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Antibodies for Western Blot

Western blot is a technique for detecting specific proteins in a complex mixture, typically a cell or tissue extract. It involves separating the sample components by polyacrylamide gel electrophoresis (PAGE), before transferring them to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane for antibody-based detection.

Advantages of Western blot are that it is relatively easy to perform and analyze, and uses only small quantities of reagents. On the flipside, Western blot has limited throughput due to the amount of hands-on time required.

Western blot detection can be either direct (with labeled primary antibodies) or indirect (with unlabeled primary antibodies and labeled secondary antibodies). Direct detection offers the advantage of fewer protocol steps than indirect detection. However, the commercial availability of conjugated primary antibodies can be a limiting factor.

Indirect detection provides signal amplification, due to polyclonal secondary antibodies binding to multiple epitopes on the primary antibody. It also increases flexibility for assay design, since a vast array of conjugated secondary antibodies is available commercially. Drawbacks of indirect detection include the increased number of protocol steps compared with direct detection and the potential for secondary antibody cross-reactivity.

Readouts for Western blot depend on the antibody label. Enzyme labels, such as horseradish peroxidase (HRP) and alkaline phosphatase (AP), can be used for chromogenic detection, whereby a chromogenic substrate (e.g., TMB or NBT/BCIP) is converted to an insoluble, colored product that precipitates onto the membrane. Advantages of chromogenic detection are that it generates a stable signal and does not require special equipment for processing or visualizing. However, the low sensitivity of chromogenic substrates means that chromogenic detection may not be suitable for detecting proteins of low abundance.

Enzyme labels can also be used for enhanced chemiluminescence (ECL) detection. This requires a chemiluminescent substrate and involves exposing the membrane to X-ray film or a digital charge-coupled device (CCD) imager. ECL detection provides high sensitivity, but produces a signal that fades over time.

Fluorescent labels, such as fluorescein (FITC) and the Alexa Fluor® dyes, are used for fluorescence detection with a fluorescent imaging instrument. These types of labels simplify multiplexing, but their use requires that blots be shielded from light. Critically, whichever detection method is chosen, staining conditions should be thoroughly optimized using relevant controls prior to testing experimental samples.

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Frequently Asked Questions

When choosing a primary antibody for Western blot, it is important to confirm that it has been validated for the Western blot application and that it recognizes the target of interest in the intended sample species. The immunogen used for antibody production is another key consideration. If the antibody was raised against a native protein, it may not be able to recognize its target under denaturing Western blot conditions. Likewise, if the antibody was raised against an N-terminal epitope, it will be unlikely to detect a C-terminal protein fragment.

When it comes to secondary antibody selection, the host species of the primary antibody is a critical factor. For example, when using a primary antibody from a mouse host, an anti-mouse secondary antibody is required for detection. The range of available conjugates should also be considered. Enzyme labels, such as horseradish peroxidase (HRP) and alkaline phosphatase (AP), allow for enhanced chemiluminescence (ECL) detection. Fluorophores, such as fluorescein (FITC) and the Alexa Fluor® dyes, are used for fluorescence detection. If the Western blot will involve using secondary antibodies for multiplexed detection, researchers are advised to choose secondaries that have been cross-adsorbed against off-target species and/or isotypes to minimize the potential for non-specific background signal.

Enzymes and fluorophores are the most common labels used with secondary antibodies in Western blotting. Of these, enzymes are typically used for enhanced chemiluminescence (ECL) detection, which benefits from high sensitivity but produces a relatively short-lived signal, while fluorophores are used for fluorescence detection, which is increasingly popular for its capacity to simplify multiplexing. Secondary antibodies may also be labeled with biotin, allowing for detection with a streptavidin conjugate (e.g., streptavidin-HRP or streptavidin-FITC), although this approach is more often used for signal amplification during immunohistochemical (IHC) staining.

Loading controls serve to confirm that any observed differences in signal intensity between samples are genuine and not due to uneven gel loading or protein transfer. They are typically proteins that exhibit abundant, constitutive expression in the sample type being studied, such as many so-called ‘housekeeping proteins’, which are required to maintain basic cell functions. Common loading controls include β-actin, α-tubulin, GAPDH, vinculin, and various mitochondrial proteins.

When choosing a loading control, it is important to confirm that it is highly expressed in your sample type and that protein expression will not be altered by experimental conditions. The loading control should also have a different molecular weight than your target of interest. For multiplexed fluorescent Western blotting, it is recommended to use a dim fluorophore for the loading control and bright fluorophores for less abundant proteins.

The benefits of using fluorophore-labeled antibodies for Western blot detection include the following:

  • Simpler multiplexing – unlike enzyme-based multiplexing, which involves repeated cycles of stripping and reprobing, the use of fluorophore-labeled antibodies allows for measuring several targets simultaneously
  • Broader dynamic range – both low and high abundance proteins can be detected at the same time without signal saturation
  • Proteins of similar molecular weight are easily distinguished – for example, different color fluorophores can be assigned to a protein and its phosphorylated isoform
  • More accurate quantitation – the amount of signal produced is proportional to the amount of protein present, and samples are not compromised by stripping and reprobing
  • Signal stability – fluorescent signals have a long lifetime, provided the blot is shielded from light, allowing for analysis at a later date
  • Reduced chemical waste – no requirement for processing x-ray film in a dark room

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