The Application of KillerRed for Acute Protein Inactivation in Living Cells

Timothy S. Jarvela1, Adam D. Linstedt1

1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.35
DOI:  10.1002/0471142956.cy1235s69
Online Posting Date:  July, 2014
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Generating loss of protein function is a powerful investigatory tool particularly if carried out on a physiologically relevant timescale in a live‐cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live‐cell imaging of cultured cells. Curr. Protoc. Cytom. 69:12.35.1‐12.35.10. © 2014 by John Wiley & Sons, Inc.

Keywords: KillerRed; CALI; acute inactivation; protein inactivation; ROS

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Table of Contents

  • Introduction
  • Strategic planning and Controls for KillerRed Experiments
  • Basic Protocol 1: Inactivation of KillerRed Using Epifluorescent Light
  • Alternate Protocol 1: Inactivation of KillerRed Constructs Using a Scanning Laser Capable of Photobleaching a Region of Interest
  • Support Protocol 1: Construct Generation and Cell Transfection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Inactivation of KillerRed Using Epifluorescent Light

  • Cells expressing KillerRed and reporter constructs (see the protocol 3Support Protocol)
  • Inverted confocal microscope
  • High numerical aperture objective
  • Lasers for desired fluorophores—561 nm or 535 nm for KillerRed imaging
  • Epifluorescent light source (e.g., Mercury arc lamp, high‐powered LEDs)
  • Excitation filter for KillerRed—between 540 to 580 nm (e.g., Cy3, TRITC, or TexasRed filter sets)
  • Stage incubator with gas control by injection of CO 2 or flow of 95% air, 5% CO 2 mixture through the incubator (see Critical Parameters)
  • Gas tank (100% CO 2 or 95% air/5% CO 2 depending on setup)

Alternate Protocol 1: Inactivation of KillerRed Constructs Using a Scanning Laser Capable of Photobleaching a Region of Interest

  Additional Materials (also see protocol 1Basic Protocol)
  • Steerable focused laser system [e.g., FRAPPA (Andor), Direct FRAP (Zeiss), or cellˆfrap (Olympus)]

Support Protocol 1: Construct Generation and Cell Transfection

  • Cell line of interest (e.g., HeLa cells)
  • Cell culture medium (e.g., MEM with 10% fetal bovine serum) for cell line used
  • Expression vector containing KillerRed fused in frame with the coding sequence of the protein of interest (see Commentary for designing fusion proteins)
  • Vector containing a relevant reporter construct (e.g., encoding GFP‐tagged Golgi protein for Golgi experiments]
  • Imaging medium (see recipe)
  • Additional reagents for tissue culture maintenance
  • Transfection reagent [e.g., JetPRIME (PolyPlus), Oligofectamine, LipofectAMINE2000 (Invitrogen)]
  • Additional reagents for knockdown of endogenous protein (if desired)
  • 60‐mm tissue culture dishes
  • Imaging chamber [metal chamber with 12‐mm glass coverslips or glass‐bottom dishes (MaTek)]
  • 35‐mm tissue culture dish lid
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Literature Cited

Literature Cited
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