In Vivo Imaging and Biochemical Characterization of Protease Function Using Fluorescent Activity‐Based Probes

Laura E. Edgington1, Matthew Bogyo2

1 Department of Pathology, Stanford University School of Medicine, Stanford, California, 2 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120235
Online Posting Date:  March, 2013
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Abstract

Activity‐based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this article, detailed protocols are provided for using fluorescent ABPs to biochemically characterize the activity of proteases in vitro. Furthermore, descriptions are provided of how these probes can be applied to image protease activity in live animals and tissues along with subsequent analysis by histology, flow cytometry, and SDS‐PAGE. Curr. Protoc. Chem. Biol. 5:25‐44 © 2013 by John Wiley & Sons, Inc.

Keywords: proteases; activity‐based probes; imaging; caspases

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Labeling Cell Lysates with ABPs
  • Support Protocol 1: Fluorescent SDS‐PAGE Analysis
  • Basic Protocol 2: Labeling Intact Cells with ABPs
  • Basic Protocol 3: Immunoprecipitations with ABPs to Confirm Targets
  • Basic Protocol 4: Noninvasive In Vivo Imaging with ABPs
  • Basic Protocol 5: Ex Vivo Imaging with ABPs
  • Basic Protocol 6: Biochemical Analysis of Probe‐Labeled Tissues
  • Basic Protocol 7: Histological Analysis of Probe‐Labeled Tissues
  • Basic Protocol 8: Flow Cytometry of Probe‐Labeled Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Labeling Cell Lysates with ABPs

  Materials
  • Cells (chosen based on model)
  • Medium for chosen cell type
  • Stimulant of caspase activity
  • Cold PBS, pH 7.4 (Invitrogen)
  • Hypotonic lysis buffer or low‐pH buffer for legumain or cathepsins (see reciperecipes)
  • Bicinchoninic acid (BCA) kit or equivalent
  • ABP (100× stock solution in DMSO)
  • Protease inhibitor (optional)
  • 4× sample buffer (see recipe)
  • 6‐well plates
  • Cell scraper or P200 pipet tip

Support Protocol 1: Fluorescent SDS‐PAGE Analysis

  Materials
  • Probe‐labeled protein in sample buffer (from Basic Protocol protocol 11, protocol 32, protocol 43, or protocol 76)
  • Appropriate running buffer (Tris‐glycine works well)
  • Protein ladders (both colored and fluorescent), such as Seeblue Plus2 prestained marker (Invitrogen) and ECL Plex Fluorescent Rainbow Markers (Amersham)
  • SDS‐PAGE gels (15% acrylamide is ideal for caspase separation)
  • Gel running apparatus
  • Typhoon flatbed laser scanner or comparable device that detects wavelengths appropriate for the probe's fluorophore
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and western blotting (Gallagher, )

Basic Protocol 2: Labeling Intact Cells with ABPs

  Materials
  • Cells (chosen based on model)
  • Medium for chosen cell type
  • Stimulant of caspase activity
  • Protease inhibitor (optional)
  • ABP (1000× stock solution in DMSO)
  • Cold PBS, pH 7.4
  • Hypotonic lysis buffer (see recipe)
  • BCA kit or equivalent
  • 4× sample buffer
  • 6‐well dishes
  • Cell scraper or P200 pipet tip

Basic Protocol 3: Immunoprecipitations with ABPs to Confirm Targets

  Materials
  • Probe‐labeled lysate (Basic Protocol protocol 11 or protocol 32)
  • IP buffer (see recipe)
  • Specific antibody towards protease of interest
  • Acetone
  • 1× and 2× sample buffer (see recipe)
  • 0.9% sodium chloride
  • Protein A/G agarose beads
  • Nutator
  • 2‐ml microcentrifuge tubes
  • Insulin syringe
  • Additional reagents and equipment for SDS‐PAGE ( protocol 2; Gallagher, )

Basic Protocol 4: Noninvasive In Vivo Imaging with ABPs

  Materials
  • Mice (strain chosen based on model); for this representative protocol, use nude mice, 6 weeks old
  • Nonfluorescent (alfalfa‐free) chow [Purified diet (alfalfa‐free), Harlan Laboratories] (optional)
  • COLO205 human colorectal tumor cells (ATCC)
  • Cold PBS, pH 7.4
  • Anti‐DR5 monoclonal antibody (Genentech)
  • Anti‐DR5 vehicle (see recipe)
  • Activity‐based probe: for this representative protocol, use LE22 (available upon request from M. Bogyo)
  • DMSO
  • Isoflurane
  • Insulin syringes (used for all injections)
  • Beard trimmer (if non‐nude mice are used)
  • Nair hair removal lotion (if non‐nude mice are used)
  • Mouse restrainer for tail vein injections
  • Anesthesia vaporizer
  • Fluorescent mouse imager such as the IVIS or FMT (both from Perkin Elmer)

Basic Protocol 5: Ex Vivo Imaging with ABPs

  Materials
  • Labeled mouse ( protocol 5) and unlabeled control
  • Cold PBS, pH 7.4
  • Dissection tools
  • Petri dishes large enough to hold tissue samples
  • Fluorescence imager (IVIS or FMT)
  • Black paper (optional)

Basic Protocol 6: Biochemical Analysis of Probe‐Labeled Tissues

  Materials
  • Probe‐labeled tissues with weights recorded ( protocol 6)
  • Muscle lysis buffer (see recipe)
  • 4× sample buffer (see recipe)
  • Low‐pH buffer for legumain or cathepsins (optional; see recipe)
  • 15‐ml conical tubes (optional)
  • Sonicator with tip for microcentrifuge tubes or other homogenizer (e.g., glass douncer, BeadBeater)
  • Additional reagents and equipment for protein quantitation ( protocol 1, step 9), probe labeling ( protocol 1, steps 10 to 13), and SDS‐PAGE ( protocol 2; Gallagher, )

Basic Protocol 7: Histological Analysis of Probe‐Labeled Tissues

  Materials
  • Probe‐labeled tissues and unlabeled control tissues (freshly prepared from protocol 6)
  • 4% paraformaldehyde in PBS
  • 30% sucrose in PBS
  • Tissue‐Tek OCT Compound
  • PBS, pH 7.4
  • Tissue molds

Basic Protocol 8: Flow Cytometry of Probe‐Labeled Tissues

  Materials
  • Probe‐labeled tissues and unlabeled control tissues (freshly prepared from protocol 6)
  • Digestion buffer (see recipe)
  • ACK lysing buffer (Invitrogen)
  • FACS buffer (see recipe)
  • Razor blades
  • 40‐µm nylon mesh filters
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Figures

Videos

Literature Cited

Literature Cited
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