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Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization
采用链霉亲和素珠Pulldown实验测定蛋白质同源寡聚   

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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

Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads.

Keywords: Pulldown assay (Pulldown 实验), Protein-protein interactions (蛋白质-蛋白质相互作用), Streptavidin bead (链霉亲和素珠), Biotinylated protein (生物素蛋白), Homooligomeric complex (同源寡聚复合物)

Background

The amyloid precursor protein (APP) can form a homodimer through its large extracellular domain as well as its transmembrane domain, which plays an important role in biological function. The current protocol has been used in characterizing homo-dimerization of the APP transmembrane C-terminal 99 amino acid fragment (C99) (Yan et al., 2017). The basic principle of this assay is shown in Figure 1: The streptavidin-coated MagBeads can trap the biotinylated protein, which can pull down the interaction protein and detected by anti-FLAG antibody.


Figure 1. The principle of MagBeads-based pull-down assay. In this particular protocol, biotinylated Avi-tagged C99 proteins and associated C99-TEV site-rTA-Flag protein were used.

Materials and Reagents

  1. Pipette tips (VWR)
  2. 6-well plate (Corning, Costar®, catalog number: 3506 )
  3. 1.5 ml Eppendorf tube
  4. 96-well plate (Corning, catalog number: 3595 )
  5. Cell culture flask (Corning, catalog number: 430639 )
  6. 96-well OptiPlate (PerkinElmer, catalog number: 6005290 )
  7. Gel loading pipette tips
  8. InvitrolonTM PVDF/Filter Paper Sandwich (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC2005 )
  9. PS1/PS2-deleted HTL cells (PS1/PS2 gene were deleted by CRISPR/Cas9 from HTL cells [Xu et al., 2016])
  10. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  11. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  12. Trypsin 0.25%-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  13. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  14. Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  15. Streptavidin MagBeads (GenScript, catalog number: L00424 )
  16. CelLyticTM M (Sigma-Aldrich, catalog number: C2978 )
  17. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 11836153001 )
  18. 2x SDS loading buffer (Bio-Rad Laboratories, catalog number: 1610737 )
  19. β-Mercaptoethanol (Bio-Rad Laboratories, catalog number: 1610710 )
  20. BoltTM 4-12% Bis-Tris Plus Gels, 15-well (Thermo Fisher Scientific, catalog number: NW04125BOX )
  21. NovexTM Tris-Glycine Transfer Buffer (25x) (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC3675 )
  22. Precision plus Protein prestained standard (Bio-Rad Laboratories, catalog number: 1610374 )
  23. Methanol (EMD Millipore, catalog number: MX0485-5 )
  24. Seppro® stripping buffer (Sigma-Aldrich, catalog number: S4324 )
  25. Biotin (Sigma-Aldrich, catalog number: B4501 )
  26. Sodium hydroxide (NaOH) solution (Fisher Scientific, catalog number: SS255 )
  27. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-1 )
  28. Potassium phosphate monobasic (KH2PO4) (EMD Millipore, catalog number: PX1565-1 )
  29. Sodium chloride (NaCl) (EMD Millipore, catalog number: SX0420-5 )
  30. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-500 )
  31. Tris base (Fisher Scientific, catalog number: BP152-5 )
  32. Glycine (Fisher Scientific, catalog number: BP381-5 )
  33. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  34. Hydrochloric acid (HCl) (EMD Millipore, catalog number: HX0603-75 )
  35. Tween 20 (EMD Millipore, catalog number: 9480-OP )
  36. Milk powder (Bio-Rad Laboratories, catalog number: 1706404 )
  37. Anti-FLAG-peroxidase antibody (Sigma-Aldrich, catalog number: A8592 )
  38. Anti-beta actin antibody (Abcam, catalog number: ab6276 )
  39. Anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, catalog number: 7076S )
  40. SuperSignal West Pico substrate (Thermo Fisher Scientific, catalog number: 34080 )
    Note: This product has been discontinued.
  41. Highly pure water (ddH2O) (produced by PURELAB Ultra system)
  42. Biotin solution (see Recipes)
  43. Phosphate buffered saline (PBS buffer) 10x (see Recipes)
  44. Electrophoresis buffer (see Recipes)
  45. Tris-buffered saline (TBS buffer) 10x (see Recipes)
  46. TBST buffer (see Recipes)
  47. Blocking solution (see Recipes)
  48. Anti-FLAG antibody solution (see Recipes)
  49. Anti-beta actin primary antibody solution (see Recipes)
  50. Anti-mouse IgG secondary antibody solution (see Recipes)
  51. Substrate solution (see Recipes)

Equipment

  1. Pipettes
  2. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 Water-Jacketed)
  3. Cell culture microscope (Nikon Instruments, model: Eclipse TS100 )
  4. Biosafety cabinet (The Baker, model: SterilGARD® e3 )
  5. Standard tabletop centrifuge (Eppendorf, model: 5417 R )
  6. Invitrogen SDS-PAGE running cassette (Thermo Fisher Scientific, catalog number: A25977 )
  7. Tank blot device (Bio-Rad Laboratories, model: Mini Trans-Blot® Module, catalog number: 1658029FC )
  8. ChemiDocTM XRS+ Imager System (Bio-Rad Laboratories, model: ChemiDocTM XRS+ )
  9. Shaker (Fisher Scientific, model: Chemistry Mixer 346 )
  10. Rocking platform (ARMA LAB, model: Orbital Shaker 100 )
  11. Magnetic separation rack (Thermo Fisher Scientific, catalog number: 12321D )

Software

  1. Image Lab 5.2.1 software (http://www.bio-rad.com/en-ch/product/image-lab-software)

Procedure

  1. Cell culture
    1. The PS1/PS2-deleted HTL cells are routinely grown in DMEM supplemented with 10% (v/v) fetal bovine serum at 37 °C under a humidified 5% CO2 atmosphere.
    2. Treat the cells with 0.25% trypsin-EDTA at 37 °C for about 2 min. Dilute the cells to 0.4 x 106/ml with DMEM medium (supplemented with 10% [v/v] fetal bovine serum) and split at 0.8 x 106/well into a 6-well plate one day prior to transfection.

  2. Transfection
    1. Prepare a master mix for one sample:
      To a sterile 1.5 ml Eppendorf tube, add:
      50 µl Opti-MEM medium
      2.6 µl Lipofectamine 2000 Transfection Reagent.
      Notes:
      1. Avoid touching the side of the tube while adding reagent.
      2. Make a Master Mix for larger sample size: for example, for 10 samples, multiply by 11 to prepare some extra mix (50 x 11 = 550 µl medium; 2.6 x 11 = 28.6 µl Lipofectamine 2000 Transfection Reagent).
    2. Add 1.3 µg total DNA to 50 µl Opti-MEM medium per well in a 96-well plate.
      Note: 1.3 µg total DNA contains 300 ng BirA biotin ligase encoding DNA, 500 ng Avi-C99 encoding DNA, and 500 ng C99-TEV site-rTA-Flag encoding DNA.
    3. Add 50 µl master mix to DNA mix per well of the 96-well plate and mix gently by pipetting.
    4. Incubate at room temperature for about 30 min.
    5. Transfer 100 µl mix into each well of cells cultured in a 6-well plate.
      Note: Carefully add the 100 µl mix drop by drop with a pipette.
    6. Add biotin solution (see Recipes) to a final concentration of 40 µM just after the transfection using a standard 0.1-2.5 µl pipettor.
      Note: We usually culture 2 ml cells in a 6-well plate, so just add 2 µl biotin solution. Make sure you add the small amount of biotin.
    7. Culture the cells in DMEM supplemented with 10% (v/v) fetal bovine serum at 37 °C under a humidified 5% CO2 atmosphere.

  3. Prewash the MagBeads
    1. Completely resuspend the beads by shaking.
    2. Add 20 µl 25% slurry of MagBeads (Beads with their storage buffer) into each sample in a new 1.5 ml tube.
      Note: Prepare a total volume of MagBeads for all samples in one tube.
    3. Place the tube on a magnetic separation rack to collect the beads. Remove and discard the supernatant by pipetting.
      Note: Use gel loading pipette tips to avoid accidental pipetting of beads.
    4. Add 0.1 ml/sample PBS buffer (see Recipes) to the tube and invert the tube several times to mix. Use the magnetic separation rack to collect the beads and discard the supernatant by pipetting with loading tips from the very bottom. Repeat this step twice

  4. Cell lysis and protein purification
    1. One day after transfection, remove the medium from the cultured cells by vacuum pump using loading pipette tips and gently rinse with a sufficient volume of PBS (i.e., 500 µl/well).
    2. Dispense 150 µl CelLyticTM M lysis buffer supplemented with 1x protease inhibitor cocktail in each well.
    3. Lyse the cells on ice for about 30 min.
    4. Transfer the lysate to 1.5 ml Eppendorf tubes with a pipette and spin at full speed (about 20,000 x g) in a microcentrifuge for 10 min at 4 °C to remove insoluble material.
    5. Take 10 µl supernatant and mix with 10 µl SDS loading buffer supplemented with 100 mM β-mercaptoethanol as input.
    6. The crude supernatant protein extracts are incubated with prewashed Streptavidin MagBeads for about 30 min at 4 °C on a shaker.
    7. Use the magnetic separation rack to collect the beads and discard the supernatant.
      Note: If necessary, keep the supernatant for analysis.
    8. Add 0.5 ml CelLyticTM M lysis buffer to the tube and gently vortex to mix. Use the magnetic separation rack to collect the beads and discard the supernatant. Repeat this step twice.
    9. Elute the sample with 20 µl SDS loading buffer containing 100 mM β-mercaptoethanol at 54 °C for 10 min.

  5. SDS-PAGE and Western blot analysis
    1. Place a premade 4-20% SDS PAGE gel in Invitrogen SDS-PAGE cassette.
    2. Load 10 µl prestained protein standard and 25 µl samples with beads into the wells of the gel and run the gel in electrophoresis buffer (see Recipes) at 200 V for 1 h.
    3. Soak the PVDF membrane with methanol and transfer to transfer buffer.
    4. Soak the sponge and filter paper in transfer buffer.
    5. Prepare the blot sandwich in the following order: anode (-, black), sponge, filter paper, SDS gel, PVDF membrane, filter paper, sponge, anode (+, white).
    6. Perform protein transfer in a tank blot device at constant 90 V and 4 °C for about 90 min.
    7. Disassemble the blot-sandwich and immediately transfer membrane to 1x TBST (see Recipes) for a brief rinse.
      Note: Check the protein standard to see the transfer efficiency.
    8. Incubate the membrane with 20 ml blocking solution (see Recipes) at room temperature for 1 h on a shaker.
    9. Wash the membrane 3 x with 10 ml TBST at room temperature, 10 min each time.
    10. Incubate with 10 ml/membrane anti-Flag antibody solution (see Recipes) for 1 h at room temperature or overnight at 4 °C.
    11. Wash the membrane 3 x with 10 ml TBST at room temperature, 10 min each time.
    12. Incubate the membrane with enough freshly prepared substrate solution (2 ml for each membrane, see Recipes) at room temperature.
    13. Transfer the membrane between 2 layers of transparent sheets of plastic wrap and apply to Bio-Rad Imager system using the blots-chemi application from 1 sec to 120 sec to get 20 pictures.
    14. Strip the primary antibody with stripping buffer at room temperature for about 15 min.
    15. Repeat the Western blot starting from the blocking step, using anti-beta Actin primary antibody solution (see Recipes) for 1 h.
    16. After 3 x washing with 10 ml TBST (at room temperature, 10 min each time), incubate with 10 ml/membrane anti-mouse IgG secondary antibody solution (see Recipes) for about 45 min.
    17. After 3 x washing with 10 ml TBST (at room temperature, 10 min each time), record images using Bio-Rad Imager system.
      Note: Image Lab 5.2.1 software setting for Western blot: choose Blots Chemi application; signal accumulation with the first image starts 1 sec, the last image ends 120 sec, total number of images is 20; select Position Gel and use ‘+’ or ‘-’ button to adjust the image area; run protocol to get images. Choose the best one for data analysis

Data analysis

Anti-FLAG Western blot analysis reveals the presence of C99-TEV site-FLAG in the input lysate and in the Streptavidin Bead pulldown material (Figure 2), suggesting C99 homooligomerization. A practical example for Streptavidin Bead Pulldown Assay was previously shown in Yan et al., 2017.


Figure 2. Pull down assay validation of C99 oligomerization. Biotinylated Avi-tagged C99 proteins and associated C99(WT)-TEV site-rTA-Flag were recovered on Streptavidin MagBeads (GenScript) and eluted with SDS sample buffer, while the dimer interface mutants (T43P/V45P: ctrl1 and I45P/V46P: ctrl2) (Yan et al., 2017) were not. Co-purified C99-TEV site-rTA-Flag proteins were detected by anti-FLAG immunoblotting. β-actin input levels serve as loading controls.

Recipes

  1. Biotin solution
    Add 0.486 g biotin powder to 50 ml ddH2O and slowly add 10 N sodium hydroxide solution drop by drop until biotin is dissolved to make 40 mM biotin solution
  2. Phosphate buffered saline (PBS buffer) 10x (1 L)
    11.5 g Na2HPO4
    2 g KH2PO4
    80 g NaCl
    2 g KCl
    Dissolve in 1 L of ddH2O
    The pH of 1x PBS should be 7.4
  3. Electrophoresis buffer (1 L)
    3.03 g Tris
    14.4 g glycine
    1 g SDS
    Add ddH2O to 1 L
  4. Tris-buffered saline (TBS buffer) 10x (1 L)
    24.228 g Tris-base
    87.66 g NaCl
    Dissolve in 1 L of sterile, deionized water and adjust pH to 7.4 with HCl
  5. TBST buffer
    Add 1 ml Tween 20 to 1 L TBS buffer
  6. Blocking solution
    1x TBST supplemented with 5% (w/v) milk powder
  7. Anti-FLAG antibody solution
    15 ml TBST with 3 μl primary anti-FLAG-peroxidase antibody
  8. Anti-beta Actin primary antibody solution
    15 ml TBST with 7.5 μl primary anti-beta Actin antibody
  9. Anti-mouse IgG secondary antibody solution
    15 ml TBST with 15 μl anti-mouse IgG, HRP-linked antibody
  10. Substrate solution
    1 volume of SuperSignal West Pico A and 1 volume of SuperSignal West Pico B, mix well

Acknowledgments

This work was supported by 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.). The authors declare no conflict of interest.

References

  1. 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.
  2. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem.

简介

Pulldown分析是一种常规的方法来确定蛋白质在体外的相互作用。 用两种不同的标签表达感兴趣的蛋白质可以检测两种标签是否可以通过两种标签之一作为同低聚体复合物来捕获。 该方案基于使用链霉抗生物素蛋白MagBeads的链霉抗生物素蛋白珠捕获生物素化蛋白质和共结合Flag标记蛋白质。
【背景】淀粉样前体蛋白(APP)可以通过其大的胞外结构域及其跨膜结构域形成同型二聚体,在生物学功能中起重要作用。 目前的方案已被用于表征APP跨膜C-末端99个氨基酸片段(C99)的同二聚化(Yan等人,2017)。 该检测的基本原理如图1所示:链霉亲和素包被的MagBeads可以捕获生物素化的蛋白质,这可以拉下相互作用蛋白质,并通过抗FLAG抗体检测。

“”src
图1.基于MagBeads的pull-down测定的原理在该特定的方案中,使用生物素化的Avi-标记的C99蛋白和相关的C99-TEV位点-rTA-Flag蛋白质。

关键字:Pulldown 实验, 蛋白质-蛋白质相互作用, 链霉亲和素珠, 生物素蛋白, 同源寡聚复合物

材料和试剂

  1. 移液枪头(VWR)
  2. 6孔板(Corning,Costar ®,产品目录号:3506)
  3. 1.5 ml Eppendorf管
  4. 96孔板(康宁,目录号:3595)
  5. 细胞培养瓶(康宁,目录号:430639)
  6. 96孔OptiPlate(PerkinElmer,目录号:6005290)
  7. 凝胶加样移液枪头
  8. Invitrol TM TM PVDF /滤纸三明治(Thermo Fisher Scientific,Invitrogen TM,目录号:LC2005)
  9. 来自HTL细胞的CRISPR / Cas9缺失了PS1 / PS2缺陷的HTL细胞(PS1 / PS2基因)[Xu等,2016] )
  10. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,产品目录号:11965092)
  11. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  12. 胰蛋白酶0.25%-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  13. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:31985062)
  14. Lipofectamine 2000转染试剂(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  15. 链霉亲和素MagBeads(金斯瑞,目录号:L00424)
  16. CelLytic TM M(Sigma-Aldrich,目录号:C2978)
  17. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11836153001)
  18. 2x SDS上样缓冲液(Bio-Rad Laboratories,目录号:1610737)
  19. β-巯基乙醇(Bio-Rad Laboratories,目录号:1610710)
  20. Bolt TM 4-12%Bis-Tris Plus凝胶,15孔(Thermo Fisher Scientific,目录号:NW04125BOX)
  21. Novex TM Tris-甘氨酸转移缓冲液(25x)(Thermo Fisher Scientific,Invitrogen TM,目录号:LC3675)
  22. 精密加蛋白质预染标准(Bio-Rad Laboratories,目录号:1610374)
  23. 甲醇(EMD Millipore,目录号:MX0485-5)
  24. Seppro®剥离缓冲液(Sigma-Aldrich,目录号:S4324)
  25. 生物素(Sigma-Aldrich,目录号:B4501)
  26. 氢氧化钠(NaOH)溶液(Fisher Scientific,目录号:SS255)
  27. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S374-1)
  28. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:PX1565-1)
  29. 氯化钠(NaCl)(EMD Millipore,目录号:SX0420-5)
  30. 氯化钾(KCl)(Fisher Scientific,目录号:BP366-500)
  31. Tris碱(Fisher Scientific,目录号:BP152-5)
  32. 甘氨酸(Fisher Scientific,目录号:BP381-5)
  33. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  34. 盐酸(HCl)(EMD Millipore,目录号:HX0603-75)
  35. 吐温20(EMD Millipore,目录号:9480-OP)
  36. 奶粉(Bio-Rad Laboratories,目录号:1706404)
  37. 抗FLAG-过氧化物酶抗体(Sigma-Aldrich,目录号:A8592)
  38. 抗β肌动蛋白抗体(Abcam,目录号:ab6276)
  39. 抗小鼠IgG,HRP连接的抗体(Cell Signaling Technology,目录号:7076S)
  40. SuperSignal West Pico底物(Thermo Fisher Scientific,目录号:34080)
    注:此产品已停产。
  41. 高纯水(ddH2O)(由PURELAB Ultra系统生产)
  42. 生物素溶液(见食谱)
  43. 磷酸盐缓冲盐水(PBS缓冲液)10倍(见食谱)
  44. 电泳缓冲液(见食谱)
  45. Tris缓冲盐水(TBS缓冲液)10倍(见食谱)
  46. TBST缓冲液(见食谱)
  47. 阻止解决方案(见食谱)
  48. 抗FLAG抗体溶液(见食谱)
  49. 抗β肌动蛋白初级抗体溶液(见食谱)
  50. 抗小鼠IgG二抗溶液(见食谱)
  51. 底物溶液(见食谱)

设备

  1. 移液器
  2. 37℃,5%CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Series 3110水套)
  3. 细胞培养显微镜(尼康仪器,型号:Eclipse TS100)
  4. 生物安全柜(贝克,型号:SterilGARD e3)
  5. 标准台式离心机(Eppendorf,型号:5417 R)
  6. 英杰公司SDS-PAGE电泳盒(赛默飞世尔科技,产品目录号:A25977)
  7. Tank-blot装置(Bio-Rad Laboratories,型号:Mini Trans-Blot Module,目录号:1658029FC)
  8. ChemiDoc TM XRS +成像仪系统(Bio-Rad Laboratories,型号:ChemiDoc TM TM XRS +)
  9. 振荡器(Fisher Scientific,型号:化学混合器346)
  10. 摇摆平台(ARMA LAB,型号:轨道摇床100)
  11. 磁分离架(Thermo Fisher Scientific,目录号:12321D)

软件

  1. Image Lab 5.2.1软件( http://www.bio- rad.com/en-ch/product/image-lab-software

程序

  1. 细胞培养
    1. 在添加有10%(v / v)胎牛血清的DMEM中,在37℃,潮湿的5%CO 2下,常规地使PS1 / PS2-去除的HTL细胞生长,大气。
    2. 用0.25%胰蛋白酶-EDTA在37℃处理细胞约2分钟。用DMEM培养基(补充有10%[v / v]胎牛血清)将细胞稀释至0.4×10 6 / ml,并以0.8×10 6 /孔在转染前一天进入6孔板。

  2. 转染
    1. 准备一个样本的主混合:
      向无菌1.5 ml Eppendorf管中加入:
      50μlOpti-MEM培养基
      2.6μlLipofectamine 2000转染试剂。
      注意:
      1. 避免在添加试剂时触摸试管的一侧。
      2. 制备更大样品量的主混合物:例如,对于10个样品,乘以11以制备一些额外的混合物(50×11 =550μl培养基; 2.6×11 =28.6μlLipofectamine 2000转染试剂) em>
    2. 在96孔板中,每孔加入1.3μl总DNA到50μlOpti-MEM培养基中。
      注意:1.3μg总DNA含有300ng BirA生物素连接酶编码DNA,500ng Avi-C99编码DNA和500ng C99-TEV位点-rTA-Flag编码DNA。

    3. 在96孔板的每个孔中加入50μl主混合物到DNA混合物中,轻轻混匀

    4. 在室温下孵育约30分钟

    5. 转移100μl混合物到6孔板中培养的细胞的每个孔中 注意:用移液器小心地加入100μl的混合物。
    6. 转染后,使用标准的0.1-2.5μl移液器加入生物素溶液(参见配方)至终浓度为40μM。
      注意:我们通常在6孔板中培养2ml细胞,所以只需要加入2μl生物素溶液。确保你添加了少量的生物素。
    7. 将培养细胞在补充有10%(v / v)胎牛血清的DMEM中在37℃,5%CO 2的潮湿空气中培养。

  3. 预先清理MagBeads

    1. 完全重新悬浮珠子

    2. 在一个新的1.5毫升试管中加入20μlMagBeads(带有储存缓冲液的珠子)的25%浆液到每个样品中。
      注意:为一个试管中的所有样品准备一个总体积的MagBeads。
    3. 将试管放在磁力分离架上收集珠子。
      移除并丢弃上清液 注意:使用凝胶加样移液枪头,以避免意外移液珠。
    4. 向管中加入0.1ml /样品PBS缓冲液(参见食谱)并倒转管几次以混合。使用磁性分离架收集珠子,并从最底部用加样尖吸取上清液。重复这个步骤两次

  4. 细胞裂解和蛋白质纯化
    1. 转染一天后,使用加样枪头通过真空泵从培养细胞中除去培养基,并用足够体积的PBS(即,500μl/孔)轻轻冲洗。
    2. 在每个孔中分配150μlCelLytic TM裂解缓冲液,补充有1x蛋白酶抑制剂混合物。

    3. 在冰上溶解细胞约30分钟
    4. 用移液管将裂解物转移到1.5ml Eppendorf管中,并在4℃下在微型离心机中全速旋转(约20,000xg)10分钟以除去不溶物质。
    5. 取10μl上清液,加入10μlSDS上样缓冲液,并加入100 mMβ-巯基乙醇作为输入。
    6. 将粗上清蛋白提取物与预洗过的链霉抗生物素蛋白MagBeads一起在摇床上在4℃温育约30分钟。
    7. 使用磁力分离架收集珠子并丢弃上清液。
      注意:如有必要,保留上清液进行分析。
    8. 添加0.5毫升CelLytic TM M裂解缓冲液的管,轻轻涡旋混合。使用磁力分离架收集珠子并丢弃上清液。重复这个步骤两次。
    9. 用含有100mMβ-巯基乙醇的20μlSDS上样缓冲液在54℃洗脱样品10分钟。

  5. SDS-PAGE和Western印迹分析
    1. 将预制的4-20%SDS PAGE凝胶置于Invitrogen SDS-PAGE盒中。
    2. 将10μl预染蛋白质标准物和25μl含珠的样品加入到凝胶的孔中,并在200V的电泳缓冲液(参见配方)中运行凝胶1小时。
    3. 用甲醇浸泡PVDF膜,然后转移到转移缓冲液中。
    4. 浸泡海绵,并在转印缓冲液中过滤纸张。
    5. 按以下顺序准备印迹三明治:阳极( - ,黑色),海绵,滤纸,SDS凝胶,PVDF膜,滤纸,海绵,阳极(+,白色)。

    6. 在恒定的90 V和4°C条件下,在槽式印迹装置中进行蛋白质转移约90分钟
    7. 拆开印迹三明治,立即转移膜到1x TBST(见食谱)进行短暂的冲洗。
      注意:检查蛋白质标准以查看转移效率。

    8. 在室温下用摇动器将20毫升阻断溶液(参见食谱)孵育1小时

    9. 在室温下用10ml TBST洗膜3次,每次10分钟
    10. 孵育10毫升/膜抗Flag抗体溶液(见食谱)在室温1小时或过夜在4°C。

    11. 在室温下用10ml TBST洗膜3次,每次10分钟
    12. 在室温下用足够新鲜制备的底物溶液(每个膜2ml,参见食谱)孵育膜。
    13. 在2层透明塑料薄膜之间转移薄膜,并使用1秒至120秒的印迹 - 化学法应用于Bio-Rad成像仪系统,以获得20张照片。

    14. 在室温下用剥离缓冲液剥离第一抗体约15分钟
    15. 使用抗β肌动蛋白初级抗体溶液(见食谱)重复从阻断步骤开始的Western印迹1小时。
    16. 用10ml TBST(室温,每次10分钟)洗涤3次后,与10ml /膜抗小鼠IgG二抗溶液(参见食谱)一起温育约45分钟。
    17. 用10ml TBST(室温,每次10分钟)洗3次后,用Bio-Rad Imager系统记录图像。
      注意:Image Lab 5.2.1 Western blot软件设置:选择Blots Chemi应用程序;第一个图像的信号积累开始1秒,最后一个图像结束120秒,图像总数为20;选择位置凝胶,使用“+”或“ - ”按钮调整图像区域;运行协议来获取图像。选择最适合数据分析的

数据分析

抗FLAG蛋白质印迹分析显示输入裂解物中和链霉抗生物素蛋白珠下拉物质中存在C99-TEV位点-FLAG(图2),表明C99均低聚。链霉亲和素珠子下拉测定的一个实际例子以前在Yan等人的<2017>中显示。


图2.下拉测定C99寡聚化验证。在链霉亲和素MagBeads(GenScript)上回收生物素化的Avi-标记的C99蛋白和相关的C99(WT)-TEV位点-rTA-Flag,并用SDS样品缓冲液洗脱,而二聚体界面突变体(T43P / V45P:ctrl1和I45P / V46P:ctrl2)(Yan等人,2017)不是。通过抗FLAG免疫印迹检测共纯化的C99-TEV位点-rTA-Flag蛋白质。 β-肌动蛋白输入水平作为加载对照。

食谱

  1. 生物素溶液
    将0.486克生物素粉末加入到50毫升ddH2O中,并逐滴缓慢加入10N氢氧化钠溶液,直至生物素溶解,制成40毫克生物素溶液。
  2. 磷酸盐缓冲盐水(PBS缓冲液)10倍(1升)
    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ddH 2 O
    1x PBS的pH应该是7.4
  3. 电泳缓冲液(1 L)
    3.03克Tris
    14.4克甘氨酸
    1克SDS
    将ddH <2> O添加到1L
  4. Tris缓冲盐水(TBS缓冲液)10×(1 L)

    24.228克Tris-碱 87.66克NaCl
    溶于1L无菌去离子水中,用HCl调pH至7.4
  5. TBST缓冲区

    加入1 ml吐温20至1 L TBS缓冲液
  6. 阻止解决方案
    1x TBST补充有5%(w / v)奶粉
  7. 抗FLAG抗体溶液
    15毫升TBST与3微升主要抗FLAG-过氧化物酶抗体
  8. 抗β肌动蛋白一抗溶液
    15毫升的TBST与7.5微升的初级抗β肌动蛋白抗体
  9. 抗鼠IgG二抗溶液
    15 ml TBST与15μl抗鼠IgG,HRP连接的抗体
  10. 底物溶液
    1卷SuperSignal West Pico A和1卷SuperSignal West Pico B混合使用

致谢

国家自然科学基金面上项目(31300607,31300245和91217311),科技部2012ZX09301001,2012CB910403,2013CB910600,XDB08020303,2013ZX09507001,上海市科委(13ZR1447600),国家自然科学基金),上海新星计划(14QA1404300),国家卫生研究院赠款DK071662(HEX),GM102545和GM104212(KM)。作者宣称没有利益冲突。

参考

  1. 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.
  2. Yan,Y.,Xu,T.H.,Harikumar,K.G.,Miller,L.J.,Melcher,K.and Xu,H.E。(2017)。 淀粉样蛋白前体蛋白的跨膜结构域的二聚化由γ-分泌酶切割位点周围的残基决定。 J Biol Chem 。
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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. Xu, T., Yan, Y., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization. Bio-protocol 7(22): e2901. DOI: 10.21769/BioProtoc.2901.
  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, 292: 15826-15837.
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