Time to Shine! - Developments in 30 Years of Western Blotting Technology

Mon, 09/13/2010 - 04:20

The vast majority of antibodies in our antibody catalog are suitable for Western blotting studies. Devised almost 30 years ago by W. Neal Burnette, it has become a standard assay wherever antibodies are used to detect proteins.

Western blotting was originally a method of transferring protein gels onto nitrocellulose membranes via electrophoresis - which is essentially how it remains today. However, much has also changed. Burnette’s original experiments involved the detection of murine leukaemia proteins using radioactive iodine-labelling to bind antibody molecules. Today, researchers use far safer methods!

One of the most common forms of Western blotting is detection by chemiluminescence, a technique for which many of our antibodies at Novus Biologicals have already been validated. In chemiluminescence, a secondary antibody (i.e. one targeting an area of a primary antibody, which is itself targeted to the protein of interest) is conjugated, or bound, to an enzyme. In the presence of a suitable substrate, light is generated equal to the amount of antibody bound, which is detected by an imager or on film.

A popular and highly sensitive technique, chemiluminescent protein detection nevertheless suffers several drawbacks. For example, its reliance on enzyme kinetics makes it unreliable quantitatively. Also, standard chemiluminescence is a one-color technology, meaning multiple overlapping proteins cannot be distinguished from each other.

This has led to the development of fluorescent Western blotting, which has an extensive color spectrum of fluorochromes. Researchers can now probe multiple proteins (i.e. multiplex them) simultaneously, often 2 or 3 at once. By comparing them to a control, it is possible to quantify the amount of each protein in a sample. Many of the antibodies in our catalog have already been shown to be suitable for fluorescent blotting, and researchers who would like to test any antibody in a new application may take advantage of our Innovators Reward Program.

Additionally, now almost any antibody can be fluorescently labelled in under an hour using the Lightning Link conjugation kits!

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