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Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells
采用生物发光共振能量转移(BRET)技术测定活细胞中分子的相互作用   

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参见作者原研究论文

本实验方案简略版
The Journal of Biological Chemistry
Aug 2017

Abstract

The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins tagged with bioluminescent Renilla luciferase (Rlu) as donor and yellow fluorescent protein (YFP) as acceptor were co-expressed in cells. This pair of donor and acceptor have an approximate Förster distance of 4.4 nm, providing the optimal working distance (Dacres et al., 2010). This technique can be used to explore the time-course of specific molecular interactions that occur in living cells.

Keywords: BRET assay(BRET测定法), Molecular approximation/interaction(分子近似/相互作用), Renilla luciferase(海肾荧光素酶), Yellow fluorescent protein(黄色荧光蛋白), Coelenterazine h(腔肠素h)

Background

Bioluminescence resonance energy transfer (BRET) studies, using a bioluminescence donor and a fluorescence acceptor, can monitor molecular interactions (such as between labeled proteins, peptides, or small molecules) occurring in real time in living cells. This approach is dependent on spatial approximation between the donor and acceptor, as well as appropriate spectral overlap to yield a meaningful signal (Figure 1). The example currently provided utilizes a Rlu-tagged protein as the donor and a YFP-tagged protein as acceptor (Harikumar et al., 2007). This has been very successfully applied to establish the presence of physiologically-relevant protein-protein interactions in the plasma membrane of living cells. It is important, however, to include controls for levels of expression that could cause non-specific protein-protein approximation and energy transfer (bystander effect), such as the use of similar levels of expression of a known non-associated protein. Also, competition with an unlabeled protein can help to establish the saturability of the interaction and the specificity of the signal.


Figure 1. Illustration of relevant events in a BRET experiment in a living cell. The energy transfer reaction is initiated by adding the luciferase substrate, coelenterazine-h, to cells expressing both molecules tagged with Rlu (donor) and with yellow fluorescent protein (acceptor). The Rlu emits light with a wavelength of approximately 475 nm that then excites the YFP to emit light at approximately 525 nm that can be quantified to represent the BRET signal. The approximate Fӧrster distance for this pair of donor-acceptor is approximately 4.4 nm.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog number: 1111-1700 )
  2. 100-mm tissue culture grade plastic plates (SARSTEDT, catalog number: 83.3902 )
  3. 5 ml tissue culture tubes (Corning, Falcon®, catalog number: 352052 )
  4. Cell culture flasks (Corning, catalog number: 3056 )
  5. 96-well OptiPlates (PerkinElmer, catalog number: 6005290 )
  6. 15 ml conical tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  7. Pasteur pipette (Fisher Scientific, catalog number: 13-678-20B )
  8. Rapid flow bottle-top filter unit with polyethersulfone (PES) membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 595-3320 )
  9. PS1/PS2-deleted HTL cells
    Note: This cell line was derived by Xu et al., 2016, utilizing CRISPR/Cas9 to delete PS1/PS2 from HTL cells, representing a cell line derived from HEK293 cells by Barnea and Axel who stably integrated a luciferase reporter under the control of the bacterial operator element tetO. These cells were used in this case to correlate with specific functional assays requiring a specialized cell type. In a general protocol, any transfectable cell type is fine.
  10. Dulbecco’s modified Eagle’s medium powder (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 12100-038 ) (see Recipe 6 for media preparation)
  11. Fetal Clone II supplement, a bovine serum product (GE Healthcare, HycloneTM, catalog number: SH30066.03 )
  12. Trypsin 0.25%-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 ) diluted to 0.05% with 1x Dulbecco’s phosphate buffered saline, pH 6.80
  13. DEAE-dextran hydrochloride (Sigma-Aldrich, catalog number: D9885 )
  14. Dimethyl sulfoxide (DMSO) (Fisher Biotech, catalog number: BP231-1 )
  15. Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628 )
  16. Enzyme-free cell dissociation solution (EMD Millipore, catalog number: S-014-C )
  17. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876 )
  18. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
  19. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-500 )
  20. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  21. HEPES (Research Products International, catalog number: H75030-1000 )
  22. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M1880 )
  23. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  24. Sodium hydroxide (NaOH) (VWR, catalog number: BDH7247-1 )
  25. Coelenterazine-h (AAT Bioquest, catalog number: 21165 ) (see Recipe 5 for preparation of stock solution)
  26. Methanol (Honeywell International, catalog number: 24229 )
  27. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  28. Sodium bicarbonate (Fisher Scientific, catalog number: BP328-500 )
  29. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
  30. 10x phosphate-buffered saline (PBS buffer) (see Recipes)
  31. 10x Kreb’s-Ringers-HEPES stock solution (10x KRH) (see Recipes)
  32. 50x CaCl2 stock solution (see Recipes)
  33. 1x KRH working solution (see Recipes)
  34. Coelenterazine-h solution (see Recipes)
  35. 1x DMEM tissue culture medium (see Recipes)

Equipment

  1. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 Water-Jacketed)
  2. Cell culture microscope (Fisher Scientific)
  3. Micropipettes (Gilson)
  4. 2103 EnVision Plate Reader (PerkinElmer, model: 2103 EnVisionTM )
  5. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM XT/XF centrifuge , catalog number : 75216362)
  6. 500 ml screw-cap autoclaved glass bottles (WHEATON, catalog number: 219759 )

Software

  1. GraphPad Prism 6 (GraphPad Software, Inc. USA)

Procedure

  1. Cell culture
    1. The PS1/PS2-deleted HTL cells are routinely grown in DMEM medium supplemented with 10% (v/v) Fetal Clone II and including 1x penicillin-streptomycin (see Recipes) at 37 °C under a humidified 5% CO2 atmosphere.
    2. Treat the cells with 0.05% trypsin-EDTA for 5 min at 37 °C in an incubator to lift the cells. Wash the cells with medium, and count them to allow a plating density of 1.5 x 106 cells per 100 mm dish one day prior to transfection.
    3. In preparation for transfection, visualize the confluence of the cells and their health using a cell culture microscope with phase optics.

  2. Transfection
    1. To a sterile 5 ml tissue culture tube, add 1.5 ml DMEM medium without serum.
    2. Add 75 µl DEAE-dextran solution and mix well.
    3. Add 1.0 µg of total DNA (0.5 µg of donor + 0.5 µg of acceptor prepared using standard DNA isolation techniques) and mix well.
    4. Use a micropipette to transfer 1.575 µl of DNA-DEAE-dextran complex to each plate.
    5. Incubate at 37 °C in a tissue culture incubator for 2 h without shaking.
    6. Aspirate the medium from each plate, add 4 ml of DMEM containing 10% DMSO and incubate at room temperature for 2 min without shaking.
    7. Aspirate the medium from each plate, add 4 ml of DMEM containing 0.1 µM chloroquine and incubate for 2 h at 37 °C without shaking.
    8. Aspirate the medium from each plate, and add 8 ml of serum-containing complete DMEM medium (see Recipes).
    9. Incubate the cells in the incubator at 37 °C in a humidified atmosphere containing 5% CO2 for 48 h.

  3. Luminescence and fluorescence measurement
    1. 48 h post-transfection, remove the medium from the cultured cells using a Pasteur pipette attached to a vacuum source and gently add 5 ml of PBS (see Recipes) to each plate to rinse the cells.
    2. Dispense 1 ml of enzyme-free cell dissociation solution into each plate and incubate without shaking for 5 min at 37 °C in tissue culture incubator to release the cells.
    3. Transfer the cells into a 15 ml conical tube and centrifuge the cells at 175 x g for 5 min at room temperature.
    4. Resuspend the cell pellet in approximately 1 ml of Kreb’s-Ringers-HEPES medium, pH 7.4 (see Recipes) to achieve a density of 0.20 x 106 cells per ml. Keep this tube at room temperature in preparation for the assay.
    5. Dispense 100 µl of cell suspension (approximately 20,000 cells) into a white 96-well OptiPlate, add 5 µl coelenterazine h (see Recipes), mix the cells by swirling the plate, and measure both the luminescence signal and the YFP fluorescence signal using an Envision Plate Reader with a BRET module.



    6. Luminescence and fluorescence signals should be collected immediately after the addition of coelenterazine h solution. The spectrofluorometer instrument should be set up for the specific pair of donor and acceptor chosen. Here, for the proposed BRET studies, luminescence emission should be collected using a dedicated filter set (460 nm, with bandwidth 25 nm) and the fluorescence emission should be collected simultaneously using a dedicated filter set (535 nm, bandwidth 25 nm). The measurement time is 0.2 sec and measurement height is 6.5 mm. (Harikumar et al., 2007). In the example, YFP fluorescence was measured by exciting the sample at 475 nm (bandwidth 25 nm) and the emission was collected at 535 nm (bandwidth 25 nm), with detector gain 150 and number of flashes 20.
    7. A representative example of data collection and analysis follows:


      Calculated BRET ratio: 1.23 - 0.70 = 0.53

Data analysis

Data should be collected in at least duplicate conditions in a minimum of three independent experiments. The statistical analysis of data can be accomplished with GraphPad Prism 6, using a t-test with the Mann-Whitney post-test.

Notes

Controls for levels of expression of donor and acceptor are important to eliminate bystander energy transfer. A significant BRET signal should be observed at levels of expression that exist physiologically. Additionally, saturability and specificity of the BRET signal are important, and can be established using competing non-labeled protein of interest. For positive control, a construct encoding a fusion protein containing both luciferase and yellow fluorescent protein (Rlu-YFP) was used to transfect the cells, and the BRET signal was measured.

Recipes

  1. 10x phosphate-buffered saline (10x PBS buffer) (1 L)
    11.5 g Na2HPO4
    2 g KH2PO4
    80 g NaCl
    2 g KCl
    Dissolve in 1 L of sterile, deionized water
    Adjust the pH of 1x PBS to 7.4
    Store at 4 °C
  2. 10x Kreb’s-Ringers-HEPES stock solution (10x KRH) (1 L)
    59.58 g HEPES
    60.74 g NaCl
    3.72 g KCl
    1.36 g KH2PO4
    2.96 g MgSO4
    Dissolve the components in deionized water and bring the volume to 1 L, do not adjust the pH
    Store at 4 °C
  3. 50x CaCl2 stock solution (100 mM) (100 ml)
    1.47 g CaCl2
    Bring the volume to 100 ml with deionized water; do not adjust pH
    Store at 4 °C
  4. 1x KRH working solution (100 ml)
    10 ml 10x KRH stock solution
    2 ml 50x CaCl2 stock solution
    Bring the volume to 100 ml with deionized water; adjust the pH to 7.4 with NaOH
  5. Coelenterazine-h solution
    0.5 mg coelenterazine-h powder
    Dissolve in 12.5 ml of 5% methanol/95% water
    Store at -20 °C
  6. 1x DMEM tissue culture medium (1 L)
    1. One 1-L packet of Dulbecco’s modified Eagle medium (13.4 g) and 3.7 g of sodium bicarbonate are added to 1 L of deionized water
    2. Adjust the pH to 6.90 with HCl, filter into 500 ml screw-cap autoclaved glass bottles using a 0.2 µm rapid flow bottle-top filter unit containing a polyethersulfone (PES) membrane
    3. Store at 4 °C

Acknowledgments

This work was supported by Mayo Clinic (L.J.M.), the Van Andel Research Institute, the National Natural Science Foundation of China (31300607, 31300245 and 91217311), Ministry of Science and Technology grants 2012ZX09301001, 2012CB910403, and 2013CB910600, XDB08020303, 2013ZX09507001, Shanghai Science and Technology Committee (13ZR1447600), Shanghai Rising-Star Program (14QA1404300), and the National Institute of Health grants DK071662 (H.E.X.), GM102545 and GM104212 (K. M.). Authors have no conflicts of interest or competing interests.

References

  1. Dacres, H., Wang, J., Dumancic, M. M. and Trowell (2010). Experimental determination of the Förster distance for two commonly used Bioluminescence resonance energy transfer pairs. Anal Chem 82: 432-435.
  2. Harikumar, K. G., Pinon, D. I. and Miller, L. J. (2007). Transmembrane segment IV contributes a functionally important interface for oligomerization of the Class II G protein-coupled secretin receptor. J Biol Chem 282: 30363-30372.
  3. Xu, T. H., Yan, Y., Kang, Y., Jiang, Y., Melcher, K. and Xu, H. E. (2016). Alzheimer's disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio. Cell Discov 2: 16026.

简介

生物发光共振能量转移(BRET)测定可以用作分子近似和/或相互作用的指标。 当具有与供体发射的适当光谱重叠的受体与供体近似时,产生显着的共振能量转移信号。 在所提供的例子中,用生物发光的海肾荧光素酶(Rlu)作为供体和作为受体的黄色荧光蛋白(YFP)标记的蛋白在细胞中共表达。 这对供体和受体具有4.4nm的近似Forster距离,提供了最佳的工作距离(Dacres et。,2010)。 这种技术可以用来探索在活细胞中发生的特定分子相互作用的时间过程。
【背景】使用生物发光供体和荧光受体的生物发光共振能量转移(BRET)研究可以监测在活细胞中实时发生的分子相互作用(例如标记的蛋白质,肽或小分子之间)。这种方法取决于供体和受体之间的空间近似,以及适当的光谱重叠以产生有意义的信号(图1)。目前提供的实例使用Rlu-标记的蛋白质作为供体和YFP-标记的蛋白质作为受体(Harikumar等人,2007)。这已被非常成功地应用于建立活细胞质膜中生理相关的蛋白质 - 蛋白质相互作用的存在。然而,重要的是包括可能导致非特异性蛋白质 - 蛋白质近似和能量转移(旁观者效应)的表达水平的对照,例如使用相似水平的已知非相关蛋白质的表达。此外,与未标记蛋白质的竞争可以帮助建立信号的相互作用的可饱和性和特异性。

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图1.在活细胞中的BRET实验中的相关事件的图解能量转移反应通过将荧光素酶底物腔肠素-h添加到表达两种标记有Rlu(供体)的分子的细胞中,和黄色荧光蛋白(受体)。 Rlu发射约475nm波长的光,然后激发YFP发射约525nm的光,其可被量化以代表BRET信号。这对供体 - 受体的近似Fsrster距离约为4.4 nm。

关键字:BRET测定法, 分子近似/相互作用, 海肾荧光素酶, 黄色荧光蛋白, 腔肠素h

材料和试剂

  1. 移液器吸头(USA Scientific,目录号:1111-1700)
  2. 100毫米组织培养级塑料板(SARSTEDT,目录号:83.3902)
  3. 5ml组织培养管(Corning,Falcon,产品目录号:352052)
  4. 细胞培养瓶(康宁,目录号:3056)
  5. 96孔OptiPlates(PerkinElmer,目录号:6005290)
  6. 15ml锥形管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339650)。
  7. 巴斯德吸管(Fisher Scientific,目录号:13-678-20B)
  8. 具有聚醚砜(PES)膜的快速流动瓶顶过滤器单元(Thermo Fisher Scientific,Thermo Scientific TM,目录号:595-3320)
  9. PS1 / PS2删除的HTL单元
    注意:该细胞系由Xu等人,2016利用CRISPR / Cas9从HTL细胞中删除PS1 / PS2得到,代表来自Barnea和Axel的来自HEK293细胞的细胞系,其稳定地整合了萤光素酶报道基因细菌操作元件tetO的控制。在这种情况下使用这些细胞来与需要特化细胞类型的特定功能测定相关联。在一般的协议中,任何转染的细胞类型都是可以的。
  10. Dulbecco改良的Eagle's中等粉末(DMEM)(Thermo Fisher Scientific,Gibco TM,产品目录号:12100-038)(参见配方6,用于培养基制备)
  11. 胎牛血清II补充物,牛血清产品(GE Healthcare,Hyclone TM,目录号:SH30066.03)
  12. 用1×Dulbecco's磷酸缓冲盐溶液(pH6.8)稀释至0.05%的胰蛋白酶0.25%-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  13. DEAE-葡聚糖盐酸盐(Sigma-Aldrich,目录号:D9885)
  14. 二甲基亚砜(DMSO)(Fisher Biotech,目录号:BP231-1)
  15. 氯喹二磷酸盐(Sigma-Aldrich,目录号:C6628)
  16. 无酶细胞解离液(EMD Millipore,目录编号:S-014-C)
  17. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S0876)
  18. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285)
  19. 氯化钠(NaCl)(Fisher Scientific,目录号:S671-500)
  20. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  21. HEPES(Research Products International,目录号:H75030-1000)
  22. 硫酸镁七水合物(MgSO 4•7H 2 O)(Sigma-Aldrich,目录号:M1880)
  23. 氯化钙二水合物(CaCl 2•2H 2 O)(Fisher Scientific,目录号:C79-500)
  24. 氢氧化钠(NaOH)(VWR,目录号:BDH7247-1)
  25. Coelenterazine-h(AAT Bioquest,目录号:21165)(见配方5制备原液)
  26. 甲醇(Honeywell International,目录号:24229)
  27. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  28. 碳酸氢钠(Fisher Scientific,目录号:BP328-500)
  29. 盐酸(HCl)(Fisher Scientific,目录号:A144-212)
  30. 10倍磷酸盐缓冲盐水(PBS缓冲液)(见食谱)
  31. 10倍Kreb's-Ringers-HEPES储备液(10倍KRH)(见食谱)
  32. 50x CaCl 2储备液(见食谱)
  33. 1x KRH工作解决方案(请参阅食谱)
  34. Coelenterazine-h溶液(见食谱)
  35. 1x DMEM组织培养基(见食谱)

设备

  1. 37℃,5%CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Series 3110水套)
  2. 细胞培养显微镜(Fisher Scientific)
  3. Micropipettes(吉尔森)
  4. 2103 EnVision Plate Reader(PerkinElmer,型号:2103 EnVision TM)
  5. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall TM Legend TM XT / XF离心机,目录号:75216362) >
  6. 500毫升螺帽蒸压玻璃瓶(WHEATON,目录号:219759)

软件

  1. GraphPad Prism 6(GraphPad Software,Inc.USA)

程序

  1. 细胞培养
    1. PS1 / PS2缺陷的HTL细胞通常在补充有10%(v / v)胎儿克隆II和包括1x青霉素 - 链霉素(参见配方)的DMEM培养基中在37℃和潮湿的5%CO 2气氛。
    2. 在37℃的培养箱中用0.05%胰蛋白酶-EDTA处理细胞5分钟以提起细胞。用培养基清洗细胞,并在转染前一天对它们进行计数以使得每100mm培养皿的电镀密度为1.5×10 6个细胞。
    3. 在准备转染时,使用具有相位光学元件的细胞培养显微镜观察细胞汇合和它们的健康。

  2. 转染
    1. 向无菌的5ml组织培养管中加入1.5ml无血清的DMEM培养基。
    2. 加入75μlDEAE-葡聚糖溶液并充分混合。
    3. 加入1.0μg总DNA(0.5μg供体+0.5μg使用标准DNA分离技术制备的受体)并充分混合。
    4. 使用微量移液器将1.575μl的DNA-DEAE-葡聚糖复合物转移到每个平板上。

    5. 在组织培养箱中37°C孵育2小时,不摇动。
    6. 从每个平板吸取培养基,加入4毫升含有10%DMSO的DMEM,室温孵育2分钟,不摇动。
    7. 从每个平板上吸取培养基,加入4毫升含有0.1μM氯喹的DMEM,37℃孵育2小时,不摇动。
    8. 从每个平板吸取培养基,并加入8毫升含血清的完整DMEM培养基(见食谱)。
    9. 将培养箱中的细胞在含有5%CO 2的潮湿气氛中于37℃孵育48小时。

  3. 发光和荧光测量
    1. 转染48小时后,使用连接到真空源的巴斯德吸管从培养细胞中除去培养基,并轻轻地向每个板中加入5ml PBS(参见食谱)以冲洗细胞。
    2. 将1ml无酶的细胞解离溶液分配到每个平板中,并在37℃下在组织培养箱中振荡孵育5分钟以释放细胞。
    3. 将细胞转移到15ml锥形管中,并在室温下以175gxg离心细胞5分钟。
    4. 在约1ml pH7.4的Kreb's-Ringer-HEPES培养基(参见食谱)中重悬细胞沉淀,以达到0.20×10 6个细胞/ ml的密度。保持这个管在室温下准备测定。
    5. 将100μl细胞悬浮液(大约20,000个细胞)分配到白色96孔OptiPlate中,加入5μl腔肠素h(参见食谱),通过旋转板混合细胞,并且使用测量法测量发光信号和YFP荧光信号使用BRET模块的Envision Plate Reader。



    6. 应当在添加腔肠素溶液之后立即收集发光和荧光信号。荧光分光光度计仪器应设置为选择特定的供体和受体对。在这里,对于所提出的BRET研究,应使用专用滤光片组(460nm,具有25nm的带宽)收集发光发射,并应使用专用滤光片组(535nm,带宽25nm)同时收集荧光发射。测量时间是0.2秒,测量高度是6.5mm。 (Harikumar et al。,2007)。在该实例中,通过在475nm(带宽25nm)激发样品测量YFP荧光,并且在535nm(带宽25nm)收集发射,检测器增益150和闪光次数为20次。
    7. 数据收集和分析的代表性例子如下:


      计算的BRET比率:1.23 - 0.70 = 0.53

数据分析

应至少在三个独立的实验中收集数据至少重复的条件。数据的统计分析可以用GraphPad Prism 6完成,使用Mann-Whitney后期检验进行检验。

笔记

控制供体和受体的表达水平对于消除旁观者的能量转移是重要的。在生理上存在的表达水平应该观察到显着的BRET信号。另外,BRET信号的饱和性和特异性是重要的,并且可以使用竞争性的未标记的感兴趣的蛋白质来建立。对于阳性对照,使用编码含有萤光素酶和黄色荧光蛋白(Rlu-YFP)的融合蛋白的构建体转染细胞,并测量BRET信号。

食谱

  1. 10x磷酸盐缓冲液(10x PBS缓冲液)(1 L)
    11.5克Na 2 HPO 4 4 2克KH 2 PO 4 4克/克 80克NaCl
    2克KCl
    溶于1升无菌去离子水中 调整1x PBS的pH值至7.4
    在4°C储存
  2. 10x Kreb's Ringer- HEPES储备液(10x KRH)(1L)
    59.58克HEPES
    60.74克NaCl
    3.72克KCl
    1.36克KH 2 PO 4 4 2.96g MgSO 4 将组分溶解在去离子水中,使体积达到1L,不要调节pH值 在4°C储存
  3. 50x CaCl 2储备溶液(100mM)(100ml)
    1.47克CaCl 2 2 /
    用去离子水定容至100 ml;不要调整pH值
    在4°C储存
  4. 1x KRH工作解决方案(100毫升)
    10毫升10倍KRH储备液
    2ml 50x CaCl 2储备液
    用去离子水定容至100 ml;用NaOH调节pH值至7.4
  5. Coelenterazine-h溶液
    0.5毫克coelenterazine-h粉

    溶于12.5毫升5%甲醇/ 95%水中 在-20°C储存
  6. 1x DMEM组织培养基(1L)
    1. 向1L去离子水中加入1L Dulbecco改良Eagle培养基(13.4g)和3.7g碳酸氢钠。
    2. 用HCl调节pH至6.90,使用含有聚醚砜(PES)膜的0.2μm快速流动瓶顶过滤器单元过滤到500ml螺旋盖高压灭菌的玻璃瓶中。
    3. 在4°C储存

致谢

这项工作由Mayo Clinic(LJM),Van Andel研究所,国家自然科学基金(31300607,31300245和91217311),科技部2012ZX09301001,2012CB910403和2013CB910600,XDB08020303,2013ZX09507001,上海科学技术委员会(13ZR1447600),上海新星计划(14QA1404300)和国家卫生研究院拨款DK071662(HEX),GM102545和GM104212(KM)。作者没有利益冲突或利益冲突。

参考

  1. Dacres,H.,Wang,J.,Dumancic,M.M。和Trowell(2010)。 两种常用生物发光共振能量转移对的实验确定Förster距离 < em> Anal Chem 82:432-435。
  2. Harikumar,K.G.,Pinon,D.I。和Miller,L.J。(2007)。 跨膜片段IV为II类G蛋白偶联分泌素受体的寡聚化提供了一个功能重要的界面。 J Biol Chem 282:30363-30372。
  3. Xu,T.H.,Yan,Y.,Kang,Y.,Jiang,Y.,Melcher,K。和Xu,H.E。(2016)。 阿尔茨海默氏病相关突变增加淀粉样蛋白前体蛋白对γ-分泌酶切割的抗性,而Aβ42/Aβ40比率。 Cell Discov 2:16026.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Harikumar, K. G., Yan, Y., Xu, T., Melcher, K., Xu, H. E. and Miller, L. J. (2017). Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells. Bio-protocol 7(22): e2904. DOI: 10.21769/BioProtoc.2904.
  2. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017a). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem.
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