搜索

Protocol for Notch-ligand Binding Assays Using Dynabeads
使用免疫磁珠进行Notch配体结合分析的实验方案   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
Apr 2017

Abstract

This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated beads, but not JAG1-coated beads. The Notch-ligand assay using Dynabeads suggested that Eogt facilitates DLL4-Notch1 interaction (Sawaguchi et al., 2017).

Keywords: Notch (Notch), Ligand binding assay (配体结合试验), Dynabeads (磁珠), Dll4-Fc (DLL4-Fc)

Background

The Notch signal pathway regulates many types of cellular events such as proliferation, cell fate determination, and cellular differentiation in all metazoans (Mumm and Kopan, 2000). To initiate Notch signaling, extracellular domains of Notch receptors engage their ligands, Delta like (DLL) ligands or Jagged (JAG) ligands, presented on opposing cells.

Epidermal growth factor (EGF)-like domains of Notch receptors are critical for the ligand binding and modified by specific glycans including O-fucose, O-glucose, and O-GlcNAc glycans (Stanley and Okajima, 2010). Some of these glycans serve as regulators of Notch signaling pathway by modulating physical interaction between Notch receptors and ligands (Moloney et al., 2000). To investigate whether O-GlcNAc regulates Notch-ligand interaction, we developed a novel Dynabeads-based Notch-ligand binding assay. In this assay, Notch receptors expressed on HEK293T cells are incubated with Dynabeads Protein A coated with DLL4-Fc or JAG1-Fc. Unlike soluble ligands used for other binding assay, direction of Notch ligands is fixed on the beads so that they behave like ligand-expressing cells. Thus, the detected binding represents trans-binding rather than cis-binding, which occurs when Notch receptors and their ligands are expressed in the same cells. This assay demonstrated that O-GlcNAc modification of Notch1 by Eogt potentiates Notch1 binding to DLL4 without affecting JAG1 binding (Sawaguchi et al., 2017).

Materials and Reagents

  1. Microtube (INA•OPTIKA, Bio-Bik, catalog number: ST-0150F )
  2. Multiwell culture plates 6 wells (Greiner Bio One International, catalog number: 657160 )
  3. 15 ml conical centrifuge tubes (Greiner Bio One International, catalog number: 188271 )
  4. Syringe filter with a 0.22 µm pore size membrane (Pall, catalog number: 4192 )
  5. HEK293T cell
  6. Notch1 expressing vector (pTracer-CMV/Notch1) (Sawaguchi et al., 2017)
    Note: NOT commercially available.
  7. Eogt expressing vector (pSecTag2/Hygro/Eogt) (Sawaguchi et al., 2017)
    Note: NOT commercially available.
  8. GFP expressing vector (pMAX-GFP) (Addgene, catalog number: VDF-1012 )
  9. Dynabeads Protein A (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10002D )
  10. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  11. 4% paraformaldehyde (Wako Pure Chemical Industries, catalog number: 163-20145 )
  12. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: 172012-500ML )
  13. Sodium phosphate dibasic (Na2HPO4) (Wako Pure Chemical Industries, catalog number: 196-02835 )
  14. Potassium phosphate monobasic (KH2PO4) (Wako Pure Chemical Industries, catalog number: 164-04295 )
  15. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 197-01667 )
  16. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
  17. DLL4-Fc (Thermo Fisher Scientific, catalog number: 10171H02H )
  18. JAG1-Fc (Thermo Fisher Scientific, catalog number: 11648H02H )
  19. Dulbecco’s modified Eagle medium (NISSUI PHARMACEUTICAL, catalog number: 05915 )
  20. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  21. Polyethylenimine, linear, MW 25,000 (PEI 25000) (Polysciences, catalog number: 23966 )
  22. Fetal bovine serum (FBS) (see Recipes)
  23. Phosphate-buffered saline (PBS) (see Recipes)
  24. 100 ng/µl DLL4-Fc/PBS and 100 ng/µl JAG1-Fc/PBS (see Recipes)
  25. Complete culture media (see Recipes)
  26. PEI solution (see Recipes)

Equipment

  1. Pipettes (various sizes) (GILSON)
  2. 6-Tube magnetic separation rack (New England Biolabs, catalog number: S1506S )
  3. Tube rotator (Taiyo, model: RT-50 ) (Figure 1A)
  4. CO2 incubator (SANYO, catalog number: MCO-175 )
  5. Orbital rotator (Oriental Instruments, catalog number: KS-6300 ) (Figure 1B)
  6. Self-made aspirator (Figures 1C and 1D)
  7. Box-type fluorescence imaging device (Olympus, catalog number: FSX100 )


    Figure 1. Equipment. A. Tube rotator RT50; B. Orbital rotator; C and D. Self-made aspirators.

Procedure

  1. Preparation of ligand-coated Dynabeads
    1. 20 µl of Dynabeads Protein A is washed with 1 ml of PBS (see Recipes) in a microtube.
      Note: The standard procedure can be scaled up for multiple samples.
    2. Beads are collected using Magnetic Separation Rack.
    3. The solution is removed from the beads.
    4. The beads are resuspended in 100 µl of PBS.
    5. 2 µl of 100 ng/µl DLL4-Fc/PBS (see Recipes) or JAG1-Fc/PBS (see Recipes) is added to the tube
    6. The beads are incubated for 20 min at room temperature or overnight at 4 °C using a tube rotator (Video 1).

      Video 1. Dynabeads Protein A was incubated at room temperature using a tube rotator. Note that Dynabeads have brown color in the video.

    7. Beads are collected using a Magnetic Separation Rack.
    8. The solution is removed from the beads.
    9. Beads are washed 3 times with 1 ml of PBS.
    10. The solution is removed from the beads.
    11. 100 µl of PBS is added to the tube.
    12. Just before use, 900 µl of ice-cold complete culture media is added to the tube.

  2. Preparation of Notch-expressing cells
    1. HEK293T cells are seeded in a 6-well plate at 6 x 105 cells per well in complete culture media (see Recipes).
    2. Next day, the culture media are replaced with 800 µl of Opti-MEM.
    3. Cells are incubated for 30 min at 37 °C in a CO2 incubator.
    4. Plasmid DNA (2 µg) is diluted into 200 µl of Opti-MEM in a microtube.
      Note: To identify transfected cells, GFP expression vector (e.g., pMAX-GFP) is included at 1/8 amount of total plasmids.
    5. The tube is vortexed gently.
    6. 6 µl of 1 mg/ml PEI solution (see Recipes) is added to the DNA solution.
    7. The mixture is incubated for 30 min at room temperature.
    8. The DNA/PEI mixtures are gently dropped onto each well of the plate.
    9. Cells are incubated for 4 h at 37 °C in a CO2 incubator.
    10. The culture media are replaced with 2 ml of complete culture media.
    11. Cells are incubated for 48 h at 37 °C in a CO2 incubator.

  3. Binding assay (Figure 2)


    Figure 2. Binding assay. A. Complete culture media in a 15 ml tube; B. Dynabeads resuspended in complete culture media; C. HEK293T cells in a 6-well plate were incubated with ligand-coupled beads in a cold room. D. The culture plate during fixation with paraformaldehyde; E. The culture plate before removing PBS; F. PBS was removed from the culture plate using self-made aspirator. G. The culture plate after removing PBS; H. The culture plate after washing three times with PBS; I. The culture plate after washing five times with PBS.

    1. The culture media are replaced with 1 ml of ice-cold complete culture media containing DLL4-Fc or JAG1-Fc beads (Figure 2C).
    2. The cells are incubated in cold room for 30 min.
    3. The culture media are removed from the culture dish using aspirator.
    4. Cells are fixed with 4% paraformaldehyde for 20 min at room temperature (Figure 2D).
    5. The fixed cells are gently washed with 5 ml of PBS for 5 min using an orbital rotator (Video 2).

      Video 2. The fixed cells were washed with PBS for using an orbital rotator

    6. PBS is removed from the culture plate using aspirator (Video 3).
      Note: The beads accumulated in the center of each well can be removed using aspirator.

      Video 3. PBS was removed from the culture plate using aspirator

    7. Cells are washed 5 times in total by repeating steps C5 and C6.
    8. 1 ml of PBS is added to each well of the dish (Figure 2I).
    9. Phase contrast and fluorescence images are captured using FSX100 (Figure 3).
    10. The number of bound beads on GFP-positive cell is counted (Figure 4).
      Note: 50 GFP-positive cells were counted for quantification. Floating beads were excluded for counting.


      Figure 3. Counting of Dynabeads-bound cells. A-C. HEK293T cells transfected to express GFP were cultured with control Dynabeads. D-F. HEK293T cells transfected to express GFP, Notch1 were incubated with DLL4-Fc-bound beads. G-I. HEK293T cells transfected to express GFP, Eogt, and Notch1 were incubated with DLL4-Fc-bound beads. A, D and G. Phase contrast images; B, E and H. Fluorescent Images; C, F and I. Merged images. Scale bars = 60 µm. C’, F’ and I’. Higher magnification of boxed area in images (C, F, I). Scale bars = 60 µm. C”, F” and I”. Same as C’, F’, and I’. Red dots represent bound beads on GFP-positive cells. The dotted line shows outline of the cells. Scale bars = 60 µm.


      Figure 4. Analysis of counting of Dynabeads-bound cells. HEK293T cells or cells transiently transfected with Notch1 with or without Eogt, were incubated with DLL4 or JAG1 beads. The number of Dynabeads bound per transfected cell marked by GFP expression was determined (n = 50). Data are mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; bar by Welch’s t-test.

Data analysis

In the previously published experiments (Sawaguchi et al., 2017), data were shown as mean ± SD from three independent experiments. In each experiment, 50 GFP-positive cells are analyzed. Welch’s t-test was used.

Notes

This protocol provides high reproducibility. Counting 50 GFP-positive cells gives low variability in most experiments.

Recipes

  1. Fetal bovine serum (FBS)
    The bottle containing FBS is incubated at 56 °C for 30 min before use
  2. Phosphate-buffered saline (PBS)
    10 mM Na2HPO4
    1.8 mM KH2PO4
    137 mM NaCl
    2.7 mM KCl
  3. 100 ng/µl DLL4-Fc/PBS and 100 ng/µl JAG1-Fc/PBS
    DLL4-Fc or JAG1-Fc (5 µg) is dissolved in 50 µl of PBS
    Note: 100 ng/µl DLL4-Fc/PBS and 100 ng/µl JAG1-Fc/PBS can be stored at 4 °C for 3 months.
  4. Complete culture media
    DMEM containing 10% FBS and penicillin-streptomycin
  5. PEI solution
    1. 100 mg of PEI is dissolved in 100 ml of Milli-Q water
    2. The solution is sterilized by passing through a 0.22 µm membrane
    3. Aliquots are stored at -30 °C

Acknowledgments

This protocol was modified from the previously published article (Sawaguchi et al., 2017). This work was supported by Japan Society for the Promotion of Science grants # JP15K15064 to TO and MO, #JP26110709 to TO, #JP26291020 to TO, #JP15K18502 to MO, #JP16J00004 to MO; Takeda Science Foundation to TO; Japan Foundation for Applied Enzymology to TO; YOKOYAMA Foundation for Clinical Pharmacology #YRY-1612 to MO.

References

  1. Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. and Vogt, T. F. (2000). Fringe is a glycosyltransferase that modifies Notch. Nature 406(6794): 369-375.
  2. Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev Biol 228(2): 151-165.
  3. Sawaguchi, S., Varshney, S., Ogawa, M., Sakaidani, Y., Yagi, H., Takeshita, K., Murohara, T., Kato, K., Sundaram, S., Stanley, P. and Okajima, T. (2017). O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife 6.
  4. Stanley, P. and Okajima, T. (2010). Roles of glycosylation in Notch signaling. Curr Top Dev Biol 92: 131-164.

简介

该协议描述了如何使用Dynabeads通过基于细胞的测定来测量Notch受体及其配体之间的相互作用。 我们已经使用方案来确定Notch1转染的HEK293T细胞和配体包被的Dynabeads之间的结合能力。 在Notch1表达细胞中表达Eogt 促进了对DLL4包被的珠粒的结合,而不是JAG1包被的珠粒。 使用Dynabeads的Notch-配体测定表明,Eogt 有助于DLL4-Notch1相互作用(Sawaguchi等人,2017)。
【背景】Notch信号通路调节许多类型的细胞事件,如所有后生动物中的增殖,细胞命运测定和细胞分化(Mumm和Kopan,2000)。为了启动Notch信号传导,Notch受体的细胞外结构域与其配体结合,呈现在相对细胞上的Delta样(DLL)配体或Jagged(JAG)配体)。

Notch受体的表皮生长因子(EGF)样结构域对配体结合至关重要,并由包含O-岩藻糖,O-葡萄糖和O-GlcNAc聚糖的特定聚糖修饰(Stanley和Okajima,2010)。这些聚糖中的一些通过调节Notch受体和配体之间的物理相互作用(Moloney等人,2000)用作Notch信号传导途径的调节剂。为了研究O-GlcNAc是否调节Notch-配体相互作用,我们开发了一种基于Dynabeads的Notch-配体结合测定。在该测定中,在HEK293T细胞上表达的Notch受体与用DLL4-Fc或JAG1-Fc包被的Dynabeads蛋白A孵育。与用于其他结合测定法的可溶性配体不同,Notch配体的方向固定在珠上,使得它们表现得像配体表达细胞。因此,检测到的结合表示反式结合而不是顺式结合,当Notch受体及其配体在相同的细胞中表达时发生。该测定表明,通过Eogt 的Notch1的O-GlcNAc修饰增强Notch1与DLL4的结合,而不影响JAG1结合(Sawaguchi等人,2017)。

关键字:Notch, 配体结合试验, 磁珠, DLL4-Fc

材料和试剂

  1. Microtube(INA•OPTIKA,Bio-Bik,目录号:ST-0150F)
  2. 多孔培养板6孔(Greiner Bio One International,目录号:657160)
  3. 15ml锥形离心管(Greiner Bio One International,目录号:188271)
  4. 具有0.22μm孔径膜的注射器过滤器(Pall,目录号:4192)
  5. HEK293T细胞
  6. Notch1表达载体(pTracer-CMV / Notch1 )(Sawaguchi等人,2017)
    注意:不可商业使用。
  7. 表达载体(pSecTag2 / Hygro / Eogt)的Eogt表达载体(Sawaguchi等人,2017)
    注意:不可商业使用。
  8. GFP表达载体(pMAX-GFP)(Addgene,目录号:VDF-1012)
  9. Dynabeads蛋白A(Thermo Fisher Scientific,Invitrogen TM,目录号:10002D)
  10. Opti-MEM(Thermo Fisher Scientific,Gibco TM ,目录号:31985070)
  11. 4%多聚甲醛(和光纯药,目录号:163-20145)
  12. 胎牛血清(FBS)(Sigma-Aldrich,目录号:172012-500ML)
  13. 磷酸二氢钠(Na 2 HPO 4)(和光纯药,目录号:196-02835)
  14. 磷酸二氢钾(KH 2 PO 4)(和光纯药,目录号:164-04295)
  15. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:197-01667)
  16. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:163-03545)
  17. DLL4-Fc(Thermo Fisher Scientific,目录号:10171H02H)
  18. JAG1-Fc(Thermo Fisher Scientific,目录号:11648H02H)
  19. Dulbecco改良Eagle培养基(NISSUI PHARMACEUTICAL,目录号:05915)
  20. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  21. 聚乙烯亚胺,线性,MW 25,000(PEI 25000)(Polysciences,目录号:23966)
  22. 胎牛血清(FBS)(见食谱)
  23. 磷酸缓冲盐水(PBS)(见食谱)
  24. 100ng /μlDLL4-Fc / PBS和100ng /μlJAG1-Fc / PBS(参见食谱)
  25. 完成文化媒体(见食谱)
  26. PEI解决方案(见配方)

设备

  1. 移液器(各种尺寸)(GILSON)
  2. 6管磁选机架(New England Biolabs,目录号:S1506S)
  3. 管旋转器(Taiyo,型号:RT-50)(图1A)
  4. CO 2培养箱(SANYO,目录号:MCO-175)
  5. 轨道旋转器(东方仪器,目录号:KS-6300)(图1B)
  6. 自制吸气器(图1C和1D)
  7. 盒式荧光成像装置(Olympus,目录号:FSX100)


    图1.设备。 A.管旋转器RT50;轨道旋转器C和D.自制吸气器。

程序

  1. 配体涂层Dynabeads的制备
    1. 将20μlDynabeads蛋白A用1ml PBS(见食谱)在微管中洗涤。
      注意:标准程序可以针对多个样本进行放大。
    2. 使用磁分离架收集珠粒。
    3. 将溶液从珠粒中取出。
    4. 将珠子重新悬浮于100μlPBS中
    5. 将2μl100ng /μl的DLL4-Fc / PBS(参见食谱)或JAG1-Fc / PBS(参见食谱)加入到管中
    6. 珠子在室温下孵育20分钟或在4℃下过夜,使用管旋转器(视频1)。

    7. 使用磁选机架收集珠粒。
    8. 将溶液从珠粒中取出。
    9. 用1ml PBS洗涤珠子3次。
    10. 将溶液从珠粒中取出。
    11. 将100μlPBS加入管中。
    12. 在使用前,将900μl冰冷的完整培养基加入管中
  2. Notch表达细胞的制备
    1. 将HEK293T细胞以6×10 5个细胞/孔接种在6孔板中,在完全培养基中(参见食谱)。
    2. 第二天,用800μl的Opti-MEM代替培养基。
    3. 将细胞在37℃下在CO 2培养箱中孵育30分钟
    4. 将质粒DNA(2μg)在微管中稀释成200μl的Opti-MEM 注意:为了鉴定转染的细胞,将GFP表达载体(例如,pMAX-GFP)包含在1/8总量质粒中。
    5. 管子轻轻旋涡。
    6. 将6μl1mg / ml PEI溶液(参见食谱)加入到DNA溶液中
    7. 混合物在室温下孵育30分钟
    8. 将DNA / PEI混合物轻轻滴在板的每个孔上
    9. 将细胞在37℃下在CO 2培养箱中孵育4小时
    10. 用2ml的完整培养基代替培养基。
    11. 将细胞在37℃下在CO 2培养箱中孵育48小时

  3. 结合测定(图2)


    图2.结合测定。 A.在15ml管中完成培养基;将动物重悬于完全培养基中;将6孔板中的HEK293T细胞与冷室中的配体偶联珠孵育。 D.用多聚甲醛固定培养板; E.去除PBS前的培养板;使用自制抽吸器从培养板中取出PBS。 G.去除PBS后的培养板; H.用PBS洗涤培养板三次; I.用PBS洗涤五次后的培养板。

    1. 用含有DLL4-Fc或JAG1-Fc珠的1ml冰冷的完全培养基代替培养基(图2C)。
    2. 细胞在冷室中孵育30分钟
    3. 使用吸气器将培养基从培养皿中取出
    4. 细胞在室温下用4%多聚甲醛固定20分钟(图2D)
    5. 使用轨道旋转器将固定的细胞用5ml PBS洗涤5分钟(视频2)
    6. 使用吸气器将PBS从培养皿中取出(视频3) 注意:可以使用吸气器将积聚在每个孔中心的珠子移除。

    7. 通过重复步骤C5和C6,将细胞洗涤5次。
    8. 将1ml PBS加入到培养皿的每个孔中(图2I)
    9. 使用FSX100捕获相位对比度和荧光图像(图3)
    10. 计数GFP阳性细胞上结合的珠粒数(图4) 注意:计数50个GFP阳性细胞进行定量。排除浮珠以进行计数。


      图3. Dynabeads绑定单元格的计数。 A-C。转染表达GFP的HEK293T细胞用对照Dynabeads培养。 D-F。将转染表达GFP,Notch1的HEK293T细胞与DLL4-Fc结合的珠孵育。 G-我。将转染以表达GFP,Eogt和Notch1的HEK293T细胞与DLL4-Fc结合的珠孵育。 A,D和G.相位图像; B,E和H.荧光图像; C,F和I.合并图像。刻度棒=60μm。 C',F'和I'。图像中盒装区域的放大倍数(C,F,I)。刻度棒=60μm。 C“,F”和I“。与C',F'和I'相同。红点表示GFP阳性细胞上的结合珠。虚线显示细胞的轮廓。刻度棒=60μm。


      图4. Dynabeads绑定细胞的计数分析。 HEK293T细胞或Notch1瞬时转染有或没有Eogt 的细胞与DLL4或JAG1珠孵育。确定用GFP表达标记的转染细胞结合的Dynabeads数(n = 50)。数据是来自三个独立实验的平均值±SD。 * P &lt; 0.05; ** 0.01; Welch的测试条。

数据分析

在先前发表的实验(Sawaguchi等人,2017)中,数据显示为来自三个独立实验的平均值±SD。在每个实验中,分析了50个GFP阳性细胞。 Welch的 t -test被使用。

笔记

该协议提供高重现性。计数50个GFP阳性细胞在大多数实验中提供了低变异性。

食谱

  1. 胎牛血清(FBS)
    将含有FBS的瓶子在56℃下孵育30分钟,然后使用
  2. 磷酸盐缓冲盐水(PBS)
    10mM Na 2 HPO 4
    1.8mM KH PO 4
    137 mM NaCl
    2.7 mM KCl
  3. 100ng /μlDLL4-Fc / PBS和100ng /μlJAG1-Fc / PBS 将DLL4-Fc或JAG1-Fc(5μg)溶于50μlPBS
    中 注意:100ng /μlDLL4-Fc / PBS和100ng /μlJAG1-Fc / PBS可以在4℃下储存3个月。
  4. 完成文化媒体
    含有10%FBS和青霉素 - 链霉素的DMEM
  5. PEI解决方案
    1. 将100毫升PEI溶于100ml Milli-Q水中
    2. 溶液通过0.22μm的膜进行灭菌
    3. 等分试样储存于-30°C

致谢

该协议已经从之前发表的文章(Sawaguchi等人,2017年)进行了修改。这项工作得到日本社会科学促进会授予#JP15K15064 TO和MO,#JP26110709 to TO,#JP26291020 to TO,#JP15K18502 to MO,#JP16J00004 to MO;武田科学基金会日本应用酶学基金会TO TO; YOKOYAMA临床药理学基金会#YRY-1612至MO。

参考

  1. Moloney,DJ,Panin,VM,Johnston,SH,Chen,J.,Shao,L.,Wilson,R.,Wang,Y.,Stanley,P.,Irvine,KD,Haltiwanger,RS和Vogt,TF(2000 )。 边缘是修改Notch的糖基转移酶。 自然 406 (6794):369-375。
  2. Mumm,J.S。和Kopan,R。(2000)。 Notch信号:来自外部。 Dev Biol 228(2):151-165。
  3. Sawaguchi,S.,Varshney,S.,Ogawa,M.,Sakaidani,Y.,Yagi,H.,Takeshita,K.,Murohara,T.,Kato,K.,Sundaram,S.,Stanley,冈岛,T.(2017)。 NOTCH1上的O-GlcNAc EGF重复调节哺乳动物中配体诱导的Notch信号传导和血管发育。 a> Elife 6.
  4. Stanley,P.和Okajima,T。(2010)。 Notch信号传导中糖基化的作用。 Curr Top Dev Biol 92 : 131-164.
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Sawaguchi et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sawaguchi, S., Ogawa, M. and Okajima, T. (2017). Protocol for Notch-ligand Binding Assays Using Dynabeads. Bio-protocol 7(20): e2582. DOI: 10.21769/BioProtoc.2582.
  2. Sawaguchi, S., Varshney, S., Ogawa, M., Sakaidani, Y., Yagi, H., Takeshita, K., Murohara, T., Kato, K., Sundaram, S., Stanley, P. and Okajima, T. (2017). O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals. Elife 6.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。