Protein Blotting: Immunoblotting

Sean R. Gallagher1

1 UVP, LLC, Upland, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 8.3
DOI:  10.1002/9780470089941.et0803s04
Online Posting Date:  December, 2010
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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 post‐translational modification analysis. Curr. Protoc. Essential Lab. Tech. 4:8.3.1‐8.3.36. © 2010 by John Wiley & Sons, Inc.

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

     
 
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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: 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 2: Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody
  • Basic Protocol 4: Visualization with Chromogenic Substrates
  • Alternate Protocol 3: Visualization with Luminescent Substrates
  • Alternate Protocol 4: Fluorescent Blot Preparation and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • 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): 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); 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 (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, Bio‐Rad, or Sartorius)
  • Glass test tube (for removal of air bubbles by rolling over membrane)
  • Porous cellophane (Hoefer) or dialysis membrane (Bio‐Rad or Sartorius), 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) and staining proteins in gels (unit 7.4)
     
    Table 8.3.4 Protein Standards for Western Blotting
    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 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): 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); 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, Bio‐Rad, or Invitrogen; see Fig. 8.3.5)
  • 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) and staining proteins in gels (unit 7.4)
Alternate Protocol 1:  Slot and Dot Blotting
 Additional Materials (also see Basic 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, Bio‐Rad, or Whatman)
  • Vacuum source
Support Protocol 1:  Ponceau S Staining of Transferred Proteins
 Materials
  • Membrane with transferred proteins (Basic Protocol 1 or 2 or Alternate Protocol 1)
  • Ponceau S solution (see recipe)
  • Plastic boxes
  • Pen with indelible ink
  • Additional reagents and equipment for photographing membranes (unit 7.5)
Support Protocol 2:  India Ink Staining of Transferred Proteins
 Materials
  • Membrane with transferred proteins (Basic Protocol 1 or 2 or Alternate Protocol 1)
  • 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 (Basic Protocol 1 or 2 or Alternate Protocol 1)
  • Tween 20 solution (see recipe)
  • Colloidal gold staining solution (Bio‐Rad, Sigma, GE Healthcare)
  • 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 (Basic Protocol 1 or 2 or Alternate Protocol 1)
  • 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 (Basic Protocol 1 or 2 or Alternate Protocol 1)
  • 7% (v/v) acetic acid/10% (v/v) methanol
  • SYPRO Ruby protein blot stain (purchase from Molecular Probes; 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.
Basic Protocol 3:  Immunoprobing with Directly Conjugated Secondary Antibody
 Materials
  • Membrane with transferred proteins (Basic Protocol 1 or 2 or Alternate Protocol 1)
  • 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. 8.3.6)
  • 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 (MB 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
     FigureFigure 8.3.6 Serial dilution of primary antibody directed against the 97‐kDa catalytic subunit of the plant plasma membrane ATPase. The blot was developed with HRPO‐coupled avidin‐biotin reagents according to Alternate Protocol 2 and visualized with 4‐chloro‐1‐naphthol (4CN; Basic Protocol 4). Note how background improves with dilution. In this case, a dilution of 1/400 to 1/800 is optimal, keeping the background to a minimum.
Alternate Protocol 2:  Immunoprobing with Avidin‐Biotin Coupling to Secondary Antibody
 Additional Materials (also see Basic Protocol 3)
  • 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)
Basic Protocol 4:  Visualization with Chromogenic Substrates
 Materials
  • Membrane with transferred proteins, probed with antibody‐enzyme complex (see Basic Protocol 3 or Alternate Protocol 2)
  • 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)
Alternate Protocol 3:  Visualization with Luminescent Substrates
 Additional Materials (also see Basic Protocol 3)
  • 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, 1999) or digital image capture (unit 7.5)

NOTE: See Troubleshooting section for suggestions concerning optimization of this protocol, particularly when employing AP‐based systems.
Alternate Protocol 4:  Fluorescent Blot Preparation and Analysis
 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 Basic Protocol 3 or Alternate Protocol 2).
  • 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; Cell Biosciences FluorChem)
  • Appropriate excitation and emission light filters (Tables 8.3.7, 8.3.8, and 8.3.9)

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

  •  FigureFigure 8.3.1 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. Figure courtesy of Hoefer, Inc.
    View Image
  •  FigureFigure 8.3.2 Large gel tank electrotransfer unit. Designed for larger 15 × 21–cm gels, these units will process four gels at a time. Note that the two outside panels hold the electrode grid. Figure courtesy of Hoefer, Inc.
    View Image
  •  FigureFigure 8.3.3 Slot‐blot unit. Through use of a vacuum manifold or by simple hand spotting, up to 96 samples can be applied to a single NC or PVDF membrane for immunoblotting analysis. 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 a sample. Figure courtesy of Hoefer, Inc.
    View Image
  •  FigureFigure 8.3.4 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 each 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‐size gel (14 × 14 cm), this means 60 and 200 mA, respectively.
    View Image
  •  FigureFigure 8.3.5 Immunoblotting with a tank transfer unit. The gel/membrane sandwich is held in a transfer cassette between two pads and assembled in the following order: three sheets of transfer buffer–soaked filter paper, transfer buffer–equilibrated gel, wet membrane, and three sheets of transfer buffer soaked filter paper. The sandwich is placed in the cassette and immersed in transfer buffer between the electrodes. 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.
    View Image
  •  FigureFigure 8.3.6 Serial dilution of primary antibody directed against the 97‐kDa catalytic subunit of the plant plasma membrane ATPase. The blot was developed with HRPO‐coupled avidin‐biotin reagents according to Alternate Protocol 2 and visualized with 4‐chloro‐1‐naphthol (4CN; Basic Protocol 4). Note how background improves with dilution. In this case, a dilution of 1/400 to 1/800 is optimal, keeping the background to a minimum.
    View Image
  •  FigureFigure 8.3.7 Pseudo‐coloring and combination of two separate images from a single blot into a combined image illustrates the final combined blot showing.
    View Image

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Literature Cited

Literature Cited
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