TimeSTAMP Tagging of Newly Synthesized Proteins

Michael Z. Lin1, Roger Y. Tsien2

1 Department of Pediatrics and Programs in Gene Therapy and Molecular Imaging, Stanford University, Stanford, California, 2 Department of Pharmacology and Howard Hughes Medical Institute, University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 26.5
DOI:  10.1002/0471140864.ps2605s59
Online Posting Date:  February, 2010
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Abstract

The ability to quantify or visualize newly synthesized proteins has important uses in cell biology. For example, a researcher may wish to quantify basal or inducible rates of translation of a specific gene of interest, or detect subcellular locations of newly synthesized copies of a protein in order to study the role of new protein synthesis in the growth of specialized cellular structures. In this unit, the TimeSTAMP method for labeling of newly synthesized copies of a protein of interest is described. In the TimeSTAMP method, the experimenter expresses a protein of interest as a fusion with a cis‐acting protease and an epitope tag, both of which are removed by default protease activity. Addition of a specific protease inhibitor then allows preservation of the tag on subsequently synthesized proteins. Finally, the tag is detected by immunological methods. Curr. Protoc. Protein Sci. 59:26.5.1‐26.5.11. © 2010 by John Wiley & Sons, Inc.

Keywords: protein synthesis; protease; conditional labeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Testing and Use of TimeStamp Tags by Immunoblotting
  • Support Protocol 1: Immunocytochemical Detection of Newly Synthesized Proteins by Drug Control of TimeStamp Tags
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Testing and Use of TimeStamp Tags by Immunoblotting

  Materials
  • Expression plasmid for the protein of interest
  • TimeSTAMP cassette DNA (from the authors: Michael Z. Lin, , http://linlab.stanford.edu; Roger Y. Tsien, , http://tsienlab.ucsd.edu)
  • Cell type of interest
  • Cell culture media
  • Transfection reagent
  • BILN‐2061 (Boehringer Ingelheim), 10 to 30 mM in DMSO
  • 2× SDS lysis buffer
  • 2‐mercaptoethanol
  • PBS or HBSS ( appendix 2E)
  • Ice
  • Sonicator with microtip or 25 U/µl benzonase nuclease (Novagen)
  • Mouse anti‐T7 antibody (Novagen)
  • Rat 2F2 (Roche) or mouse 12CA5 (Roche) or mouse HA‐7 (Sigma) or chicken anti‐HA (ICL) antibody
  • Secondary antibodies for detection by chemiluminescence or fluorescence
  • Chemiluminescence substrate (if performing chemiluminescence)
  • 12‐well plates
  • Appropriate incubators
  • Chemical safety flow hood
  • 1.5‐ml microcentrifuge tubes
  • Heating block
  • Microcentrifuge
  • Autoradiography film (for chemiluminescence) or immunoblot imaging system (for chemiluminescence and fluorescence)
  • Additional reagents and equipment for cloning (Ausubel et al., ), SDS‐PAGE (unit 10.1), immunoblotting (units 10.7& 10.10), and PCR ( 4.NaN)

Support Protocol 1: Immunocytochemical Detection of Newly Synthesized Proteins by Drug Control of TimeStamp Tags

  Materials
  • Cell type of interest
  • Cell culture medium
  • TimeSTAMP fusion construct (see protocol 1)
  • Transfection reagent
  • BILN‐2061 (Boehringer Ingelheim), 10 to 30 mM in DMSO
  • 8% paraformaldehyde in PBS, pH 7.2 to 7.5 (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Blocking solution (see recipe)
  • Rat 2F2 anti‐HA (Roche)
  • Mouse anti‐T7 antibody (Novagen)
  • Rabbit or chicken antibody to protein of interest (if available)
  • Alexa Fluor 488‐conjugated goat anti‐mouse IgG, highly cross‐absorbed to rat serum proteins (Invitrogen)
  • Alexa Fluor 568‐conjugated goat anti‐rat IgG, highly cross‐absorbed to mouse serum proteins (Invitrogen)
  • Alexa Fluor 647‐conjugated goat anti‐rabbit or anti‐chicken IgG (Invitrogen)
  • PBS with 0.1% Triton X‐100
  • Vectashield mounting solution (Vector Laboratories)
  • 12‐mm coverslips and 24‐well plate, or 35‐mm glass‐bottom tissue culture dishes (Mattek)
  • Fluorescence microscope with filters for fluorescein, rhodamine/Texas Red, and Cy5 wavelengths
NOTE: Donkey secondary antibodies can be used instead of goat secondary antibodies if the experimenter desires to use a goat primary antibody to the protein of interest.
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Figures

Videos

Literature Cited

   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., (eds). 2009. Current Protocols in Molecular Biology. John Wiley & Sons. New York.
   Bartenschlager, R., Lohmann, V., Wilkinson, T., and Koch, J.O. 1995. Complex formation between the NS3 serine‐type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. J. Virol. 69:7519‐7528.
   Dieterich, D.C., Lee, J.J., Link, A.J., Graumann, J., Tirrell, D.A., and Schuman, E.M. 2007. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non‐canonical amino‐acid tagging. Nat. Protoc. 2:532‐540.
   Flores, M.V., Strawbridge, J., Ciaramella, G., and Corbau, R. 2009. HCV‐NS3 inhibitors: Determination of their kinetic parameters and mechanism. Biochim. Biophys. Acta. 1794:1441‐1448.
   Ingallinella, P., Altamura, S., Bianchi, E., Taliani, M., Ingenito, R., Cortese, R., De Francesco, R., Steinkuhler, C., and Pessi, A. 1998. Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products. Biochemistry 37:8906‐8914.
   Ingallinella, P., Bianchi, E., Ingenito, R., Koch, U., Steinkuhler, C., Altamura, S., and Pessi, A. 2000. Optimization of the P′‐region of peptide inhibitors of hepatitis C virus NS3/4A protease. Biochemistry 39:12898‐12906.
   Kourtis, N., and Tavernarakis, N. 2009. Cell‐specific monitoring of protein synthesis in vivo. PLoS One 4:e4547.
   Landro, J.A., Raybuck, S.A., Luong, Y.P., O'Malley, E.T., Harbeson, S.L., Morgenstern, K.A., Rao, G., and Livingston, D.J. 1997. Mechanistic role of an NS4A peptide cofactor with the truncated NS3 protease of hepatitis C virus: Elucidation of the NS4A stimulatory effect via kinetic analysis and inhibitor mapping. Biochemistry 36:9340‐9348.
   Lin, M.Z., Glenn, J.S., and Tsien, R.Y. 2008. A drug‐controllable tag for visualizing newly synthesized proteins in cells and whole animals. Proc. Natl. Acad. Sci. U.S.A. 105:7744‐7749.
   Mili, S., and Macara, I.G. 2009. RNA localization and polarity: from A(PC) to Z(BP). Trends Cell Biol. 19:156‐164.
   Rock, K.L., York, I.A., and Goldberg, A.L. 2004. Post‐proteasomal antigen processing for major histocompatibility complex class I presentation. Nat. Immunol. 5:670‐677.
   Tong, X., Chase, R., Skelton, A., Chen, T., Wright‐Minogue, J., and Malcolm, B.A. 2006. Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034. Antiviral Res. 70:28‐38.
Key References
   Lin et al., 2008. See above.
  Describes design and use of TimeSTAMP to track newly synthesized proteins of interest by immunoblotting and immunocytochemistry.
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