Nitrocellulose is easy to use and provides suitable data for most common enzymatic reporter experiments. Most common membranes used are nitrocellulose or polyvinyldifluoride (PVDF). The choice of WB membrane depends on the type of experiment to be performed. Fluorescent WB is also ideal for quantitative analysis since detection allows for wide dynamic ranges and signal normalization. Fluorescent detection is suitable for multiplex WB experiments where multiple targets can be detected in the same assay using fluorophore conjugates with non-overlapping emission spectra. When reporter enzymes are used, chromogenic, or luminescent substrates can be applied for detection.įluorophore reporter molecules do not require substrate but they do require specialized equipment for data collection. Reporter molecules include horseradish peroxidase alkaline phosphatase enzymes, and fluorophores. Western blot experiments can be performed in several formats, most of which require a conjugated secondary antibody to act as the reporter molecule. Wash steps using a mild detergent that contains buffer are also typically performed after antibody incubations to remove any non-specific binding. This is followed by an incubation with a secondary antibody conjugated to a reporter molecule, allowing the visualization of the target protein however, primary antibodies conjugated to a reporter do not require secondary antibody usage. The proteins are then transferred from the gel onto a membrane (typically nitrocellulose or PVDF), which is then blocked with a protein blocking buffer to prevent non-specific binding and probed using a primary antibody to detect the protein of interest. The Western blot assay uses gel electrophoresis (SDS-PAGE or native PAGE) to separate proteins according to molecular weight. It is a widely used method for detection of a specific protein in a complex matrix, such as cell or tissue lysate (i.e. Specific detection of bands corresponding to the protein of interest result from successively probing your blot with primary and conjugated secondary antibodies. Western blot (WB)-or immunoblot-is a workhorse immunoassay for most labs used to demonstrate antibody specificity, confirm gene expression, detect post-translational modifications (PTMs), diagnose diseases, and more.

Lycor dot blot protocol