Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

VAMP8-3xHA Uptake Assay in HeLa Cells

HeLa 细胞中的VAMP8-3xHA摄取试验

Steve Jean Steve Jean
Amy A. Kiger Amy A. Kiger

发布: 2016年02月20日第6卷第4期 DOI: 10.21769/BioProtoc.1739 浏览次数: 8582

评审: Ralph BottcherVaibhav B ShahAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of EMBO Reports, featuring study using the protocol.

Mar 2015

Advertisement

Submit your protocol to the Special Issue on Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

嘌呤霉素近邻连接检测（Puro-PLA）评估人神经元轴突局部翻译

嘌呤霉素近邻连接检测（Puro-PLA）评估人神经元轴突局部翻译

Raffaella De Pace [...] Saikat Ghosh

2025年03月05日 3110 阅读

小鼠咬肌肌纤维类型分析的共聚焦成像方案优化

小鼠咬肌肌纤维类型分析的共聚焦成像方案优化

Catalina Matias [...] Jeffrey J. Brault

2025年04月05日 2802 阅读

基于Fiji ImageJ的全自动化流程开发：批量分析共聚焦图像数据并量化蛋白共定位的Manders系数

基于Fiji ImageJ的全自动化流程开发：批量分析共聚焦图像数据并量化蛋白共定位的Manders系数

Vikram Aditya [...] Wei Yue

2025年04月05日 2667 阅读

Abstract

Transmembrane proteins are rarely exclusively localized to a specific vesicle or an organelle. Most transmembrane proteins undergo complicated trafficking routes. Thus, transmembrane proteins are under constant flux, and at steady state, found on a variety of vesicles or organelles. This characteristic makes the study of their trafficking routes complex, since at any given moment, different molecules are often being trafficked in opposing directions. Pulse-chase experiments can temporally track a specific pool of a transmembrane protein of interest, allowing for the kinetic description of its trafficking route. This type of technique has been used extensively to follow a large array of plasma membrane localized proteins (Diril et al., 2006; Jean et al., 2010). Here, we describe a method that allows the study of VAMP8 trafficking from the plasma membrane to endolysosomal compartments. This method was used to describe a role for MTMR13 and RAB21 in the regulation of VAMP8 trafficking to endolysosomes (Jean et al., 2015).

Keywords: Membrane trafficking (膜转运) search Endosomal sorting (胞内分选) search Autophagy (自噬) search

Materials and Reagents

  1. Costar® 24 Well Clear TC-Treated Well Plates (Corning, catalog number: 3526 ) 

  2. #1.5 round glass coverslip (Ted Pella, catalog number: 260368 ) 

  3. 100 mm Tissue Culture Dish (Corning, catalog number: 430167 ) 

  4. 0.22 µm filtering unit (Genesee Scientific Corporation, catalog number: 25-227 )
  5. HeLa cells (ATCC, catalog number: CCL-2 ) 

  6. JetPRIME (Polyplus-transfection, catalog number: 114-07 ) 

  7. pCDNA3-VAMP8-3xHA expression plasmid (self-made) (Jean et al., 2015)
  8. Dulbecco’s Modified Eagle Medium with High glucose with 4 mM L-Glutamine and sodium pyruvate (GE Healthcare, HycloneTM, catalog number: SH30243.FS ) (see Recipes for complete DMEM) 

  9. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml ) 

  10. Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  11. Trypsin-EDTA (0.25%) (Life Technologies, catalog number: 25200-056 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  12. 0.4% Trypan-Blue (Life Technologies, catalog number: 15250-061 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  13. Rabbit anti-HA antibody (Abcam, catalog number: ab9110 ) 

  14. Earl’s Balanced Salt Solution (with sodium bicarbonate, without phenol red, liquid, sterile- filtered) (Sigma-Aldrich, catalog number: E3024-500 ml ) 

  15. Bovine Serum Albumin (Fraction V, Heat shock treated) (Thermo Fisher Scientific, catalog number: BP1600-100 ) 

  16. Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S671-3 )

  17. Potassium Chloride (KCl) (VWR International, catalog number: BDH9258-500 g ) 

  18. Sodium Phosphate Dibasic Anhydrous (Na2HPO4) (Thermo Fisher Scientific, catalog number: S374500 )

  19. Potassium Phosphate Monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: P285-S500 ) 

  20. Paraformaldehyde (Thermo Fisher Scientific, catalog number: AC41678-5000 ) 

  21. Goat Serum (Life Technologies, catalog number: 16210-064 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 16210-064”.
  22. Triton X-100 (Sigma-Aldrich, catalog number: X100-500 ml ) 

  23. Mouse anti-human Lamp1 (Developmental Studies Hybridoma Bank, catalog number: H4A3 ) 

  24. Mouse anti-EEA1 (BD Biosciences, catalog number: 610457 ) 

  25. Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Life Technologies, catalog number: A-11029 ) 

    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11029”.
  26. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 546 conjugate (Life Technologies, catalog number: A11035 ) 

    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: A11035”.
  27. [4’, 6-Diamidino-2-Phenylindole, Dihydrochloride] (DAPI) (Life Technologies, catalog number: D1306 ) 

    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306”.
  28. FluorSave reagent (Merck Millipore Corporation, Calbiochem®, catalog number: 345789 ) 

  29. Complete DMEM (see Recipes) 

  30. 1x Phosphate Buffer Saline (PBS) (see Recipes) 

  31. 4% Paraformaldehyde solution (see Recipes) 

  32. Blocking buffer (see Recipes) 

  33. Antibody incubation buffer (see Recipes) 


Equipment

  1. 37 °C, 5% CO2 cell culture incubator [similar to Thermo Scientific Heracell VIOS 160i CO2 incubator (Thermo Fisher Scientific, catalog number: 51030285 )]

  2. Tissue culture biosafety cabinet [similar to Thermo Scientific 1300 Series Class II (Thermo Fisher Scientific, catalog number: 1323)] 

  3. Hemacytometer (VWR International, catalog number: 100498-470 ) 

  4. Pipettes 10 μl, 100 μl and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
  5. Vacuum Flask (self-made)
    Note: Required to remove media from the 24 well plates during the immunofluorescence protocol.
  6. Microcentrifuge (Eppendorf, catalog number: 5415D ) 


Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Jean, S. and Kiger, A. A. (2016). VAMP8-3xHA Uptake Assay in HeLa Cells. Bio-protocol 6(4): e1739. DOI: 10.21769/BioProtoc.1739.
  2. Jean, S., Cox, S., Nassari, S. and Kiger, A. A. (2015). Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion. EMBO Rep 16(3): 297-311.

    3. ris ris Download Citation in RIS Format

分类

细胞生物学 > 基于细胞的分析方法 > 转运

细胞生物学 > 细胞成像 > 共聚焦显微镜

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件