Immunoprecipitation

Juan S. Bonifacino1, David C. Gershlick1, Esteban C. Dell'Angelica1

1 National Institute of Child Health and Human Development, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.2
DOI:  10.1002/cpcb.3
Online Posting Date:  June, 2016
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Abstract

Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein‐protein interactions. Clear step‐by‐step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody. © 2016 by John Wiley & Sons, Inc.

Keywords: immunoprecipitation; immunoprecipitation‐recapture; antigen; antibody; agarose bead; magnetic bead

     
 
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Table of Contents

  • Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution
  • Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution
  • Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions
  • Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent
  • Alternate Protocol 4: Immunoprecipitation Using Yeast Cells Disrupted with Glass Beads
  • Alternate Protocol 5: Immunoprecipitation of a Tagged Protein Using Antibody‐Conjugated Magnetic Beads
  • Basic Protocol 2: Immunoprecipitation‐Recapture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunoprecipitation Using Cells in Suspension Lysed with a Nondenaturing Detergent Solution

  Materials
  • Unlabeled or labeled cells in suspension (unit 7.1; Bonifacino, )
  • PBS ( appendix 2A), ice cold
  • Nondenaturing lysis buffer (see recipe), ice cold
  • 50% (v/v) protein A–Sepharose bead (Sigma, Pharmacia Biotech) slurry in PBS containing 0.1% (w/v) BSA and 0.01% (w/v) sodium azide (NaN 3)
  • Specific polyclonal antibody (antiserum or affinity‐purified immunoglobulin) or monoclonal antibody (ascites, culture supernatant, or purified immunoglobulin)
  • Control antibody of same type as specific antibody (e.g., preimmune serum or purified irrelevant immunoglobulin for specific polyclonal antibody; irrelevant ascites, culture supernatant, or purified immunoglobulin for specific monoclonal antibody; see Critical Parameters)
  • 10% (w/v) BSA ( appendix 2A)
  • Wash buffer (see recipe), ice cold
  • Microcentrifuge with fixed‐angle rotor (Eppendorf 5415C or equivalent)
  • Tube rotator (capable of end‐over‐end inversion)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer (also see appendix 1D; Meisenhelder and Semba, ).NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 1: Immunoprecipitation Using Adherent Cells Lysed with a Nondenaturing Detergent Solution

  • Unlabeled or labeled cells grown as a monolayer on a tissue culture plate (unit 7.1; Bonifacino, )
NOTE: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 2: Immunoprecipitation Using Cells Lysed with Detergent Under Denaturing Conditions

  • Denaturing lysis buffer (see recipe)
  • Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent)
  • 25‐G needle attached to 1‐ml syringe

Alternate Protocol 3: Immunoprecipitation Using Cells Lysed Without Detergent

  • Detergent‐free lysis buffer (see recipe)
  • 25‐G needle attached to 3‐ml syringe
NOTE: All solutions should be ice‐cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 4: Immunoprecipitation Using Yeast Cells Disrupted with Glass Beads

  • Unlabeled or radiolabeled yeast cells
  • Lysis buffer, ice cold: nondenaturing, denaturing, or detergent‐free lysis buffer (see reciperecipes)
  • Glass beads (acid‐washed, 425‐ to 600‐µm diameter; Sigma)
NOTE: All solutions should be ice‐cold and procedures should be carried out at 4°C or on ice.

Alternate Protocol 5: Immunoprecipitation of a Tagged Protein Using Antibody‐Conjugated Magnetic Beads

  Materials
  • Cells expressing the tagged protein of interest, as well control cells, plated in 100‐mm‐diameter cell culture dish
  • PBS ( appendix 2A), ice cold
  • Co‐immunoprecipitation lysis buffer (see recipe), ice cold
  • Dilution buffer: wash buffer (see recipe) supplemented with protease inhibitors (e.g., Roche Complete Mini, EDTA‐free, 1 tablet per 10 ml buffer), ice cold
  • Magnetic beads conjugated to antibody against GFP or epitope tag of interest (e.g., GFP‐Trap‐M; Cromotek; usually provided as a slurry with concentrations ranging from ∼3 to 10 mg/ml, depending on the manufacturer)
  • Wash buffer (see recipe), ice cold
  • 1× SDS‐PAGE sample buffer (unit 6.1; Gallagher, )
  • Rubber policeman
  • Round‐bottomed 2‐ml microcentrifuge tubes and 1.5‐ml conical‐bottom microcentrifuge tubes (Eppendorf)
  • Microcentrifuge with fixed‐angle rotor (Eppendorf 5415 C or equivalent)
  • Magnetic separation rack
  • Tube rotator (capable of end‐over‐end inversion)
  • 95°C heat block
NOTE: Before starting the protocol, carefully read the product sheet for the magnetic beads to check for any specific adjustments recommended by the manufacturer.

Basic Protocol 2: Immunoprecipitation‐Recapture

  Materials
  • Elution buffer (see recipe)
  • Beads containing bound antigen (see protocol 1, step 26)
  • 10% (w/v) BSA ( appendix 2A)
  • Nondenaturing lysis buffer (see recipe)
  • Heating block set at 95°C (Eppendorf Thermomixer 5436 or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
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