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Western Biot Detection Methods

Introduction

Western blotting is a routine yet powerful tool for detection and characterization of proteins. The technológy takes advantage of the specificity of nntibodies to provide definitive Identification of proteins that have been separated by polyacrylamide gei electrophorcsis and transferred to a membranę. Western blotting involves sequential recognition between three components: the target protein, a primary antibody that rccognizes the target protein, and a secondary antibody that ix)th rccognizes the primary antibody and generates a detcctable signal. Aftcr proteins have been bound to a membnine, the membranę is blocked, ineubated with primary antibody, then secondary antibody, and washed. The primary antibody binds to the protein of interest, and the secondary antibody enablcs its detection.

The secondary antibody can be labeled with a radioisotope, a fluorophore, or an enzyme, typically horseradish peroxidase (HRP) or alkaline phosphacase (AP).

For many years, radioisotopes were the method of choice to label secondary antibody probes for blotting applications. Ncwer methods arc less hazardous and easier to use chan radioactivity, but give cómparablę sensitivity. Current western blotting methods include light-based (chemi-luminescent, bioluminescent, chemifluorescent and fluorescent) detection, autoradiography, and colorimetric detection (Table 1 and Figurę 1).

Chemiluminescent Detection Chemiluminescence is the production of detcctable light from a Chemical reaction. For western biot detection, such reactions can be catalyzed by an enzyme such as AP or HRP conjugated to an antibody. The light signal can be captured on X-ray film or on CCD instruments such as the Bio-Rad VersaDoc “ and ChemiDoc” XRS systems.

The benefits of detecting chemiluminescent signals with a CCD system are numerous. The linear dynamie rangę of CCD systems is 2 to 4.8 orders of magnitudc, comparable to that of

Table 1. Comparison of western biot detection methods.

phosphor imager*. CCD system* also offer eonvenlence and efficicncy by providlng a digital record of expcrimcnts for s ha ring and data analysis and by eliminaring the necd to continually purcha.se consumablcs for film deveIopment.

Chemiluminescent technology ls easily adapted to cxisting western blotring procedurę* because it uses antibody-conjugated enzymes to generale the light signal. The hlocking and wash procedures are familiar western blotting steps. Most chemiluminescent methods require only a few Chemical components to generate light. For luminol chemi-luminescence, a subtrate is chemically oxidized by HRP to producc an cxcited-state product. In most Iuminol-bascd methods, induding the Immun-Star" HRP kit, light is emitted when the excited product rćturns to the ground State. The signal of the biot is visunlized hy exposing the biot to film or using imaging systems as described above. Besides luminol, some other commercially available chemiluminescent subtrates include acridan-based subtrates like Lumigen PS-3 (which use AP or HRP as a reporter enzyme)"and l'2'dioxetane-based subtrates_(which use AP as a reporter enzyme). The emission of light can be enhanced up to l ,000-fold by the addition ofenhancers such as phenols. The enhancer used differentiates one suppliers kit from another.

Chemiluminescent western biot detection has several advantages over other methods. including speed and sensitivity. Average exposure times are 30 seconds to 15 minutes. This is a large improvement over ^l2Sl detection, which can require up to 48 hours of film exposure. Detection of Iow picogram amounts of protein is typical of chemiluminescent systems, which are morę sensitive than most colonmetric systems, and approximately equal to that of radioisotopic detection. It is important to notę that che detect ion sensitivity is somewhat dependent on the affinity of the antigen and primary and secondary anrihodies and can vary considerably from one protein samplc to another.

Chemiluminescent detection does not have the disadvantages related to isotope detection, such as

Sensitivity

Economy oł antibody use Speed of detection Ease of reprobing Ease of quantitation Durability of results Suitable detection system

Luminescent

(Chemi- and Blo<)

Excellent

Excellent

Excellent

Very good

Very good

Excellent

ChemiDoc XRS

YersaDoc 3000/4000

Chemifluorescent

Very good

Very good

Excollent

Fair

Fair

Fair

Molecular Imager FX Pro Plus

Fluorescent Very good Excellent Excellent Fair

Excellent Excellent Molecular Imager

FX Pro Plus ...

Radioisotopic

Exc#n

Excellen^;.

Poor

Fair

Exceflent Bceellent Molecular Imager FX Pro Rus

Colorimetric

Verygood

Good

Good

Poor

Far

Good .

GS-800