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Once your samples are separated and transferred to a membrane, the target protein is probed by using a specific antibody.
Western blotting protocols most frequently utilize a non-labeled primary antibody directed against the target protein and a species-speciﬁc, labeled secondary antibody directed against the primary antibody. The signal emitted by the label is then measured and its strength indicates the quantity of target protein on the membrane.
Before probing, the membrane is incubated in a blocking solution to prevent unspecific binding of the antibodies.
Western blotting involves the immobilization, via hydrophobic interactions, of biomolecules on a membrane made of an inert material such as polyvinylidene diﬂuoride (PVDF) or nitrocellulose. As non-speciﬁc antibody binding to non-occupied spaces on the surface of the membrane is detrimental to the speciﬁcity and sensitivity of the assay, it is essential to "block" the membrane.
The choice of blocking agent, usually proteins or non-ionic detergents, is guided by the detection system in use and by empirical testing. For example, Bovine Serum Albumin (BSA) is commonly used as a blocking reagent.
The choice of primary antibody depends on how the target protein is folded, as different epitopes are exposed under different conditions. Although internal epitopes are normally concealed in proteins, antibodies against internal epitopes are advantageous when probing denatured proteins.
A wide variety of secondary antibodies are commercially available and the choice depends on the species in which the primary antibody was produced, while the choice of labeling method depends on parameters such as the desired level of sensitivity. Four kinds of labeling systems are in common use: enzymes, fluorophores, biotinylation, and radioisotopes.
Streptavidin has an extraordinarily high affinity for biotin, making the biotin/streptavidin interaction useful for several techniques including Western blotting. Typically, biotinylated secondary antibodies are used with streptavidin-HRP, which produces signal intensity and allows detection of low abundance targets.
Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two most commonly used enzymes for protein detection in Western blotting. Both can be used with either chemiluminescent, chemiﬂuorescent, or chromogenic substrates.
|Size (Mr)||140 000||40 000|
|Stability||Unstable below 0°C||Stable below 0°C|
|Number of substrates||Few||Many|
|Optimal pH range||8 to 10||5 to 7|
Due to the high sensitivity of digital imaging techniques, the use of ﬂuorophores in Western blotting systems is appealing. The benefits of fluorescence detection include high sensitivity, detection across a wide linear dynamic range of protein quantities, and signal stability over time.
These factors make fluorescence suitable for quantitative Western blotting. In addition, the use of multiple fluorophores that emit light of distinct, characteristic wavelengths allows the simultaneous detection of several proteins on a single blot (multiplexed detection).
The most common radioactive labels for antibodies are 35S and 125I. While expensive imaging systems are not required, radioactivity-based detection nevertheless requires special consideration for handling radioactive waste and a dedicated fume hood, especially when using 125I. Radioisotopes can be highly sensitive but require relatively long exposure.
Amersham Imager 600 is a sensitive and robust chemiluminescence imager for high-resolution digital imaging of protein and DNA samples in gels and membranes.
Blocking the membrane increases assay specificity and sensitivity by reducing non-specific antibody binding.
Sensitive and convenient enzyme-based detection of proteins.
No additional reagents required after secondary antibody probing.
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