子Brd4与p53肿瘤抑制蛋白的功能联系(吴等人。2013年)。 BiFC首先由Hu等人(2002)使用增强的黄色荧光蛋白(EYFP)的两种非荧光蛋白片段描述,所述荧光蛋白是Aequorea victoria GFP变体蛋白,分别融合Rel家族蛋白和bZIP家族转录因子,以调查这两个家庭成员在活细胞之间的相互作用。通过引入突变以降低其对pH和氯离子的敏感性,从而产生称为金星荧光蛋白的超增强YFP,而在37℃下不显示减少的荧光,如通常用EYFP观察到的,YFP被改进(Nagai等al。,2006)。荧光信号通过两个非荧光片段(例如,金黄N末端1-158个氨基酸残基,称为Venus-N)及其C-末端159-239个氨基酸的互补而再生残基,命名为Venus-C;参见图1A和Gully等人,2012; Ding等人,2006; Kerppola,2006),它们通过它们各自的融合伴侣(例如,Venus-N到p53和Venus-C到人Brd4的PDID结构域;参见图1B和1C)。再生的荧光信号的强度和细胞位置可以通过荧光显微镜检测。基于邻近的BiFC测定法的优点是:首先,其允许在体内两个配偶体蛋白之间的时间和空间相互作用的直接可视化;第二,荧光信号提供用于检测蛋白质 - 蛋白质相互作用的灵敏读数,甚至在与内源蛋白质的表达水平相当的低表达水平;第三,荧光信号的强度与蛋白质 - 蛋白质相互作用的强度成比例(Morell等人,2008);第四,BiFC信号源自内在蛋白质 - 蛋白质相互作用,而不是来自外源性荧光团,由于它们与细胞大分子或亚细胞区室的非特异性缔合,可能不反映真实的蛋白质 - 蛋白质相互作用。然而,BiFC的一些限制包括最终稳定的BiFC复合物的缓慢成熟(T 1/2 1/2小时)(Hu等人,2002),使其不适合用于实时观察在BiFC检测前消失的瞬时相互作用,以及由于两个非荧光片段缔合之间的融合非依赖性缔合而导致的高表达水平下的增强的BiFC背景。通过体内蛋白质 - 蛋白质相互作用产生的BiFC信号可以通过在蛋白质 - 蛋白质接触表面处引入的氨基酸突变来验证。这种成像技术已广泛用于不同的细胞类型和生物(Kerppola,2006)。

关键字:互补试验, 金星, BRD4, p53, HCT116



图1.金星(超增强YFP)构建体的蛋白片段。将金星蛋白(氨基酸1-239;登录号:CAO79509)在残基158处切割成两个片段以产生金星N- (顶部)和金星C-末端(底部)。 Venus-N-p53和Venus-C-PDID融合片段的示意图。 Venus-N-p53和Venus-C-PDID含有分别与p53融合的Venus-N末端和Venus-C末端(氨基酸1-393; Gully等人,2012)磷酸化依赖性相互作用结构域(PDID,氨基酸287-530)(Wu等人,2013),其中包含两个拷贝的Gly 4亚单位的柔性接头>引入Ser肽以允许Venus-N末端和Venus-C末端之间的最佳空间接触,并且还防止Venus片段与其感兴趣的融合蛋白之间的空间位阻。 AscI和XbaI表示用于从PCR扩增的DNA片段产生融合物的限制性酶切位点的位置。加入甲硫氨酸(M)的起始密码子以允许Venus-C-PDID的翻译。应当注意,尽管经常使用范围从5至17个氨基酸的接头肽(Remy和Michnick,2007),但是没有系统地分析接头的确切长度和序列性质(Kerppola,2013)。 C.当Venus-N和Venus-C靠近时,通过细胞中的p53-PDID相互作用汇集在一起产生BiFC荧光信号。

材料和试剂

  1. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442)
  2. 抗生素(青霉素/链霉素)(Sigma-Aldrich,目录号:P0781)
  3. 细胞培养基(完全:含10%FBS和抗生素;无抗生素:仅含10%FBS)
  4. 甲醛(Thermo Fisher Scientific,目录号:F79-500)
  5. Triton X-100(Sigma-Aldrich,目录号:79284)
  6. BSA(Sigma-Aldrich,目录号:A3059)
  7. Lipofectamine 2000(Life Technologies,Invitrogen TM,目录号:11668-019)
  8. Venus-N-p53(Gully等人,2012)(Santa Cruz,目录号:sc8334)和Venus-C-PDID(Wu等人,2013) 质粒(Santa Cruz,目录号:sc5384)(参见图1B)
  9. 针对Venus的主要抗体
    例如抗全长GFP抗体(Santa Cruz,目录号:sc8334或sc9996)
    抗C末端GFP抗体(Santa Cruz,目录号:sc5384)
    或β-肌动蛋白(Sigma-Aldrich,目录号:A5441)
  10. 与荧光染料发射波长不同于Venus(激发488nm,发射515±15nm)或Hoechst 33258(激发350nm,发射461nm)的荧光染料结合的二抗,例如Alexa Fluor 来自Life Technologies
  11. 氯化钠(Thermo Fisher Scientific,目录号:7647-14-5)
  12. 氯化钾(Thermo Fisher Scientific,目录号:7447-40-7)
  13. 磷酸氢二钠七水合物(Na 2 HPO 4+,7H 2 O 2)(Thermo Fisher Scientific,目录号: S373500)
  14. 磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:7778-77-0)
  15. 铝箔(杂货店)
  16. 永久固定介质(Vector Laboratories,目录号:H-5000)
  17. 显微镜载玻片(Thermo Fisher Scientific,目录号:12-544-7)
  18. 指甲油(杂货店)
  19. 10x磷酸盐缓冲盐水(PBS)(见配方)
  20. 3.7%甲醛(新鲜制备)(参见配方)
  21. 磷酸盐缓冲盐水与Triton X-100和BSA(PBSTB)(参见配方)
  22. Hoechst 33258(Sigma-Aldrich,目录号:861405)(参见Recipes)

设备

  1. 玻璃底培养皿(含有14-mm中心微孔,涂有聚-D-赖氨酸的35-mm玻璃底板)(MatTek,目录号:P35GC-1.5-14-C)
  2. 组织培养罩(NuAire,型号:II类,A2型)
  3. 37℃细胞培养孵育器(Thermo Fisher Scientific,Forma II型,系列II,水夹套和HEPA过滤)
  4. 共聚焦荧光显微镜(Nikon,型号:Eclipse TE-2000E/C1)
  5. Rocker(Labnet International,型号:Rocker 25)

软件

  1. NIS元素基本研究(版本2.2)
  2. 尼康EZ-C1免费浏览器(版本3.90)

程序

     :步骤1至7在组织培养罩中进行; 步骤8至10在常规工作台上完成。

  1. 转染前一天:在35-mm玻璃底培养皿中种子对数期生长的感兴趣细胞(2×10 5个细胞在2ml中),并允许过夜孵育以适当的细胞附着和扩增 37℃的细胞培养箱 注意:在转染当天,最佳细胞培养物为30%至40%汇合,且具有低百分比的重叠细胞。
    注意:预热培养基和1x PBS至37℃。
  2. 用2ml 37℃1x PBS冲洗细胞两次 注意:在所有步骤中移液解决方案时,避免使用中心玻璃区域。
  3. 更换1毫升无抗生素培养基。
  4. 根据制造商的说明,用Lipofectamine 2000共转染Venus-N-p53和Venus-C-PDID构建体。
    注意:在25μlOpti-MEM中,0.5μg每种构建体(总共1μgDNA)加上2.5μlLipofectamine 2000在HCT116细胞中效果很好。
    注意:阴性对照例如具有与非相互作用蛋白(或结构域,例如,Wu等人,2013中描述的Brd4氨基酸149-284)的Venus-C的Venus-N-p53,Venus-具有Venus-C的Venus-N,具有Venus-C-PDID的Venus-N或具有Venus-C的Venus-N的N-p53应当并行包括以进行比较。
  5. 在37℃下在细胞培养箱中离开细胞
  6. 在转染后6小时用完全培养基更换转染培养基。
  7. 孵育细胞在细胞培养箱37℃下24小时 注意:转染后的孵育时间可随蛋白质表达水平而变化。用于测试Venus-N-p53和Venus-C-PDID的最佳表达时间和蛋白质表达水平的试验性实验是有益的(参见 图2)。
    注意:瞬时表达的蛋白质的量应当滴定至内源蛋白质的水平,反映体内内源蛋白质的相互作用。


    图2. Venus-N-p53和Venus-C-PDID蛋白表达。转染后24小时,p53-空(p53 -/- )HCT116细胞中金星蛋白表达的Western印迹分析。抗体:Venus-N-p53,Venus-C-PDID和β-肌动蛋白
  8. 在摇床上以10-20rpm的速度用2ml的1×PBS洗涤细胞5分钟,共3次。
    注意:这一步骤对于减少Hoechst 33258的背景信号是重要的,因为转染DNA沉积物非特异性地粘附到板表面。
  9. 准备进行细胞成像:

    对于活细胞成像:
    注意:在摇杆上以10-20 rpm的速度执行步骤a到c(见下文)。
    注意:在所有步骤移液解决方案时,避免使用中心玻璃区域。
    1. 在室温下用Hoechst 33258,2ml(5μg/ml)在细胞核中染色DNA 30分钟。
      注意:对于多个菜肴,在下次Hoechst 33258染色前一次准备一个培养皿,以便有足够的时间进行荧光显微镜可视化。
    2. 除去染色溶液,用2ml 1×PBS洗涤细胞5分钟,共3次
    3. 加入1ml 1×PBS进行荧光检测
    4. 在荧光显微镜下可视化荧光信号
    5. 获取较低放大倍数的图像,然后是较亮的视场,金星和Hoechst 33258的高倍放大图像。
      注意:此步骤最多可能需要30至60分钟,具体取决于调整位置/焦点和所需图像的更高分辨率。
    6. 典型结果如图3所示。


      图3. BiFC结果。 通过使用瞬时表达Venus-N-p53和Venus-C的p53-无效(p53 -/- )HCT116细胞进行的BiFC活细胞成像,直接观察p53-PDID在体内的相互作用-PDID。通过使用与20x或60x物镜组合的10x目镜来获得两个不同的放大图像。 20倍物镜通常用于观察大量细胞并提供细胞定位的一般瞥见,而60倍物镜允许在亚细胞区室中更详细的定位(Kerppola,2006)。合并:组合金星(伪色绿色),Hoechst 33258(伪色蓝色)和明场信号。 B.来自600x图像的A(i和ii)中显示的p53-PDID相互作用的放大图像。 BiFC信号与核DNA染色(Hoechst 33258)共定位,如伪色青色中所示。使用NIS Element Basic Research软件通过Nikon Eclipse TE-2000E/C1共聚焦荧光显微镜获得图像,并通过Nikon EZ-C1软件进一步处理。
      注意:固定的细胞图像实际上与活细胞成像相同(数据未显示)。

    对于固定细胞成像:
    注意:以10-20 rpm的速度在摇杆上执行步骤a到k(见下文)。
    注意:在所有步骤移液解决方案时,避免使用中心玻璃区域。
    1. 用1ml 3.7%甲醛的PBS溶液在室温下固定细胞15分钟 注意:3.7%甲醛必须新鲜制备。
    2. 除去甲醛溶液,用2ml 1×PBS洗涤固定的细胞5分钟,共3次
    3. 取出洗涤溶液,并用2ml含有0.25%Triton X-100的1×PBS孵育固定细胞,在室温下透化细胞30分钟。
    4. 用2ml 1×PBS洗涤细胞5分钟,共3次 注意:如果仅需要来自Venus和Hoechst 33258的信号(即,没有荧光团偶联的抗体扩增),则跳过抗体孵育程序(步骤e至i)。然而,这些步骤有助于验证转染细胞中的蛋白质表达。
    5. 用2ml PBSTB孵育透化细胞30分钟以阻断非特异性抗体结合
    6. 将PBSTB用1ml PBSTB中的一抗(针对目的蛋白或GFP)在室温下1小时或在4℃下过夜替换。
      注意:从1:500抗体稀释开始,以确定最佳条件。两种一抗可同时使用。
    7. 用2ml 1×PBS洗涤细胞5分钟,共3次
    8. 在室温下在黑暗中与1ml PBSTB中的二抗(与荧光团结合)孵育1小时(用铝箔包裹)。
      注意:从1:1,000抗体稀释开始,以确定最佳状态。
    9. 用2ml 1×PBS洗涤细胞5分钟,共3次
    10. 用Hoechst 33258(2ml(0.5μg/ml))染色细胞核DNA,10分钟。
    11. 用2ml 1×PBS洗涤细胞5分钟,共三次。
    12. 加入1ml 1×PBS进行荧光检测
    13. 在荧光显微镜下可视化重构的荧光信号
    14. 获取较低放大倍数的图像,然后较高放大倍率的明场,金星,Hoechst 33258或来自荧光团共轭二抗的信号。
  10. 需要时,可通过以下步骤进行样品的永久保存:
    1. 取出PBS,并用刀片将底部玻璃(,即,盖玻片)与培养皿分开(见图4A)。
      注意:请勿触摸盖玻片中心圆圈中的细胞附着侧。
    2. 在显微镜载玻片上滴50μl固定介质(见图4B)
    3. 轻轻地倾斜盖玻片(参见图4C),细胞附着面朝下,用安装介质安装在显微镜载玻片上(见图4D)。
    4. 用移液管尖端轻轻按下中心的盖玻片以除去气泡(见图4E),并用纸巾除去边缘周围多余的固定介质(见图4F)。
    5. 标记显微镜载玻片和密封盖玻片边缘指甲油15分钟,或直到干燥,以防止样品移动和干燥(见图4G)。
    6. 储存于-20°C或4°C在黑暗中。


      图4.  永久保存BiFC样品的示例

食谱

  1. 10x PBS(1L)
    80克NaCl
    2克KCl
    21.7g Na 2 HPO 4 sub 7H。 O
    2g KH sub 2 PO 4 sub
    将ddH <2> O添加到1 L
    高压灭菌并在室温下贮存
    用ddH 2 O稀释以制备1×PBS(需要高压灭菌器,然后在室温下贮存)
  2. 3.7%甲醛(新鲜制备10ml) 1ml 37%甲醛
    1ml 10x PBS
    8ml灭菌的ddH 2 O 2 /
  3. PBSTB(新鲜制备100ml)
    10ml 10×PBS
    250微升Triton X-100 1 g BSA
    将灭菌的ddH 2 O加至100ml ml
  4. Hoechst 33258
    溶解在1×PBS中,使最终的原液浓度为10mg/ml
    用1x PBS稀释至所需浓度
    储存在4°C

致谢

我们感谢Wu Shwu-Yuan Wu博士在本协议开发和编写过程中的技术帮助和讨论。 这里详述的方案主要从Wu等人(2013)中描述的程序延伸。 这项工作部分由NIH赠款(CA103867和CA124760),CPRIT赠款(RP110471和RP120340)和Welch基金会赠款(I-1805)支持。

参考文献

    1. Ding,Z.,Liang,J.,Lu,Y.,Yu,Q.,Songyang,Z.,Lin,S.Y.and Mills,G.B。(2006)。 基于逆转录病毒的蛋白质互补测定屏幕显示功能性AKT1结合伴侣。 Proc Natl Acad Sci USA 103(41):15014-15019。
    2. Gully,CP,Velazquez-Torres,G.,Shin,JH,Fuentes-Mattei,E.,Wang,E.,Carlock,C.,Chen,J.,Rothenberg,D.,Adams,HP,Choi,HH, Guma,S.,Phan,L.,Chou,PC,Su,CH,Zhang,F.,Chen,JS,Yang,TY,Yeung,SCand Lee,MH(2012)。 Aurora B激酶磷酸化并引发p53的降解。 Proc Natl Acad Sci U S A 109(24):E1513-1522。 
    3. Hu,C.D.,Chinenov,Y.and Kerppola,T.K。(2002)。 使用双分子荧光互补,可视化活细胞中bZIP和Rel家族蛋白之间的相互作用。 Mol Cell 9(4):789-798。
    4. 凯波拉(2006)。 用于活细胞中蛋白质相互作用的可视化的双分子荧光互补(BiFC)测定的设计和实施。 Nat Protocols 1(3):1278-1286。
    5. Kerppola,T.K。(2013)。 双分子荧光互补的融合蛋白设计(BiFC)。冷春天Harb Protoc 2013(8):714-718。
    6. Morell,M.,Espargaro,A.,Aviles,F.X。,和Ventura,S。(2008)。 通过偶联双分子荧光互补和流式细胞术研究和选择体内蛋白质相互作用。 Nat Protocols 3(1):22-33。
    7. Nagai,T.,Ibata,K.,Park,E.S.,Kubota,M.,Mikoshiba,K.and Miyawaki,A。(2002)。 一种黄色荧光蛋白的变体,快速有效地用于细胞 - 生物应用。 Nat Biotechnol 20(1):87-90。
    8. Remy,I.and Michnick,S.W。 (2007)。蛋白质片段互补测定在细胞生物学中的应用。 BioTechniques 42(2):137-145。
    9. Wu,S.Y.,Lee,A.Y.,Lai,H.T.,Zhang,H.and Chiang,C.M。(2013)。 Phospho开关触发Brd4染色质结合和激活剂募集用于基因特异性靶向。 Mol Cell 49(5):843-857。

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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lai, H. and Chiang, C. (2013). Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo. Bio-protocol 3(20): e935. DOI: 10.21769/BioProtoc.935.
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