Immunoblotting and Immunodetection

Sean Gallagher1, Scott E. Winston (tank transfer systems)2, Steven A. Fuller (tank transfer systems)2, John G.R. Hurrell (tank transfer systems; reversible staining of proteins)3

1 UVP, Inc., Upland, California, 2 Nabi, Rockville, Maryland, 3 FluorRx, Carmel, Indiana
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.8
DOI:  10.1002/0471142727.mb1008s83
Online Posting Date:  July, 2008
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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 horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. Curr. Protoc. Mol. Biol. 83:10.8.1-10.8.28. © 2008 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: Slot and Dot Blotting of Proteins
  • Alternate Protocol 3: 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 4: Immunoprobing with Avidin-Biotin Coupling to Secondary Antibody
  • Basic Protocol 3: Visualization with Chromogenic Substrates
  • Alternate Protocol 5: Visualization with Luminescent Substrates
  • Alternate Protocol 6: Signal Amplification with Avidin-Biotin Visualization Reagents
  • Support Protocol 3: Stripping and Reusing Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • 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 10.8.1; UNIT 10.2A): 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)
  • Electroblotting apparatus (Bio-Rad, Invitrogen, Amersham, or Hoefer; e.g., Figs. 10.8.1 and 10.8.2)
  • Indelible pen (e.g., Paper-Mate) or soft lead pencil
  • Additional reagents and equipment for one- and two-dimensional gel electrophoresis (UNITS 10.2-10.4) and staining proteins in gels (UNIT 10.6) and on membranes (see Support Protocol 1 and UNIT 10.7)

NOTE: Deionized, distilled water should be used throughout this protocol.
 
Table 10.8.1 Protein Standards for Immunoblotting
Protein standardApplication
UnstainedMolecular weight calibration and transfer efficiency; can be visualized with total protein stains
TaggedMolecular 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
PrestainedExcellent 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
 FigureFigure 10.8.1 Immunoblotting with a tank blotting unit. The polyacrylamide gel containing the protein is laid on a sheet of filter paper. The uncovered side of the gel is overlaid with a sheet of membrane precut to the size of the gel plus 1 to 2 mm on each edge, then this membrane is overlaid with another sheet of filter paper. The filter paper containing the gel and membrane is sandwiched between Scotch-Brite pads. This sandwich is placed in a plastic support, and the entire assembly is placed in a tank containing transfer buffer. For transfer of negatively charged protein, the membrane is positioned on the anode side of the gel. For transfer of positively charged protein, the membrane is placed on the cathode side of the gel. Charged proteins are transferred electrophoretically from the gel onto the membrane. Transfer is achieved by applying a voltage of 100 V for 1 to 2 hr (with cooling) or 14 V overnight.
 FigureFigure 10.8.2 Minigel tank electrotransfer unit. Designed for smaller (8 × 10–cm) gels, these units will process four gels at a time. Note that the two outside panels hold the electrode grid.

Alternate Protocol 1: Protein Blotting with Semidry Systems

 Additional Materials (also see Basic 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, Bio-Rad, or Invitrogen)

Alternate Protocol 2: Slot and Dot Blotting of Proteins

 Additional Materials (also see Basic Protocol 1)
  • <10 µg protein sample in <100 µl water or TBS (no detergent)
  • Tris-buffered saline (TBS; APPENDIX 2)
  • Slot or dot blotting apparatus (e.g., Hoefer, Bio-Rad, or Whatman; Fig. 10.8.4)
  • Vacuum source
     FigureFigure 10.8.4 (A) Slot-blot unit. Through use of a vacuum manifold or by simple hand spotting, up to 96 samples can be applied to a single nitrocellulose or PVDF membrane for immunoblotting. Although this approach cannot discriminate between the protein of interest and a cross-reactive antigen, it is a quick way to perform preliminary characterization and high-volume, routine quantitation of samples. (B) Dilution series of immunoprobed BSA developed with peroxidase-tagged secondary antibody and DAB/NiCl2.

Alternate Protocol 3: Blotting of Stained Gels

 Materials
  • Destained gel containing proteins of interest (UNIT 10.6)
  • 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 Basic Protocol 1)
  • Ponceau S solution (see recipe)
  • Additional reagents and equipment for photographing membranes (UNIT 10.6)

Basic Protocol 2: Immunoprobing with Directly Conjugated Secondary Antibody

 Materials
  • Membrane with transferred proteins (see Basic Protocol 1 or Alternate Protocols 1 to 3)
  • Blocking buffer appropriate for membrane and detection protocol (see recipes)
  • Primary antibody specific for protein of interest
  • TTBS (nitrocellulose or PVDF) or TBS (nylon; see APPENDIX 2 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 bag
  • Powder-free gloves
  • Plastic box

Alternate Protocol 4: Immunoprobing with Avidin-Biotin Coupling to Secondary Antibody

 Additional Materials (also see Basic Protocol 2)
  • 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 Basic Protocol 2 or Alternate Protocol 4)
  • TBS (APPENDIX 2)
  • Chromogenic visualization solution (Table 10.8.2)
  • Additional reagents and equipment for gel photography (UNIT 10.6)

Alternate Protocol 5: Visualization with Luminescent Substrates

 Additional Materials (also see Basic Protocol 3)
  • Luminescent substrate buffer: 50 mM Tris·Cl, pH 7.5 (for HRP; APPENDIX 2) 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 10.8.2)
  • Clear plastic wrap
  • Additional reagents and equipment for autoradiography (APPENDIX 3A)

NOTE: See Troubleshooting section for suggestions concerning optimization of this protocol, particularly when employing AP-based systems.

Alternate Protocol 6: Signal Amplification with Avidin-Biotin Visualization Reagents

 Materials
  • Membrane with transferred proteins (see Basic Protocol 1 or Alternate Protocols 1 to 3)
  • 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 (APPENDIX 2) containing 150 mM NaCl
  • 0.1 M Tris·Cl, pH 9.5 (APPENDIX 2)
  • 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
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Figures

  •  FigureFigure 10.8.1 Immunoblotting with a tank blotting unit. The polyacrylamide gel containing the protein is laid on a sheet of filter paper. The uncovered side of the gel is overlaid with a sheet of membrane precut to the size of the gel plus 1 to 2 mm on each edge, then this membrane is overlaid with another sheet of filter paper. The filter paper containing the gel and membrane is sandwiched between Scotch-Brite pads. This sandwich is placed in a plastic support, and the entire assembly is placed in a tank containing transfer buffer. For transfer of negatively charged protein, the membrane is positioned on the anode side of the gel. For transfer of positively charged protein, the membrane is placed on the cathode side of the gel. Charged proteins are transferred electrophoretically from the gel onto the membrane. Transfer is achieved by applying a voltage of 100 V for 1 to 2 hr (with cooling) or 14 V overnight.
    View Image
  •  FigureFigure 10.8.2 Minigel tank electrotransfer unit. Designed for smaller (8 × 10–cm) gels, these units will process four gels at a time. Note that the two outside panels hold the electrode grid.
    View Image
  •  FigureFigure 10.8.3 Immunoblotting with a semidry transfer unit. Generally, the lower electrode is the anode, and one gel is transferred at a time. A Mylar mask (optional in some units) is put in place on the anode. This is followed by three sheets of transfer buffer–soaked filter paper, the membrane, the gel, and finally, three more sheets of buffer-soaked filter paper. To transfer multiple gels, construct transfer stacks as illustrated, and separate with a sheet of porous cellophane. For transfer of negatively charged protein, the membrane is positioned on the anode side of the gel. For transfer of positively charged protein, the membrane is placed on the cathode side of the gel. Transfer is achieved by applying a maximum current of 0.8 mA/cm2 of gel area. For a typical minigel (8 × 10 cm) and standard-sized gel (14 × 14 cm), this means 60 and 200 mA, respectively.
    View Image
  •  FigureFigure 10.8.4 (A) Slot-blot unit. Through use of a vacuum manifold or by simple hand spotting, up to 96 samples can be applied to a single nitrocellulose or PVDF membrane for immunoblotting. Although this approach cannot discriminate between the protein of interest and a cross-reactive antigen, it is a quick way to perform preliminary characterization and high-volume, routine quantitation of samples. (B) Dilution series of immunoprobed BSA developed with peroxidase-tagged secondary antibody and DAB/NiCl2.
    View Image
  •  FigureFigure 10.8.5 Serial dilution of primary antibody directed against the 97-kDa catalytic subunit of the plant plasma membrane ATPase. Blot was developed with HRPO-coupled avidin-biotin reagents according to Alternate Protocol 4 and visualized with 4-chloro-1-naphthol (4CN). Note how background improves with dilution.
    View Image
  •  FigureFigure 10.8.6 Chemiluminescent blot captured with a 5-min exposure using a 1-megapixel cooled (–100°C) CCD scientific camera. In this case the reaction had decayed to a point where film was not able to record an image, while the CCD camera shows the dilution series. The blot, a two-fold IgG dilution series from 500 to 2 ng, was probed using peroxidase-conjugated affinity-purified sheep anti-mouse IgG (H&L; Rockland Immunochemicals) and visualized with luminol.
    View Image

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Literature Cited

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.
    Carr, S.A. and Annan, R.S. 1996. Overview of peptide and protein analysis by mass spectrometry. Curr. Protoc. Prot. Sci. 4:16.1.1-16.1.27.
    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.
    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.
    Harlow, E. and Lane, D. 1999. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 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.
    Kurien, B.T. and Scofield, R.H. 2006. Western blotting. Methods 38:283-293.
    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. Immunol. Methods 135:277-280.
    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.
    Pluskal, M.G., Przekop, M.B., Kavonian, M.R., Vecoli, C., and Hicks, D.A. 1986. A new membrane substrate for western blotting of proteins. BioTechniques. 4:272.
    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, 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.

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