Immunoblotting and Immunodetection

Duojiao Ni1, Peng Xu2, Sean Gallagher1

1 LLC, Upland, California, 2 University of Virginia School of Medicine, Charlottesville, Virginia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.10
DOI:  10.1002/cpim.10
Online Posting Date:  August, 2016
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Abstract

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS‐PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody‐antigen complexes are tagged with fluorophores, horseradish peroxidase, or alkaline phosphatase coupled to a secondary anti‐IgG antibody, and detected using appropriate fluorescent imaging technologies or with chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. © 2016 by John Wiley & Sons, Inc.

Keywords: immunoblot; western blot; horseradish peroxidase; alkaline phosphatase; antibodies

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Protein Blotting with Tank Transfer Systems
  • Alternate Protocol 1: Protein Blotting with Semidry Systems
  • Alternate Protocol 2: Rapid Western Transfer with iBlot Dry Blotting System
  • Alternate Protocol 3: Slot and Dot Blotting of Proteins
  • Alternate Protocol 4: Blotting of Stained Gels
  • Support Protocol 1: Reversible Staining of Transferred Proteins with Ponceau S
  • Support Protocol 2: Quantitation of Transferred Proteins with Ponceau S
  • Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody
  • Alternate Protocol 5: Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody
  • Basic Protocol 3: Visualization with Chromogenic Substrates
  • Alternate Protocol 6: Visualization with Luminescent Substrates
  • Alternate Protocol 7: Signal Amplification with Avidin‐Biotin Visualization Reagents
  • Alternate Protocol 8: Fluorescent Blot Preparation and Analysis
  • Support Protocol 3: Stripping and Reusing Membranes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Protein Blotting with Tank Transfer Systems

  Materials
  • Samples for analysis
  • Protein molecular weight standards [Table 8.10.1; unit (Gallagher, )]: prestained (Sigma or BioRad), biotinylated (Vector Labs or Sigma), fluorescent (e.g., BenchMark Fluorescent Protein Standards; Invitrogen), or compatible with other colorimetric and fluorescent detection methods (e.g., MagicMark and MagicMark XP Western Protein Standards; Invitrogen)
  • Transfer buffer (see recipe)
  • Powder‐free gloves
  • Scotch‐Brite pads (3M) or equivalent sponge
  • Whatman 3MM filter paper or equivalent
  • Transfer membrane: 0.45‐μm nitrocellulose (Millipore or Schleicher & Schuell), PVDF (Millipore Immobilon P), neutral nylon (Pall Biodyne A), or positively charged nylon (Pall Biodyne B; BioRad Zetabind)
  • Electroblotting apparatus (BioRad, Invitrogen, Amersham, or Hoefer; e.g., Figs and )
  • Indelible pen (e.g., Paper‐Mate) or soft lead pencil
  • Additional reagents and equipment for one‐ and two‐dimensional gel electrophoresis [unit (Gallagher, ), and unit (Adams and Gallagher, ), and Adams ( )] and staining proteins in gels (unit ; Sasse and Gallagher, ) and on membranes [see protocol 6 and Sasse and Gallagher ( )]
  • NOTE: Deionized, distilled water should be used throughout this protocol.
Table 8.0.1   MaterialsProtein Standards for Immunoblotting

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 visually inspecting the blot; typically does not produce as sharp a band as other standards, making precise molecular weight calculations difficult. Both colored and fluorescent tags are available.

Alternate Protocol 1: Protein Blotting with Semidry Systems

  Additional Materials (also see protocol 1)
  • Six sheets of Whatman 3MM filter paper or equivalent, cut to size of gel and saturated with transfer buffer
  • Semidry transfer unit (Hoefer, BioRad, or Invitrogen)

Alternate Protocol 2: Rapid Western Transfer with iBlot Dry Blotting System

  Materials
  • Pre‐run gel containing protein samples and protein standard
  • iBlot transfer device (Thermo Fisher; see Fig.  )
  • Blotting roller (included in the iBlot transfer device package)
  • Gel Transfer Stacks Mini (including both bottom stack, top stack and sponge) for blotting one minigel
  • Forceps
  • Additional reagents and equipment for visualization with chromogenic ( protocol 10), chemiluminescent ( protocol 11), or fluorescent substrates ( protocol 13), and signal amplification with avidin‐biotin visualization reagents ( protocol 12)

Alternate Protocol 3: Slot and Dot Blotting of Proteins

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

Alternate Protocol 4: Blotting of Stained Gels

  Materials
  • Destained gel containing proteins of interest (unit ; Sasse and Gallagher, )
  • 25 mM Tris base/192 mM glycine/1% SDS
  • 25 mM Tris base/192 mM glycine/0.1% SDS
  • 25 mM Tris base/192 mM glycine/0.1% SDS with 6 M urea (optional)

Support Protocol 1: Reversible Staining of Transferred Proteins with Ponceau S

  Additional Materials (also see protocol 1)
  • Ponceau S solution (see recipe)
  • Additional reagents and equipment for photographing membranes (unit ; Sasse and Gallagher, )

Support Protocol 2: Quantitation of Transferred Proteins with Ponceau S

  Additional Materials (also see protocol 6)
  • UV/vis spectrophotometer
  • 2‐ml cuvette

Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody

  Materials
  • Membrane with transferred proteins (see protocol 1 or Alternate Protocols protocol 21 to protocol 43)
  • Blocking buffer (see recipe) appropriate for membrane and detection protocol
  • Primary antibody specific for protein of interest
  • TTBS (nitrocellulose or PVDF) or TBS (nylon; see for recipes)
  • Secondary antibody conjugate: species‐specific anti‐Ig conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP; e.g., Cappel, Vector Labs, Kirkegaard & Perry, or Sigma; dilute as indicated by manufacturer and store frozen in 25‐μl aliquots until use)
  • Heat‐sealable plastic bags
  • Plastic bag heat‐sealer
  • Orbital shaker or rocking platform
  • Incubation trays for membrane strips
  • Powder‐free gloves
  • Plastic box

Alternate Protocol 5: Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody

  Additional Materials (also see protocol 8)
  • Vectastain ABC (HRP) or ABC‐AP (AP) kit (Vector Labs) containing the following: reagent A (avidin), reagent B (biotinylated HRP or AP), and biotinylated secondary antibody (request membrane immunodetection protocols when ordering)

Basic Protocol 3: Visualization with Chromogenic Substrates

  Materials
  • Membrane with transferred proteins, probed with antibody‐enzyme complex (see protocol 8 or protocol 5)
  • TBS ( )
  • Chromogenic visualization solution (Table 8.10.3; also see recipes for 4CN and DAB/NiCl 2 visualization solutions)
  • Additional reagents and equipment for gel photography (unit ; Sasse and Gallagher, )

Alternate Protocol 6: Visualization with Luminescent Substrates

  Additional Materials (also see protocol 10)
  • Luminescent substrate buffer: 50 mM Tris·Cl, pH 7.5 (for HRP; ) or dioxetane phosphate substrate buffer (for alkaline phosphatase; see recipe)
  • Nitro‐Block solution (AP reactions only): 5% (v/v) Nitro‐Block (Applied Biosystems) in dioxetane phosphate substrate buffer, prepared just before use
  • Luminescent visualization solution (Table 8.10.3; also see recipes for luminol visualization solution and dioxetane phosphate visualization solution)
  • Clear plastic wrap
  • Additional reagents and equipment for autoradiography ( ; Voytas and Ke, )
  • NOTE: See Troubleshooting for suggestions concerning optimization of this protocol, particularly when employing AP‐based systems.

Alternate Protocol 7: Signal Amplification with Avidin‐Biotin Visualization Reagents

  Materials
  • Membrane with transferred proteins (see protocol 1 or Alternate Protocols protocol 21 to protocol 43)
  • Primary antibody (from mouse or rabbit) specific for protein of interest
  • Vectastain ABC‐AmP Chromogenic or Chemiluminescent Western Blotting Immunodetection Kit (Vector Labs) containing:
  • 10× casein solution (250 ml)
  • Biotinylated secondary antibody (0.25 ml): anti‐mouse IgG (for mouse primary antibodies) or anti‐rabbit IgG (for rabbit primary antibodies)
  • Reagents A and B (0.5 ml each)
  • Substrate: chemiluminescent/fluorescent substrate (DuoLux; 100 ml) or BCIP/NBT chromogenic substrate kit (stock reagents for 200 ml)
  • PBS: 10 mM sodium phosphate buffer, pH 7.5 ( ) containing 150 mM NaCl
  • 0.1 mM Tris·Cl, pH 9.5 ( )
  • Staining trays
  • X‐ray film (e.g., Kodak BioMax)
  • UV transilluminator or UV imaging transillumination, cooled CCD acquisition system (e.g., Biospectrum Imaging System, UVP, Inc.)
NOTE: The components supplied in each Vectastain ABC‐AmP Western Blotting Immunodetection Kit provide sufficient reagents to develop approximately twenty 100‐cm2 blots. The volumes of the reagents in the protocol below are optimized for the development of a 100‐cm2 membrane. Volumes may be proportionally adjusted for blots of a different size. All kit reagents may be used immediately following dilution. For optimal results, it is recommended that all diluted reagents from the kit be used the same day that they are prepared. Vectastain ABC‐AmP Kit stock reagents should be stored under refrigeration and kept in the box in which they are supplied.

Alternate Protocol 8: Fluorescent Blot Preparation and Analysis

  Materials
  • Membrane processed according to Basic Protocols 2 and/or 3 using fluorescently tagged ABC primary or secondary antibodies: e.g., Biotium (http://www.biotium.com/), GE Amersham, Life Technologies, LI‐COR (http://www.licor.com), Pierce (fluorescent standards are also quite useful; e.g., LI‐COR Chameleon Protein Ladders)
  • Imaging system with multiple emission filters and overhead (“epi”) variable‐excitation‐light illumination (e.g., UVP BioSpectrum with fiber optic–based quartz halogen or xenon arc light source; Protein Simple; BioRad); alternatively, dedicated laser scanning systems are available for NIR blotting imaging (Odyssey series, LI‐COR)
  • Appropriate excitation and emission light filters (Tables 8.10.4, 8.10.5, and 8.10.6)
  • NOTE: Nitrocellulose or low‐fluorescence PVDF blotting membrane (e.g., Millipore Immobilon‐FL PVDF) is required for fluorescence imaging to avoid a high background

Support Protocol 3: Stripping and Reusing Membranes

  Materials
  • 0.2 M NaOH
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Figures

Videos

Literature Cited

Literature Cited
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   Bjerrum, O.J. , Larsen, K.P. , and Heegaard, N.H.H. 1988. Nonspecific binding and artifacts‐specificity problems and troubleshooting with an atlas of immunoblotting artifacts. In CRC Handbook of Immunoblotting of Proteins, Vol. I: Technical Descriptions ( O.J. Bjerrum and N.H.H. Heegaard , eds.) pp. 227‐254. CRC Press, Boca Raton, Fla.
   Blake, M.S. , Johnston, K.H. , Russell‐Jones, G.J. , and Gotschlich, E.C. 1984. A rapid, sensitive method for detection of alkaline phosphatase–conjugated anti‐antibody on Western blots. Anal. Biochem. 136:175‐179. doi: 10.1016/0003‐2697(84)90320‐8.
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   Haan, C. and Behrmann, I. 2007. A cost effective noncommercial ECL‐solution for Western blot detections yielding strong signals and low background. J. Immunol. Methods 318:11‐19. doi: 10.1016/j.jim.2006.07.027.
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   Sandhu, G.S. , Eckloff, B.W. , and Kline, B.C. 1991. Chemiluminescent substrates increase sensitivity of antigen detection in Western blots. BioTechniques 11:14‐16.
   Sasse, J. and Gallagher, S.R. 2008. Detection of proteins on blot transfer membranes. Curr. Protoc. Mol. Biol. 84:10.7.1‐10.7.6.
   Sasse, J. and Gallagher, S.R. 2004. Staining proteins in gels. Curr. Protoc. Immunol. 58:8.9.1‐8.9.25.
   Schneppenheim, R. , Budde, U. , Dahlmann, N. , and Rautenberg, P. 1991. Luminography—a new, highly sensitive visualization method for electrophoresis. Electrophoresis 12:367‐372. doi: 10.1002/elps.1150120508.
   Silva, J. M. and McMahon, M. 2014. The fastest western in town: A contemporary twist on the classic western blot analysis. J. Vis. Exp. 84:e51149.
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   Voytas, D. and Ke, N. 2002. Detection and quantitation of radiolabeled proteins and DNA in gels and blots. Curr. Protoc. Immunol. 50:A.3J.1‐A.3J.10.
Key References
   Bjerrum, O.J. and Schafer‐Nielsen, C. 1986. Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Electrophoresis ‘86 ( M.J. Dunn , ed.) pp. 315‐327. VCH Publishers, Deerfield Beach, Fla.
  Describes the semidry blotting system.
   Gillespie and Hudspeth , 1991. See above.
  Describes alkaline phosphatase–luminescent detection methods.
   Harlow and Lane , 1999. See above.
  Details alternative detection methods.
   Salinovich, O. and Montelaro, R.C. 1986. Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Anal. Biochem. 156:341‐347.
  Describes the use of Ponceau S staining for immunoblotting.
   Schneppenheim et al., 1991. See above.
  Details peroxidase‐based luminescent detection methods.
Internet Resources
   http://www.neb‐online.de/downloads/CST_wb_whitepaper_2013.pdf
  A guide to successful western blotting from cell signaling technology, 2013.
   http://www.perkinelmer.com/catalog/category/id/lightning%20blot%20system.
  Introduction to 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
  iBlotDry Blotting System quick reference.
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