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Chemiluminescent Western Blotting

Chemiluminescent substrates are popular because they offer several advantages over other detection methods. These advantages have allowed chemiluminescence to become the detection method of choice in most protein laboratories. Using chemiluminescence allows multiple exposures to be made in order to obtain the best image. The detection reagents can be removed and the entire blot reprobed to visualize another protein or to optimize detection of the first protein. A large linear response range allows detection and quantitation for a large range of protein concentrations. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method.

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Watch this video on Western blot detection using chemiluminescent substrates

Introduction

Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This is in contrast to substrates that produce a stable, colored product; these colored precipitates remain on the membrane after the enzyme-substrate reaction has terminated. On a chemiluminescent Western blot, the substrate is the limiting reagent in the reaction; as it is exhausted, light production decreases and eventually ceases. A well-optimized procedure using the proper antibody dilutions will produce a stable output of light for several hours, allowing consistent and sensitive detection of proteins.

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Stripping and Reprobing Blots

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Chemiluminescent Western Blotting Substrate Selection Guide

Luminol Reaction

Chemiluminescent reaction of luminol. Chemiluminescence is a property of chemical reactions which emit light as a byproduct. Luminol is one of the most widely used chemiluminescent reagents. The Oxidation of luminol by peroxide results in creation of an excited state product called 3-aminophthalate*. This product decays to a lower energy state by releasing photons of light.

Data Imaging for Chemiluminescence

The localized formation of peroxide through reaction of hydrogen peroxide with immobilized horseradish peroxidase makes this a popular Western blotting system. As it decays back to the ground state, the 3-aminophthalate* emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Although x-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, cooled CCD cameras offer the advantages of qualitative analysis, instant image manipulation, higher sensitivity, greater resolution and a larger dynamic range than film. Plus, you don't have to spend quality time in the darkroom.

Although electronic data capture with digital cameras and imagers is growing in popularity as the technologies improve and equipment prices decline, most of the data obtained from Western blotting with chemiluminescence is still captured on film. Often, it is necessary to expose several films for different time periods to obtain the proper balance between signal and background. The goal is to time the exposure of the membranes to the film so that the desired signal is clearly visible while the background remains low. This is difficult to accomplish since the process cannot be observed and stopped when the desired endpoint is reached. If the film is not exposed long enough (underexposed), the signal will not be visible. If the film is exposed too long (overexposed), the signal may be lost in the background or separate bands may become blurred together.

Overexposed film can be optimized after exposure by using a film treatment option that effectively reduce the film exposure time without altering the integrity of the data. This is done at the lab bench while watching the film and the process can be halted when the signal is clearly visible and background is at a minimum.

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Tech Tip #32: Guide to enzyme substrates for Western blotting

Tech Tip #67: Chemiluminescent Western blotting technical guide and protocols

Tech Tip #24: Optimize antigen and antibody concentrations for Western blots

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myECL Imager

Western Blot Kits

Filter Paper and Membranes

X-ray Film

Background Eliminator for Film

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Western Blotting Handbook

Molecular Weight Markers for Chemiluminescent Detection

For detection of any Western blot, it is desirable to use prestained molecular weight markers that are transferred to the membrane along with the protein sample. The appearance of the molecular weight markers on the membrane allows estimation of molecular weights for any protein bands that are detected as well as effective separation of the proteins of interest in the gel prior to the transfer step. When chemiluminescent detection is used for Western blotting, protein bands are detected on film or with digital imaging equipment. Unless modified, molecular weight markers do not show up on film or the imaging system since they do not produce an output of light. For this, there are several solutions described below.

One solution is the use of molecular weight markers that employ antibody binding domains from Protein A or G. The antibody capture domains of these proteins are engineered into the molecular weight markers and thus bind to the antibodies used in the Western blot and thus allow a signal to be generated and captured alongside the experimental data. The application of these markers is limited because the variable affinities of antibodies for Protein A and G result in variable levels of signal. Many antibodies such as those of the mouse IgG1 subclass, do not bind strongly to Protein A or G.

Another popular option is to use biotinylated protein molecular weight markers, which allow the use of streptavidin alongside the detection antibodies or when a biotinylated primary detection antibodies are used.

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SuperSignal Molecular Weight Protein Ladders

Other Unstained and Prestained Protein Ladders

Written and/or reviewed by Jared Snider, Ph.D.

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