Probing Small‐Molecule Microarrays with Tagged Proteins in Cell Lysates

Marius S. Pop1, Dina Wassaf2, Angela N. Koehler3

1 The Dana‐Farber Cancer Institute, Boston, Massachusetts, 2 Broad Institute of Harvard and MIT, Cambridge, Massachusetts, 3 Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140101
Online Posting Date:  December, 2014
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Abstract

The technique of small‐molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal‐to‐noise ratios (SNRs). SMM screening is a high‐throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding‐based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment. © 2014 by John Wiley & Sons, Inc.

Keywords: small‐molecule microarray; binding assay; cellular lysate; fluorescence; protein complex

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Small‐Molecule Microarray (SMM) Screening
  • Support Protocol 1: Preparation and Quantification of Cell Lysate for Small Molecule Microarray (SMM)
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Small‐Molecule Microarray (SMM) Screening

  Materials
  • Printed small‐molecule microarray (SMM) slides, four replicate arrays (Uttamchandani and Yao, ; Casalena et al., ); store SMM slides at −20°C until ready
  • FKBP12 control protein, available in various tagged formats (e.g., His‐tag, GST‐tag, etc.) available from multiple suppliers (e.g., carrier‐free and His‐tagged, R&D Systems, cat. no. 3777‐FK100)
  • TBST buffer (see recipe)
  • HEK293T lysate containing HA‐tagged target protein (see protocol 2Support Protocol)
  • MIPP lysis buffer (see recipe)
  • Mock lysate (see protocol 2Support Protocol)
  • Anti‐HA antibody, fluorophore‐labeled (e.g., Cy5 or Cy3) (Cell Signaling, cat. no. 3444) secondary antibody.
  • Milli‐Q water or equivalent
  • Rapamycin (LC Labs, cat. no. R‐5000)
  • Alternative or additional controls:
    • Alexa647‐labeled streptavidin control protein (Life Technologies, cat. no. S21374)
    • Biotin‐cadaverine (Life Technologies, cat. no. A‐1594)
  • 4‐well dishes (VWR, cat. no. 7321‐424)
  • Rocking shakers, 4°C and room temperature
  • Laboratory Slide Spinner (Labnet, cat. no. C1303T)
  • GenePix 4200A microarray scanner (Molecular Devices)
  • GenePix Pro software (Molecular Devices)

Support Protocol 1: Preparation and Quantification of Cell Lysate for Small Molecule Microarray (SMM)

  Materials
  • HEK293T cell line (ATCC# CRL‐11268)
  • DMEM cell culture medium supplemented with 10% FBS and 1× penicillin/streptomycin
  • Protein target cloned in a mammalian expression vector (e.g., pcDNA3.1) driven by CMV promoter and harboring the HA tag at either N‐ or C‐ termini
  • OptiMEM medium (Life Technologies, cat. no. 11058‐021)
  • Fugene6 transfection reagent (Promega, cat. no. E2691)
  • PBS buffer, ice cold
  • MIPP lysis buffer (ice‐cold, see recipe) supplemented with protease inhibitors
  • BCA protein quantification reagents (Thermo Scientific, cat. no. 23225)
  • 10 mg/ml bovine serum albumin (BSA) solution
  • HA‐tagged purified protein (any protein similar in size with the desired target)
  • Anti HA antibody (Covance, cat. no. MMS‐101P)
  • Appropriate labeled secondary antibody (i.e., for Odyssey detection or HRP‐based)
  • Cell culture plates (10‐cm diameter)
  • Cell scrapers
  • 1.5‐ml tubes
  • Eppendorf 5810 refrigerated centrifuge
  • 96‐well plate (non‐sterile)
  • Spectrophotometer
  • Densitometry software (ImageQuant, ImageJ, Odyssey)
  • Additional reagents and equipment for western blot analysis (i.e., SDS protein gel, protein markers, buffers, transfer membrane, gel running chamber, power supply, gel transfer apparatus, etc.)
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Figures

Videos

Literature Cited

Literature Cited
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Internet Resources
  http://chembank.broadinstitute.org/
  A valuable resource is the Chembank database. It is a repository of existing SMM data sets performed for various types of targets and executed in either lysate format or with purified proteins. It can be especially beneficial in identifying and eliminating SMM hits with a promiscuous binding spectrum.
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