Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

Dani L. Bodor1, Mariluz Gómez Rodríguez1, Nuno Moreno1, Lars E.T. Jansen1

1 Instituto Gulbenkian de Ciência, Oeiras, Portugal
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.8
DOI:  10.1002/0471143030.cb0808s55
Online Posting Date:  June, 2012
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Abstract

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP‐tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse‐chase and quench‐chase‐pulse experiments. These time‐slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP‐tagging in fixed‐ and live‐cell studies and evaluate the recently developed fast‐acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification. Curr. Protoc. Cell Biol. 55:8.8.1‐8.8.34. © 2012 by John Wiley & Sons, Inc.

Keywords: SNAP; pulse‐chase; quench‐chase‐pulse; centromeres; automated quantification; protein dynamics; protein turnover; quantitative fluorescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Pulse‐Chase Experiments
  • Basic Protocol 2: Quench‐Chase‐Pulse Experiments
  • Basic Protocol 3: Integrating Cell Synchronization and RNAi in Snap‐Based Quench‐Chase‐Pulse Labeling Experiments
  • Alternate Protocol 1: Integrating Cell Synchronization and RNAi in Snap‐Based Pulse‐Chase Labeling Experiments
  • Basic Protocol 4: Live Imaging of Pulse‐Labeled Cells
  • Alternate Protocol 2: Live Imaging of Pulse‐Labeled Cells: 8‐Well Coverglass Method
  • Basic Protocol 5: Automated Quantification of Snap‐Tagged Protein Turnover at Centromeres
  • Support Protocol 1: Expression of Snap‐Fusion Proteins
  • Support Protocol 2: Cell Fixation and Immunofluorescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pulse‐Chase Experiments

  Materials
  • Cells expressing SNAP‐tagged fusion protein (see protocol 8)
  • Standard culture medium (CM; see recipe).
  • TMR‐Star (see recipe).
  • Sterile DMSO
  • Sterile 1× PBS (cell‐culture grade, Invitrogen)
  • Clean, sterile, poly‐lysine coated ( appendix 2A) coverslips (12 mm diameter; 1.5‐mm thickness)
  • 24‐well plates
  • Additional reagents and equipment for basic cell culture techniques including trypsinization of cells (unit 1.1)

Basic Protocol 2: Quench‐Chase‐Pulse Experiments

  Materials
  • Bromothenylpteridine (BTP; see recipe)
  • Additional reagents and equipment for setting up the pulse‐chase experiment ( protocol 1)

Basic Protocol 3: Integrating Cell Synchronization and RNAi in Snap‐Based Quench‐Chase‐Pulse Labeling Experiments

  Materials
  • Transfection reagents for siRNAs: e.g., Oligofectamine (Invitrogen), and associated products
  • siRNAs (Dharmacon)
  • 50 mM thymidine stock in H 2O
  • 24 mM deoxycytidine in H 2O
  • 5 mg/ml nocodazole stock in DMSO
  • 10 mM MG132 stock in DMSO
  • Additional reagents and equipment for pulse‐chase and quench‐pulse‐chase experiments (Basic Protocols protocol 11 and protocol 22)

Alternate Protocol 1: Integrating Cell Synchronization and RNAi in Snap‐Based Pulse‐Chase Labeling Experiments

  Materials
  • Cells expressing SNAP‐tagged fusion protein (see protocol 8)
  • Live imaging medium (LM; see recipe)
  • VALAP (see recipe)
  • 30 U/ml Oxyrase stock (Oxyrase Inc.; http://www.oxyrase.com/)
  • 6‐well plates
  • Clean, sterile, 22 × 22‐mm poly‐lysine coated ( appendix 2A) coverslips (1.5‐mm thickness)
  • Permanent double‐sided tape (Scotch)
  • Glass microscope slides
  • Additional reagents and equipment for pulse‐chase ( protocol 1) and quench‐pulse‐chase ( protocol 2) experiments

Basic Protocol 4: Live Imaging of Pulse‐Labeled Cells

  Materials
  • Cells expressing SNAP‐tagged fusion protein (see protocol 8)
  • Live imaging medium (LM; see recipe)
  • Mineral oil
  • 8‐well chambered coverglass (Lab‐Tek)
  • Additional reagents and equipment for pulse‐chase ( protocol 1) and quench‐pulse‐chase ( protocol 2) experiments

Alternate Protocol 2: Live Imaging of Pulse‐Labeled Cells: 8‐Well Coverglass Method

  Materials
  • A standard computer
  • ImageJ software (freely available from NIH, http://rsbweb.nih.gov/ij/index.html)
  • CRaQ plugin for ImageJ (go to Supplemental Files at http://www.currentprotocols.com/protocol/cb0808, or download from http://uic.igc.gulbenkian.pt/micro‐macros.htm).
  • Digital images of SNAP‐labeled cells, as described in protocol 1 or 2 after fixation and antibody staining as described in protocol 9

Basic Protocol 5: Automated Quantification of Snap‐Tagged Protein Turnover at Centromeres

  Materials
  • HEK 293‐GP cells (Burns et al., ).
  • Standard culture medium (CM; see recipe)
  • pBABE‐Puro plasmid (Addgene, plasmid 1764), expressing SNAP‐tagged protein
  • pVSV‐G plasmid (sold as part of Clontech kit, cat. no. 631530)
  • Lipofectamine LTX (Invitrogen) and associated products
  • 8 mg/ml Polybrene (hexadimethrine bromide; Sigma)
  • Selection drugs (e.g., Blasticidin S, puromycin, or hygromycin; see annotation to step 12, below)
  • Sterile PBS (cell culture grade; e.g., Invitrogen) containing 5% (w/v) bovine serum albumin (BSA)
  • Conditioned medium (see recipe)
  • 10‐cm standard cell culture dishes
  • 10‐ml syringes
  • 0.45‐µm syringe filters
  • 6‐well plates
  • 96‐well plates
  • Additional reagents and equipment for basic cell culture techniques including trypsinization (unit 1.1), and flow cytometry (Robinson et al., )

Support Protocol 1: Expression of Snap‐Fusion Proteins

  Materials
  • 4% paraformaldehyde (PFA; Thermo Scientific) in PBS, 37°C
  • 0.1 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • PBS‐TX: 1× PBS containing 0.1% (v/v) Triton X‐100
  • IF blocking buffer (see recipe)
  • Primary antibodies
  • Secondary fluorescently labeled antibodies (highly cross‐absorbed to minimize cross reactivity between antibodies; Jackson ImmunoResearch)
  • 1 mg/ml DAPI stock solution (see recipe)
  • MOWIOL (see recipe)
  • Clear nail polish
  • Clean, sterile, poly‐lysine coated ( appendix 2A) coverslips (12 mm diameter; 1.5 mm thickness)
  • Fine forceps with sharp pointed ends
  • Humid dark box: can be made from an empty micropipet tip‐box, with a small layer of water and a thick sponge on the bottom—place a smooth glass plate, covered with a sheet of parafilm, on top of the sponge—use fresh parafilm for each experiment; any transparent surface of the box is covered with aluminum foil
  • Standard glass microscope slides
  • Additional reagents and equipment for pulse‐chase ( protocol 1), quench‐chase‐pulse ( protocol 2), and integrating cell synchronization and RNAi in SNAP‐based quench‐chase pulse labeling experiments ( protocol 3)
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Figures

Videos

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Key References
   Jansen et al., 2007. See above.
  First description of use of SNAP‐tag for determination of protein turnover and assembly using pulse‐chase and quench‐chase‐pulse experiments.
   Keppler et al., 2003. See above.
  First description of use of SNAP‐tag in living cells for fluorescent labeling of proteins.
Internet Resources
   http://rsbweb.nih.gov/ij/index.html
  ImageJ software and Grouped_ZProjector plugin.
   http://uic.igc.gulbenkian.pt/micro‐macros.htm
  CRaQ macro for ImageJ.
   http://rsb.info.nih.gov/ij/plugins/shading‐corrector.html
  Shading corrector plugin for ImageJ.
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Supplementary Material

Sequence maps of SNAP- and SNAPf-tags:  sequence_maps.zip

CRaQ: Centromere Recognition and Quantification macro for ImageJ: CRaQ_v1.12.zip