Protein Blotting: Immunoblotting

Duojiao Ni1, Peng Xu2, Diviya Sabanayagam1, Sean R. Gallagher1

1 UVP, LLC, Upland, California, 2 Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 8.3
DOI:  10.1002/9780470089941.et0803s12
Online Posting Date:  May, 2016
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Abstract

Immunoblotting (also referred to as western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by the polyacrylamide gel and, to make the separated proteins accessible, the proteins need to be moved out of the gel and bound to a rectangular sheet of PVDF or nitrocellulose membrane. Specialized blotting equipment electrophoretically transfers the negatively charged proteins from the gel onto the membrane. The nitrocellulose or PVDF membrane binds the proteins as they move out of the gel, producing an exact replica, on the membrane surface, of the original protein gel separation. The membrane is then blocked to prevent any nonspecific protein binding and visualized by specific antibodies to detect the presence or absence of a particular protein. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression, and postā€translational modification analysis. Ā© 2016 by John Wiley & Sons, Inc.

Keywords: alkaline phosphatase; chemiluminescence; chromogenic; DAB; dot blot; fluorescence; nitrocellulose; peroxidase; protein blotting; PVDF; slot blot; TMB; western blotting

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Protocols
  • Basic Protocol 1: Protein Blotting with Semidry Systems
  • Basic Protocol 2: Tank Transfer
  • Alternate Protocol 1: Rapid Western Transfer with iBlot Dry Blotting System
  • Alternate Protocol 2: Slot and Dot Blotting
  • Support Protocol 1: Ponceau S Staining of Transferred Proteins
  • Support Protocol 2: India Ink Staining of Transferred Proteins
  • Support Protocol 3: Gold Staining of Transferred Proteins
  • Support Protocol 4: Alkali Enhancement of Protein Staining
  • Support Protocol 5: Fluorescent Protein Blot Staining of Transferred Proteins
  • Support Protocol 6: Viewing and Photographing SYPRO Ruby‐Stained Protein Blots
  • Basic Protocol 3: Immunoprobing with Directly Conjugated Secondary Antibody
  • Alternate Protocol 3: Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody
  • Basic Protocol 4: Visualization with Chromogenic Substrates
  • Alternate Protocol 4: Visualization with Luminescent Substrates
  • Alternate Protocol 5: Fluorescent Blot Preparation and Analysis
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Protein Blotting with Semidry Systems

  Materials
  • Samples for analysis
  • Protein molecular weight standards (unit 8.4; Haushalter, ): prestained (Sigma or BioRad), biotinylated (Vector Labs or Sigma), fluorescent (e.g., BenchMark fluorescent protein standards; Thermo Fisher Scientific/Invitrogen), or compatible with other colorimetric and fluorescent detection method (e.g., MagicMark and MagicMark XP western protein standards; Thermo Fisher/Invitrogen); see Table 8.3.4.
  • Transfer buffer (see recipe)
  • 100% methanol
  • UV transilluminator or overhead illuminator (e.g., UVP)
  • Transfer membrane: 0.45‐μm nitrocellulose (e.g., Millipore or Schleicher & Schuell) or polyvinylidene difluoride (PVDF; Millipore Immobilon P)
  • Plastic trays (polypropylene for PVDF membranes), larger than the gels
  • Razor blade or spatula
  • Six sheets of Whatman 3 MM filter paper or equivalent, cut to size of gel
  • Semidry transfer unit (Hoefer, BioRad)
  • Glass test tube (for removal of air bubbles by rolling over membrane)
  • Porous cellophane (Hoefer, Gel Company) or dialysis membrane ), equilibrated with transfer buffer (see recipe), optional
  • Indelible pen (e.g., PaperMate) or soft lead pencil
  • Additional reagents and equipment for performing one‐dimensional gel electrophoresis (unit 7.3; Gallagher, ) and staining proteins in gels (unit 7.4; Gallagher and Sasse, )
Table 8.3.4   MaterialsProtein Standards for Western Blotting

Protein standard Application
Unstained Molecular weight calibration and transfer efficiency; can be visualized with total protein stains
Tagged Molecular weight calibration and transfer efficiency; visualized during immunodetection steps; a variety of potential tags, including biotinylated and antibody‐specific amino acid sequence engineered into standard proteins
Prestained Excellent for checking transfer efficiency and visual inspection of the blot; typically do not produce as sharp a band as other standards, making precise molecular weight calculations difficult

Basic Protocol 2: Tank Transfer

  Materials
  • Samples for analysis
  • Protein molecular weight standards (unit 8.4; Haushalter, ): prestained (Sigma or BioRad), biotinylated (Vector Labs or Sigma), fluorescent (e.g., Benchmark fluorescent protein standards; Thermo Fisher/Invitrogen), or compatible with other colorimetric and fluorescent detection method (e.g., MagicMark and MagicMark XP western protein standards; Thermo Fisher/Invitrogen); see Table 8.3.4
  • Transfer buffer (see recipe)
  • 100% methanol
  • Razor blade or spatula
  • Plastic tray (polypropylene for PVDF membranes), larger than the gel
  • 0.45‐μm nitrocellulose (Millipore or Schleicher & Schuell) or polyvinylidene difluoride (PVDF; Millipore Immobilon P)
  • Transfer tank blotting apparatus and cassette with sponge (Hoefer, BioRad, or Thermo Fisher/Invitrogen; see Fig.  )
  • Six sheets of Whatman 3 MM filter paper or equivalent, cut to size of gel
  • Glass test tube (optional)
  • Heat exchanger and cooling recirculating water bath (optional)
  • Additional reagents and equipment for one‐dimensional or gradient gel electrophoresis (unit 7.3; Gallagher, ) and staining proteins in gels (unit 7.4; Gallagher and Sasse, )

Alternate Protocol 1: Rapid Western Transfer with iBlot Dry Blotting System

  Materials
  • Pre‐run gel containing protein samples and protein standard (unit 7.3; Gallagher, )
  • iBlot Dry Blotting System (Life Technologies)
  • Gel Transfer Stacks Mini (including bottom stack, top stack and sponge) for blotting one minigel (Life Technologies
  • Blotting roller (included in the iBlot transfer device package)
  • Forceps

Alternate Protocol 2: Slot and Dot Blotting

  Additional Materials (also see protocol 1)
  • <10 μg protein sample in < 100 μl water or TBS (no detergent)
  • Tris‐buffered saline (TBS; unit 3.3)
  • Slot and dot blotting apparatus (e.g., Hoefer, BioRad)
  • Vacuum source

Support Protocol 1: Ponceau S Staining of Transferred Proteins

  Materials
  • Membrane with transferred proteins ( protocol 1 or 2 or protocol 4)
  • Ponceau S solution (see recipe)
  • Plastic boxes
  • Pen with indelible ink
  • Additional reagents and equipment for photographing membranes (unit 7.5; Moomaw et al., )

Support Protocol 2: India Ink Staining of Transferred Proteins

  Materials
  • Membrane with transferred proteins ( protocol 1 or 2 or protocol 4)
  • Tween 20 solution (see recipe)
  • India ink solution (see recipe)
  • Plastic boxes

Support Protocol 3: Gold Staining of Transferred Proteins

  Materials
  • Nitrocellulose membrane with transferred proteins ( protocol 1 or 2 or protocol 4)
  • Tween 20 solution (see recipe)
  • Colloidal gold staining solution (BioRad, Sigma)
  • Plastic boxes
  • Glass dish or heat‐sealable plastic bags
  • Filter paper

Support Protocol 4: Alkali Enhancement of Protein Staining

  Materials
  • Nitrocellulose membrane with transferred proteins ( protocol 1 or 2 or protocol 4)
  • 1% (w/v) KOH
  • Phosphate‐buffered saline (PBS; unit 3.3)
  • Glass or Pyrex dish

Support Protocol 5: Fluorescent Protein Blot Staining of Transferred Proteins

  Materials
  • Nitrocellulose or PVDF membrane with transferred proteins ( protocol 1 or 2 or protocol 4)
  • 7% (v/v) acetic acid/10% (v/v) methanol
  • SYPRO Ruby protein blot stain (Sigma, Thermo Fisher Scientific; also see recipe)
  • 150 mM Tris·Cl, pH 8.8 (unit 3.3)/20% (v/v) methanol
  • Small polypropylene staining dish
  • Orbital shaker
  • Forceps
NOTE: Perform all washing, staining, and other incubation steps with continuous, gentle agitation (e.g., on an orbital shaker at 50 rpm). For PVDF membranes, be sure to float the membrane face down on the solution.

Support Protocol 6: Viewing and Photographing SYPRO Ruby‐Stained Protein Blots

  Materials
  • Membrane with transferred proteins ( protocol 1 or 2 or protocol 4)
  • Blocking buffer for colorimetric detection (see recipe) or blocking buffer for luminescence detection (see recipe)
  • Primary antibody specific for protein of interest (working concentration optimized; see Fig.  )
  • TTBS (nitrocellulose or PVDF membranes) or TBS (nylon membranes; see unit 3.3 for recipes)
  • Secondary antibody conjugate: horseradish peroxidase (HRPO)‐ or alkaline phosphatase (AP)‐anti‐Ig conjugate (MP Biomedical, Vector Labs, KPL, or Sigma‐Aldrich; dilute as indicated by manufacturer)
  • Heat‐sealable plastic bag, plastic box, or slotted incubation tray
  • Orbital shaker or rocking platform

Basic Protocol 3: Immunoprobing with Directly Conjugated Secondary Antibody

  Additional Materials (also see protocol 11)
  • Vectastain ABC (HRPO) or ABC‐AP (AP) kit (Vector Labs) containing:
  • Reagent A (avidin)
  • Reagent B (biotinylated HRPO or AP)
  • Biotinylated secondary antibody (request membrane immunodetection protocols when ordering)

Alternate Protocol 3: Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody

  Materials
  • Membrane with transferred proteins, probed with antibody‐enzyme complex (see protocol 11 or protocol 12)
  • Tris‐buffered saline (TBS; unit 3.3)
  • Chromogenic visualization solution (see Table 8.3.3)
  • Additional reagents and equipment for photographing gels (see unit 7.5; Moomaw et al., )

Basic Protocol 4: Visualization with Chromogenic Substrates

  Additional Materials (also see protocol 11)
  • Luminescent substrate buffer: 50 mM Tris·Cl, pH 7.5 (for HRPO; unit 3.3)
  • Luminescent visualization solution or kit for HRPO or AP (Table 8.3.3)
  • Clear plastic wrap
  • Film cassette
  • Additional reagents and equipment for autoradiography (Voytas and Ke, ) or digital image capture (unit 7.5; Moomaw et al., ) and digital image processing ( appendix 3C; Gallagher, )
NOTE: See Troubleshooting section for suggestions concerning optimization of this protocol, particularly when employing AP‐based systems.

Alternate Protocol 4: Visualization with Luminescent Substrates

  Materials
  • Fluorescently tagged antibodies: Biotium (http://www.biotium.com/), GE Amersham, Life Technologies, Li‐Cor (http://www.licor.com), Pierce
  • Membrane with transferred proteins, probed with antibody‐enzyme complex (see protocol 11 or protocol 12).
  • Imaging system with multiple emission filters and overhead LED or Xenon (“epi”) variable‐excitation‐light illumination (e.g., UVP BioSpectrum with fiber optic–based quartz halogen or xenon arc light source; Protein Simple; Bio Rad)
  • Appropriate excitation and emission light filters (Tables 8.3.8, 8.3.9, and 8.3.10)
  • Additional reagents and equipment for digital image processing ( appendix 3C; Gallagher, )
NOTE: NC or low‐fluorescence PVDF blotting membrane (e.g., Millipore Immobilon‐FL PVDF) is required for fluorescence imaging to avoid a high background.
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Figures

Videos

Literature Cited

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Internet Resources
  http://media.cellsignal.com/www/pdfs/resources/white‐papers/guide‐to‐successful‐wb.pdf
  A guide to successful western blotting from Cell Signaling Technology, 2013.
  http://www.perkinelmer.com/catalog/category/id/lightning%20blot%20system
  Introduction to the Lightning Blot System by Perkin Elmer
  http://www.bio‐rad.com/en‐us/product/semi‐dry‐rapid‐blotting‐systems/trans‐blot‐turbo‐transfer‐system
  Introduction to Trans‐Blot Turbo Transfer System by BioRad.
  http://www.piercenet.com/product/pierce‐power‐blotter
  Introduction to Pierce Power Blotter by Thermo Scientific.
  http://www.lifetechnologies.com/us/en/home/life‐science/protein‐expression‐and‐analysis/western‐blotting/western‐blot‐transfer/iblot‐dry‐blotting‐system.html
  Introduction of iBlot Dry Blotting System by Life Technologies.
  http://tools.lifetechnologies.com/content/sfs/manuals/iblotsystem_qrc.pdf
  iBlot Dry Blotting System quick reference.
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