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Labeling
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Labeling in Western blotting allows the user to identify his or her target protein so it can be specifically recognized during the detection step. This procedure usually involves two steps, the primary antibody and the secondary antibody.
The primary antibody is usually an unlabelled antibody directed against the target protein. Following transfer of proteins onto a membrane, saturate non-specific interactions with a blocking agent, and incubate the membrane in a solution of the primary antibody. The antibodies specifically bind to the target antigen and the excess is washed away.
After this first wash, incubate the membrane in a solution of secondary labeled antibody, which is directed against the primary antibody. Again, wash away the excess antibodies to get the membrane ready for detection.
Many variations of this standard protocol exist. You can use the Biotin-Streptavidin system, use a labeled primary antibody, or amplify your signal with a third labeled antibody. The selection of a method depends on the level of sensitivity and the signal-to-noise ratio required.
The tag used is also part of the protocol variations for labeling in Western blotting. You can use an antibody tagged with radiochemicals, horseradish peroxidase (HRP), alkaline phosphatase (AP) or fluorescent molecules.
GE Healthcare is a leading supplier of labeling products for Western blotting, offering one of the widest ranges of products on the market today.
Factors to consider for labeling :
Choosing the label
Selecting the labeling method for Western blotting will depend on many parameters including level of sensitivity and ease-of-use. There are four kinds of labelling systems that are generally used:
Chemiluminescence
Chemiluminescence is the most popular labeling system used for Western blotting. |  |
Its principle relies on the enzymatic conversion of a luminol-like molecule to a reactive molecule by horseradish peroxidase (HRP). This molecule generates light in the presence of hydrogen peroxide; the emission is usually amplified by an enhancer. This method gives a high level of sensitivity, does not require expensive instruments, and generates no hazardous wastes. In addition, the light produced by this method peaks
after 5–20 minutes and decays slowly thereafter.
Radioactivity
This method can use 35S or 125I-labeled antibodies. It does not require expensive instruments, but it still requires special consideration for handling of radioactive wastes and a dedicated fume hood when using 125I. Radioactive labeling methods can be highly sensitive but require long exposure time before detection.
Fluorescence
Fluorescence is a labeling method that can achieve levels of sensitivity comparable to radioactivity with the advantage of being detectable immediately. As the principle of fluorescence requires an excitation energy source, which is usually a laser beam, this method can be performed only with specialized scanners and image acquisition systems.
Chemifluorescence
Chemifluorescence is a labeling method where an enzyme, alkaline phosphatase (AP), converts its substrate to a fluorescent precipitate that can be detected on a fluorescence scanner. The AP is conjugated either to an antibody or a streptavidin molecule. The method is as sensitive as chemiluminescence and has a wider dynamic range than radioactivity.
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Labeling of the antibody
Using a label on either the primary or the secondary antibody is a matter of selectivity, economics, and signal-to-noise ratio. Usually, Western blotting protocols utilize a non-labeled primary antibody directed against the target protein and a species-specific labeled secondary antibody directed against the primary antibody.
Labeling the primary antibody
Primary antibodies are carefully tested for target protein selectivity. This process generates lower yields and is usually more expensive than secondary antibody production. Presumably, the cost to the end user would be even higher to engineer tagged primary antibodies. Homemade labeling methods can be performed, but quality cannot be monitored. In addition, primary antibodies act more as direct labeling, thus generating a signal of medium intensity during the detection step.
Labeling the secondary antibody
In contrast with primary antibodies, secondary antibodies are directed against all antibodies, thus are easier to produce and to engineer for any kind of label, resulting in lower costs for the end user. In addition, the recognition of primary antibodies by secondary antibodies generates a pyramidal amplification, thus giving an excellent signal level for detection and an appreciable signal-to-noise ratio.
Labeling the tertiary antibody
Protocols using a tertiary antibody usually complement a labeled secondary antibody to amplify the poor signal generated from proteins with low level of expression. This method implies an additional incubation step, which now can be avoided with the development of more powerful labeling and detection reagents for Western blotting.
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