Immunoblotting; an introduction from GE Life Sciences

 

Introduction

Western blotting, also known as immunoblotting, is a well established and widely used technique for the detection and analysis of proteins. The method is based on building an antibody:protein complex via specific binding of antibodies to proteins immobilized on a membrane and detecting the bound antibody with one of several detection methods. The Western blotting method was first described in 1979 (1) and has since become one of the most commonly used methods in life science research.

The aim of this handbook is to guide and inspire beginners as well as experts toward successful Western blotting. It provides expertise and support, taking you through the complete Western blotting workflow, from sample preparation to detection and analysis. Chapters 2 to 8 follow the Western blotting workflow step by step and describe theoretical as well as practical aspects of the technique together with useful hints and tips. Examples of typical applications as well as some new approaches to Western blotting are described in Chapter 9. Moreover, protocols and recipes are provided in Chapter 11, including a recommended standard Western blotting procedure, to help you design and run your experiments. Over the past decade, improved detection methods and software have brought quantitative analysis to Western blotting and this handbook provides examples and protocols to help you adapt your methods and obtain more quantitative data from your Western blots. Finally, a troubleshooting guide is provided in Chapter 10, just in case!

A series of methodology handbooks is available from GE Healthcare, many of which cover topics closely connected to Western blotting, such as sample preparation or 2-D gel electrophoresis. Where appropiate, we refer to these handbooks in the text and provide links to the PDF versions in the on-line version of this handbook.

The Western blotting workflow

Although the details of Western blotting protocols may vary from application to application, with adaptations to suit specific protein characteristics and the level of information required, they all follow some common basic steps.

The sample of interest, for example, must usually undergo some degree of preliminary treatment before continuing to separation by electrophoresis. A sample may consist of a complex protein mixture such as a cell or tissue extract, but it can also be a sample of purified proteins, such as a fraction from a purification procedure.

The sample is applied to gel electrophoresis for protein separation and the proteins are then immobilized on a membrane following electrophoretic transfer from the gel. Non-protein binding areas on the membrane are blocked to prevent non-specific binding of antibodies. The membrane is incubated with a primary antibody that specifically binds to the protein of interest. Unbound antibodies are removed by washing and a secondary antibody conjugated to an enzyme, a fluorophore or an isotope is used for detection. The detected signal from the protein:antibody:antibody complex is proportional to the amount of protein on the membrane.

The most commonly used method for detection is chemiluminescence, based on secondary antibodies conjugated with horseradish peroxidase enzyme. On the addition of a peroxide-based reagent, the enzyme catalyses the oxidation of luminol resulting in the emission of light. The light signal can be captured either using a charge-coupled device (CCD) camera-based imager or by exposure to X-ray film. A more recent detection method is fluorescence. The secondary antibodies are labeled with a fluorophore such as a CyDye™. A fluorescent light signal can

be detected directly using a laser scanner or a CCD camera-based imager equipped with appropriate light sources and filters. Regardless of the detection method, the signal intensity correlates with the amount of protein and can be visually estimated as well as quantitated using analysis software.

GE Healthcare and Western blotting

Since the introduction of the first enhanced chemiluminescent (ECL™) detection reagent for Western blotting – Amersham™ ECL – in 1990, the portfolio of products offered by GE Healthcare has been improved and optimized across all Western blotting requirements from electrophoresis and transfer equipment to highly sensitive detection systems and software. By selecting the optimal equipment, gels, markers, blockers, secondary antibodies, detection reagents, imaging systems and software, the portfolio of GE Healthcare products for Western blotting enables you to achieve excellent results with as little trouble as possible.



Chemiluminescence

Chemiluminescence is defined as light emission produced in a multistep reaction whereby peroxidase catalyzes the oxidation of luminol. In the presence of chemical enhancers and catalysts, the light intensity and the duration of light emission is greatly increased, in a process known as enhanced chemiluminescence, ECL. The light emission produced in the ECL reaction peaks after 5 to 20 min and decays slowly, depending on the characteristics of the detection reagent. The maximum light emission occurs at a wavelength of 425 nm and can be detected by a CCD camera or by exposure to X-ray film.

ECL based on horseradish peroxidase (HRP)-conjugated secondary antibodies has become the most commonly used detection method for Western blotting. It is a sensitive detection method, where the light emission is proportional to protein quantity. Minute quantities of proteins can be detected and quantitated.

Amersham ECL

Amersham ECL is a sensitive chemiluminescence detection reagent suitable for a wide range of Western blotting applications for detection of moderate levels of proteins. The light signal can be captured on X-ray film, or a CCD camera-based imager. It is an excellent choice for confirmatory Western blotting applications when the protein amount is not limited such as in protein purification processes or detection of the expression of recombinant proteins.

Amersham ECL Prime

Amersham ECL Prime is a chemiluminescence detection reagent, designed to provide very high sensitivity and long signal duration. The light is intense, which makes it optimal for CCD camera- based imagers, such as ImageQuant™ LAS 4000, although X-ray film can be used as well. Moreover, the high signal intensity allows the use of low antibody concentrations with sustained performance. Minute levels of protein can be detected and a linear signal response across a wide range of protein levels allows high precision quantitative analysis. Amersham ECL Prime is useful in all kind of applications but is especially recommended where high sensitivity and quantitation are required from a Western blotting experiment.

Fluorescence

Fluorescence is produced upon excitation of a fluorophore at a specific wavelength which then emits light at a distinct wavelength. Excitation of the fluorophore and detection of the light emission is done using an imager equipped with appropriate excitation light sources and emission filters.

Fluorescence detection is a relatively recent method for Western blotting and has some advantages over chemiluminescence detection. The fluorescent signal is detected directly from fluorophore-labeled secondary antibodies without the addition of any further reagents. The signal is stable and does not decay significantly. It is a very sensitive detection method where the signal is proportional to protein quantity. Moreover, it is possible to detect more than one protein at the same time by using secondary antibodies labeled with different fluorophores. These factors make fluorescence detection an excellent choice for quantitative, multiplexed analysis.

Amersham ECL Plex

Amersham ECL Plex fluorescence detection systems provide high sensitivity, as well as a broad linear dynamic range and are well adapted to quantitative Western blotting. The fluorescent signal is stable on the membrane for up to three months and is captured using a multichannel fluorescent imager or a CCD camera-based imager equipped with appropriate light sources and emission filters.

The emission of light of different wavelengths enables multiplexed detection – the simultaneous detection of up to three proteins on the same membrane. This makes Amersham ECL Plex optimal for quantitative applications where a target protein and an internal standard such as

a housekeeping protein can be detected at the same time without stripping and reprobing. In addition, two proteins of similar molecular weight, such as a phosphorylated protein and the corresponding non-modified protein, can be simultaneously detected.

Chemifluorescence

Chemifluorescence is a fluorescent light emission produced in a reaction whereby alkaline phosphatase (AP) converts a reagent to a fluorescent product. Excitation of the product and detection of the light emission is done by using an imager equipped with appropriate excitation light sources and emission filters.

Chemifluorescence detection in Western blotting is a possible option if the preferred secondary antibody is conjugated with AP instead of HRP. Sensitivity is less compared with chemiluminescence, but the signal is more stable.

Amersham ECF

Amersham ECF is a chemifluorescence detection system with similar sensitivity to Amersham ECL. The signal is more stable than the chemiluminescent signal and is detected

with a fluorescence scanner. It can be used for Western blotting applications where the protein of interest is not limited, for example, in protein purification, or detection of the expression of recombinant proteins.

 Reference

  1. Towbin, H. et al. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U S A. 76, 4350-4354 (1979).