Analysis of Membrane Proteins by Western Blotting and Enhanced Chemiluminescence

  • Samantha J. Bradd
  • Michael J. Dunn
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 19)

Abstract

The high resolution capacity of polyacrylamide gel electrophoresis (PAGE) (seeChapter 19) has resulted in the widespread application of this group of techniques to protein separations. PAGE procedures can provide characterization of proteins in terms of their charge, size, relative hydrophobicity, and abundance. However, they provide no direct clues as to the identity or function of the separated components. A powerful approach to this problem is provided by probing the separated proteins with antibodies and other ligands specific for components of the protein mixture being analyzed.

1 Introduction

1.1 Background

The high resolution capacity of polyacrylamide gel electrophoresis (PAGE) (seeChapter 19) has resulted in the widespread application of this group of techniques to protein separations. PAGE procedures can provide characterization of proteins in terms of their charge, size, relative hydrophobicity, and abundance. However, they provide no direct clues as to the identity or function of the separated components. A powerful approach to this problem is provided by probing the separated proteins with antibodies and other ligands specific for components of the protein mixture being analyzed.

Early attempts to exploit this approach involved the reaction of gels after electrophoresis with solutions of the probes being employed, this technique being termed immunofixation. However, the polyacrylamide gel matrix is restrictive to the diffusion of probe molecules, so that prolonged incubation and washing steps have to be used to achieve a strong specific signal with a low background. Unfortunately, the separated protein zones are also subject to diffusion during this time resulting in loss of resolution and merging of adjacent bands.

These problems were solved with the advent of protein-blotting techniques, based on those developed by Southern (1) for analysis of electrophoretic separations of DNA. In these procedures the proteins, after separation by electrophoresis, are transferred (“blotted”) onto the surface of a membrane such as nitrocellulose. When immobilized on the surface of a membrane, the proteins are readily accessible to interaction with probes. Moreover, relatively short incubation times can be used, and the proteins themselves are no longer subject to diffusion.

1.2 Experimental Strategy

1.2.1 Apparatus

Various techniques have been employed for the transfer of electrophoretically separated proteins to membranes. However, the most popular method is the application of an electric field perpendicular to the plane of the gel. This technique of electrophoretic transfer, first described by Towbin et al. (2), is known as Western blotting. Two types of apparatus are in routine use for electroblotting. In the first approach the sandwich assembly of gel and blotting membrane is placed vertically between two platinum wire electrode arrays contained in a tank filled with blotting buffer (3). The disadvantages of this technique are that (1) a large volume of blotting buffer must be used, (2) efficient cooling must be provided if high current settings are employed to facilitate rapid transfer, and (3) the field strength applied (V/cm) is limited by the relatively large interelectrode distance. In the second type of procedure the gel-blotting membrane assembly is sandwiched between two horizontal plate electrodes, typically made of graphite (4). The advantages of this method are that (1) relatively small volumes of transfer buffer are used, (2) special cooling is not usually required, although the apparatus can be run in a cold room if necessary, and (3) a relatively high field strength (V/cm) can be applied owing to the short interelectrode distance resulting in faster transfer times.

1.2.2 Blotting Membranes

A variety of membranes have been described for Western blotting (3), but nitrocellulose is still the most popular. Supported nitrocellulose membranes that overcome the problem of the fragility of this matrix are now available. Recently other membranes based on the use of polyvinylidine difluoride (PVDF) or polypropylene have been produced. These membranes have the advantages of robustness and high protein-binding capacity. These membranes work well in immunoblotting protocols (5) and are used extensively in techniques that allow the direct chemical characterization of proteins separated by PAGE (e.g., microsequencing, amino acid analysis, peptide mapping) (6).

1.2.3 Visualization

After transfer, total protein patterns can be visualized by total protein staining, usually with Amido black (3). Before reaction with specific ligands, blots must be reacted with a reagent to block any protein-binding sites remaining on the membrane. A variety of reagents such as bovine serum albumin, gelatin, and nonionic detergents can be used for this blocking step (3), and more recently dried nonfat milk has become popular for this purpose (7). After blocking, the blot is reacted with the specific ligand. Visualization of the bound ligand can be achieved by labeling of the primary ligand itself, e.g., by radiolabeling (usually 125I) or enzyme conjugation. However, this approach is not generally popular, as derivitization of the primary ligand can often interfere with its reactivity. Generally an indirect technique is used in which the blot is reacted with a second reagent specific for the primary ligand. For antibodies this involves the use of an anti-Ig specific for the species in which the primary antibody was produced. Again visualization can be achieved by radiolabeling, e.g., 125I-protein A (8). However, techniques in which the secondary reagent is conjugated with an enzyme (e.g., horseradish peroxidase, alkaline phosphatase) and subsequently visualized with a colored reaction product are much more popular (9). The sensitivity of these methods can be substantially increased using the avidin-biotin system (10) or colloidal gold techniques (11).

More recently, visualization techniques based on the use of chemiluminescent reactions of reagents such as cyclic diacylhydrazides have been developed for use with Western blots (12). This chapter describes the use of one such method in which horseradish peroxidase is used to catalyze the oxidation of luminol in the presence of hydrogen peroxide under alkaline conditions. This reaction results in the emission of light and can be increased up to 1000-fold by certain enhancers (e.g., para-substituted phenolic compounds). This enhanced chemiluminescent (ECL) system is used in conjunction with standard immunodetection protocols employing peroxidase-conjugated secondary antibodies as shown in Fig. 1. The image is visualized by expo- sure of the blot to an appropriate blue-sensitive film, usually for times of between 15 s and 1 h. This method provides at least a 10-fold increase in sensitivity over other detection methods, and facilitates the detection of <1 pg of protein.
Fig. 1.

Diagram showing the mechanism of detection of proteins on Western blots using the ECL system.

2 Materials

2.1 Semidry Blotting

Prepare buffers from analytical grade reagents and dissolve in deionized water. Buffers should be stored at 4°C and are stable for up to 3 mo.

  1. 1.

    Transfer/equilibration buffer: 20 mM Tris, 150 mM glycine, pH 8.3. Dissolve 2.42 g Tris base and 11.26 g glycine and make up to 1 L. The pH of the buffer is pH 8.3 and should not require adjustment.

     
  2. 2.

    Transfer membrane: Hybond-ECL nitrocellulose transfer membrane (Amersham International) cut to the size of the gel to be blotted (seeNote 1).

     
  3. 3.

    Filter paper: Whatman 3MM filter paper cut to the size of the gel to be blotted.

     
  4. 4.

    Equipment: A number of commercial companies produce semidry electroblotting apparatus and associated power supplies. We have used the NovaBlot apparatus from Pharmacia Biotechnology.

     
  5. 5.

    Rocking platform.

     
  6. 6.

    Plastic boxes for gel incubations.

     

2.2 Protein Detection Using Enhanced Chemiluminescence (ECL)

  1. 1.

    Washing solution (PBS-T): Phosphate buffered saline (PBS) containing 0.05% (w/v) Tween 20. Make 0.05 g Tween 20 up to 100 mL with PBS (8.0 g/L NaCl, 0.2 g/L KC1, 1.15 g/L Na2HPO4·2H2O, 0.2 g/L KH2PO4).

     
  2. 2.

    Blocking solution (PBS-TM): PBS-T with the addition of 3% (w/v) nonfat dried milk (Marvel). Dissolve 3 g milk powder and make up to 100 mL with PBS-T.

     
  3. 3.

    Secondary antibody reagent: Peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (DAKO, Carpinteria, CA) diluted 1:1000 in PBS-TM (seeNote 2).

     
  4. 4.

    ECL Western blotting detection kit (Amersham International).

     
  5. 5.

    Detection film: Hyperfilm ECL (Amersham International) (seeNote 3).

     
  6. 6.

    Diaminobenzidine (DAB) solution: Contains 0.05% (w/v) DAB and 0.01% (v/v) H2O2. Dissolve 0.05 g DAB in 100 mL of PBS, and add 0.01 mL H2O2 (30% stock solution).

     
  7. 7.

    Photographic equipment: Photoradiography cassettes are required for exposure of the blots with ECL film. The film can be developed using an automatic X-ray film developing unit or by hand using standard X-ray developer and fixer.

     
  8. 8.

    Rocking platform.

     
  9. 9.

    Plastic bags for incubations.

     
  10. 10.

    Glass plate. ll.SaranWrap™.

     

3 Methods

3.1 Semidry Blotting

This technique has been described in detail in vol. 3 of this series (4) and will only be briefly described below.

  1. 1.

    Following separation of the membrane proteins by SDS-PAGE (seeChapter 19), place the gel in equilibration buffer, and gently agitate on a rocking platform for 30 min at room temperature.

     
  2. 2.

    Stack six sheets of filter paper wetted with blotting buffer on the lower (anode) plate of the electroblotter, followed by the prewetted nitrocellulose transfer membrane. Then carefully place the gel on top of the membrane. Apply a further six sheets of wetted filter paper, and place the upper plate (cathode) in position (seeNote 4).

     
  3. 3.

    Transfer the proteins at 0.8 mA/cm2 of gel for 40 min (seeNote 5).

     

3.2 Protein Detection by ECL

Carry out all incubations with gentle agitation on a rocking platform at room temperature.

  1. 1.

    Following transfer, gently remove the transfer membrane from the electroblotter; seal in a plastic bag containing the blocking solution, and incubate for 1 h.

     
  2. 2.

    Remove the blocking solution and incubate the blot for 1 h with the primary antibody diluted at an appropriate concentration in PBS-TM (seeNote 6). In the example illustrated in Fig. 2 (strips 2 and 3), mouse monoclonal antibodies to the α- and β-subunits of erythrocyte spectrin were diluted 1:10,000 and 1:1000 respectively for visualization using ECL.

     
  3. 3.

    Wash the blot three times for 5 min with PBS-T.

     
  4. 4.

    Incubate the blot for 1 h with peroxidase-conjugated secondary antibody diluted at an appropriate concentration (1:1000 in this example) in PBS-TM.

     
  5. 5.

    Wash the blot three times for 5 min in PBS alone.

     
  6. 6.

    Mix the two ECL reagents in a ratio of 1:1 to give a final volume of 0.125 mL/cm2 transfer membrane. Incubate the blot in the ECL reagent mixture for 1 min.

     
  7. 7.

    Drain excess solution from the blot; then place on a clean glass plate (protein side uppermost) and cover with food wrapping film (e.g., Saran Wrap™). It is important that the blot should not be allowed to dry out at this stage. Air bubbles and creases in the film must be avoided.

     
  8. 8.

    Expose the blot to the film in the dark for 15 s to 60 min and then develop using an automated processor or by hand. In the example illustrated in Fig. 2 an exposure of 1 min was used.

     
  9. 9.

    After completion of the ECL exposure, the transfer membrane can be washed (2×15 min in PBS-T) and visualized by the addition of DAB solution for 10 min at room temperature.

     
Fig. 2.

Result of an immunoblotting experiment to detect the α- and β-subunits of spectrin after SDS-PAGE separation of human erythrocyte membrane proteins. 1, Amido Black stain of blot; 2 to 4, visualization using the ECL system; 5 and 6, visualization using DAB. Monoclonal antibodies to α-spectrin (2, diluted 1:10,000; 5, diluted 1:1000), β-spectrin (3, diluted 1:1000; 6, diluted 1:200), αβ-spectrin (4, diluted 1:500).

4 Notes

  1. 1.

    Hybond ECL is a 0.45-µm pore size nitrocellulose membrane. Although this is the recommended immobilizing matrix to be used with the ECL detection kit, other nitrocellulose membranes and nylon filters are also suitable.

     
  2. 2.

    The appropriate peroxidase-conjugated anti-Ig specific for the species in which the primary antibody was produced must be used.

     
  3. 3.

    The film used to detect the signal produced by the ECL system must be sensitive to blue light. Hyperfilm ECL has been specifically chosen to give optimum results with the ECL technique.

     
  4. 4.

    The method for setting up the semidry blotting apparatus is generally applicable and is appropriate for the equipment that we have used. Instructions provided with equipment from other manufacturers should be followed.

     
  5. 5.

    The maximum mA/cm2 of gel quoted applies to the apparatus we have used. This should be established from the manual for the particular equipment available.

     
  6. 6.

    The appropriate dilutions of the primary and secondary antibody reagents and the exposure time required to obtain optimal results will vary for each particular primary antibody. These parameters must be established using serial dilutions and varying exposure times so that a strong specific signal is obtained with minimal nonspecific background staining. For visualization using ECL, the antibodies can usually be diluted 5–10 times more than for visualization using DAB.

     

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  9. 9.
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Copyright information

© Humana Press Inc. 1993

Authors and Affiliations

  • Samantha J. Bradd
    • 1
  • Michael J. Dunn
    • 1
  1. 1.Department of Cardiothoracic SurgeryNational Heart and Lung Institute, Heart Science CentreHarefieldUK

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