Summary

Проточной цитометрии бимолекулярного флуоресценции Комплементация: Высокая пропускная способность Количественный метод изучения взаимодействия белок-белок

Published: August 15, 2013
doi:
Please note that all translations are automatically generated. Click here for the English version.

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Disclosures

The authors have nothing to disclose.

Acknowledgements

Materials

<strong>REAGENTS</strong>
Dulbecco’s modified Eagle’s media (DMEM)Invitrogen11965-092
Penicilin-Streptomycin (pen/strep), 100xInvitrogen15070-063
Fetal Bovine Serum (FBS)Invitrogen16000-044
Anti-Flag antibodySigmaM2, F1804
Anti-HA antibodyCovance16B12, MMS-101R
α-tubulinSigmaDM1A, T9026
Anti-GFP antibodyClontech632569
Cell culture plates, 6-well tissue culture treatedThermo Fisher Scientific130184
HEK 293T cellsATCCCRL-11268
Maxiprep plasmid purification kit, high speedQiagen12663
Dulbecco’s Phosphate-buffered saline (DPBS), sterile 1xInvitrogen14190-144
Trypsin-EDTA, 0.05% (w/v)Gibco25300
Polystyrene round-bottom tubes for FACS stainingBD Biosciences352052
ParaformaldehydeFisher ScientificS74337MF
Prolong gold antifade reagent with DAPIInvitrogenP-36931
Superfrost plus Microscope slidesFisher Scientific12-550-15
Fisherfinest premium cover glassFisher Scientific12-548-5P
<strong>EQUIPMENT</strong>
CO<sub>2</sub> air-jacketed IncubatorNuAIR DH autoflow
Confocal microscope LSM510 METACarl Zeiss, Inc
Electrophoresis and transfer unitBiorad
Cyan ADP Flow CytometerBeckman Coulter

References

  1. Collura, V., Boissy, G. From protein-protein complexes to interactomics. Subcell Biochem. 43, 135-183 (2007).
  2. Stumpf, M. P., et al. Estimating the size of the human interactome. Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).
  3. Shekhawat, S. S., Ghosh, I. Split-protein systems: beyond binary protein-protein interactions. Curr. Opin. Chem. Biol. 15, 789-797 (2011).
  4. Dwane, S., Kiely, P. A. Tools used to study how protein complexes are assembled in signaling cascades. Bioeng. Bugs. 2, 247-259 (2011).
  5. Piehler, J. New methodologies for measuring protein interactions in vivo and in vitro. Curr. Opin. Struct. Biol. 15, 4-14 (2005).
  6. Kerppola, T. K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Protoc. 1, 1278-1286 (2006).
  7. Shyu, Y. J., Suarez, C. D., Hu, C. D. Visualization of ternary complexes in living cells by using a BiFC-based FRET assay. Nat. Protoc. 3, 1693-1702 (2008).
  8. Wong, K. A., O’Bryan, J. P. Bimolecular fluorescence complementation. J. Vis. Exp. (50), e2643 (2011).
  9. Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. Flow cytometry: general principles and applications to selected studies in tumor biology. Int. Adv. Surg. Oncol. 2, 125-153 (1979).
  10. Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry. J. Vis. Exp. (45), e2429 (2010).
  11. Smith, C. L. Basic confocal microscopy. Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).
  12. Westwick, J. K., Lamerdin, J. E. Improving drug discovery with contextual assays and cellular systems analysis. Methods Mol. Biol. 756, 61-73 (2011).
  13. Kilpatrick, L. E., Holliday, N. D. Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation. Methods Mol. Biol. 897, 109-138 (2012).
Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Play Video

Cite This Article
Wang, L., Carnegie, G. K. Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction. J. Vis. Exp. (78), e50529, doi:10.3791/50529 (2013).

View Video