Immunoblotting and Immunodetection

Sean Gallagher1, Scott E. Winston2, Steven A. Fuller2, John G.R. Hurrell3

1 UVP, Inc., Upland, California, 2 Nabi, Rockville, Maryland, 3 FluorRx, Carmel, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.19
DOI:  10.1002/0471142301.ns0519s29
Online Posting Date:  November, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides numerous protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS‐PAGE and the antigens are then electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. Any remaining binding sites are blocked by immersing the membrane in a blocking solution. After probing with the primary antibody, the membrane is washed and the antibody‐antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti‐IgG antibody (e.g., goat anti‐rabbit IgG) and appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Protein Blotting with Tank Transfer Systems
  • Alternate Protocol 1: Protein Blotting with Semidry Systems
  • Alternate Protocol 2: Blotting of Stained Gels
  • Support Protocol 1: Reversible Staining of Transferred Proteins
  • Support Protocol 2: Quantitation of Protein with Ponceau S
  • Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody
  • Alternate Protocol 3: Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody
  • Basic Protocol 3: Visualization with Chromogenic Substrates
  • Alternate Protocol 4: Visualization with Luminescent Substrates
  • Alternate Protocol 5: Visualization with Chemiluminescent, Fluorescent, or Chromogenic Substrates
  • Support Protocol 3: Stripping and Reusing Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Protein Blotting with Tank Transfer Systems

  Materials
  • Samples for analysis
  • Protein molecular weight standards: prestained (Sigma or Bio‐Rad), biotinylated (Vector Labs or Sigma), fluorescent (e.g., BenchMark Fluorescent Protein Standards; Invitrogen), or compatible with other colorimetric and fluorescent detection method (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; Bio‐Rad Zetabind) membrane
  • Electroblotting apparatus (Bio‐Rad, Invitrogen, Amersham, or Harvard Bioscience)
  • Indelible pen (e.g., Paper‐Mate) or soft lead pencil
  • Additional reagents and equipment for one‐ and two‐dimensional gel electrophoresis (e.g., Coligan et al., ) and staining proteins in gels and on membranes (see protocol 4 and Coligan et al., )
NOTE: Deionized, distilled water should be used throughout this protocol.

Alternate Protocol 1: Protein Blotting with Semidry Systems

  • Six sheets of Whatman 3MM filter paper or equivalent, cut to size of gel and saturated with transfer buffer
  • Semidry transfer unit (Hoefer, Bio‐Rad, or Sartorius)

Alternate Protocol 2: Blotting of Stained Gels

  Materials
  • Destained gel containing proteins of interest
  • 25 mM Tris base/192 mM glycine/1% SDS
  • 25 mM Tris base/192 mM glycine/0.1% SDS

Support Protocol 1: Reversible Staining of Transferred Proteins

  • Ponceau S solution (see recipe)
  • Additional reagents and equipment for photographing membranes

Support Protocol 2: Quantitation of Protein with Ponceau S

  • Spectrophotometer and 2‐ml cuvette

Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody

  Materials
  • Membrane with transferred proteins (see protocol 1 or protocol 2)
  • Blocking buffer appropriate for membrane and detection protocol (see recipe)
  • Primary antibody specific for protein of interest
  • TTBS (for nitrocellulose or PVDF; see recipe) or TBS (for nylon; see appendix 2A)
  • Secondary antibody conjugate: horseradish peroxidase (HRP)– or alkaline phosphatase (AP)–anti‐Ig conjugate (Cappel, Vector Labs, Kirkegaard & Perry, or Sigma; dilute as indicated by manufacturer and store frozen in 25‐µl aliquots until use)
  • Heat‐sealable plastic bag
  • Powder‐free gloves
  • Plastic box

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

  • Blocking buffer appropriate for membrane and detection protocol (see recipe)
  • TTBS (for nitrocellulose or PVDF; see recipe) or TBS (for neutral or positively charged nylon; appendix 2A)
  • 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 and probed with antibody‐enzyme complex (see protocol 6 or protocol 7)
  • TBS ( appendix 2A)
  • Chromogenic visualization solution (Table 5.19.1)
  • Additional reagents and equipment for gel photography

Alternate Protocol 4: Visualization with Luminescent Substrates

  • Luminescent substrate buffer: 50 mM Tris·Cl, pH 7.5 (for HRP; appendix 2A) 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 5.19.1)
  • Clear plastic wrap
  • Additional reagents and equipment for autoradiography (see Coligan et al., )
NOTE: See section for suggestions concerning optimization of this protocol, particularly when employing AP‐based systems.

Alternate Protocol 5: Visualization with Chemiluminescent, Fluorescent, or Chromogenic Substrates

  Materials
  • Membrane with transferred proteins (see protocol 1 or protocol 2)
  • Primary antibody (from mouse or rabbit) specific for protein of interest
  • Vectastain ABC‐AmP Chromogenic or Chemiluminescent Western Blotting Immunodetection Kit (Vector Laboratories) containing:
    • 10× casein solution (250 ml)
    • Vector biotinylated secondary antibody (0.25 ml): anti–mouse IgG (for mouse primary antibodies) or anti–rabbit IgG (for rabbit primary antibodies)
    • Vectastain ABC‐AmP reagent A (0.5 ml)
    • Vectastain ABC‐AmP reagent B (0.5 ml)
    • Substrate: chemiluminescent/fluorescent substrate (100 ml) and BCIP/NBT chromogenic substrate kit (stock reagents for 200 ml)
  • PBS: 10 mM sodium phosphate buffer, pH 7.5 ( appendix 2A) containing 150 mM NaCl
  • 0.1 M Tris·Cl, pH 9.5 ( appendix 2A)
  • Staining trays
  • X‐ray film (e.g., Kodak BioMax)
  • UV transilluminator or UV imaging trans‐ and epi‐CCD acquisition 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.

Support Protocol 3: Stripping and Reusing Membranes

  Materials
  • 0.2 M NaOH
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   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.
   Bronstein, I., Voyta, J.C., Murphy, O.J., Bresnick, L., and Kricka, L.J. 1992. Improved chemiluminescent Western blotting procedure. BioTechniques 12:748‐753.
   Burnette, W.N. 1981. Western blotting: Electrophoretic transfer of proteins from sodium dodecyl sulfate‐polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195‐203.
   Coligan, J.E., Dunn, B.M., Speicher, D.W., and Wingfield, P.T. (eds.) 2004. Current Protocols in Protein Science John Wiley & Sons, Hoboken, N.J.
   Craig, W.Y., Poulin, S.E., Collins, M.F., Ledue, T.B., and Ritchie, R.F. 1993. Background staining in immunoblot assays. Reduction of signal caused by cross‐reactivity with blocking agents. J. Immunol. Methods 158:67‐76.
   Dionisi, H.M., Checa, S.K., and Viale, A.M. 1995. Protein immunoblotting of stained gels. BioTechniques 19:348‐350.
   Gillespie, P.G. and Hudspeth, A.J. 1991. Chemiluminescence detection of proteins from single cells. Proc. Natl. Acad. Sci. U.S.A. 88:2563‐2567.
   Harlow, E. and Lane, D. 1988. Immunoblotting. In Antibodies: A Laboratory Manual pp. 471‐510. CSH Laboratory, Cold Spring Harbor, N.Y.
   Harper, D.R. and Murphy, G. 1991. Nonuniform variation in band pattern with luminol/horseradish peroxidase Western blotting. Anal. Biochem. 192:59‐63.
   Kaufmann, S.H., Ewing, C.M., and Shaper, J.H. 1987. The erasable Western blot. Anal. Biochem. 161:89‐95.
   Klein, D., Kern, R.M., and Sokol, R.Z. 1995. A method for quantification and correction of proteins after transfer to immobilization membranes. Biochem. Mol. Biol. Int. 36:59‐66.
   Mandrell, R.E. and Zollinger, W.D. 1984. Use of zwitterionic detergent for the restoration of antibody‐binding capacity of electroblotted meningococcal outer membrane proteins. J. Immunol. Methods 67:1‐11.
   McKimm‐Breschkin, J.L. 1990. The use of tetramethylbenzidine for solid phase immunoassays. J. Immunological Methods 135:277‐280.
   Millipore. 1990. Protein blotting protocols for Immobilon‐P transfer membrane. Bedford, Mass.
   Moos, M. 1992. Isolation of proteins for microsequence analysis In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 8.7.1‐8.7.12. Greene Publishing Associates and John Wiley & Sons, New York.
   Pampori, N.A., Pampori, M.K., and Shapiro, B.H. 1995. Dilution of the chemiluminescence reagents reduces the background noise on Western blots. BioTechniques 18:588‐590.
   Peluso, R.W. and Rosenberg, G.H. 1987. Quantitative electrotransfer of proteins from sodium dodecyl sulfate polyacrylamide gels onto positively charged nylon membranes. Anal. Biochem. 162:389‐398.
   Perides, G., Plagens, U., and Traub, P. 1986. Protein transfer from fixed, stained, and dried polyacrylamide gels and immunoblot with protein A–gold. Anal. Biochem. 152:94‐99.
   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.
   Schneppenheim, R., Budde, U., Dahlmann, N. and Rautenberg, P. 1991. Luminography—a new, highly sensitive visualization method for electrophoresis. Electrophoresis 12:367‐372.
   Suck, R.W.L. and Krupinska, K. 1996. Repeated probing of Western blots obtained from Coomassie Brilliant Blue–stained or unstained polyacrylamide gels. BioTechniques 21:418‐422.
   Talbot, P.V., Knobler, R.L., and Buchmeier, M. 1984. Western and dot immunoblotting analysis of viral antigens and antibodies: Application to murine hepatitis virus. J. Immunol. Methods 73:177‐188.
   Tesfaigzi, J., Smith‐Harrison, W., and Carlson, D.M. 1994. A simple method for reusing western blots on PVDF membranes. BioTechniques 17:268‐269.
   Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76:4350‐4354.
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, 1988. 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library