Technical Support FAQ

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Technical Support FAQ


Question:

Can membranes be stripped after using the Immun-Star HRP chemiluminescent kits? What is the recipe for the stripping buffer?


Answer:

Membranes can be stripped and reused if kept wet after starting the western blot process. There are several protocols for stripping the membrane for a second probing with a different antibody. One of the most common calls for the use of high temperatures (70ºC) with 2% SDS and 100 mM beta-mercaptoethanol (BME) in 62.5 mM Tris-HCl, pH 6.7, for 30 min to destroy the antigen-antibody complex. Other methods use a very high (9.5) or very low (2.2) pH with the presence of detergents such as Tween 20 or SDS.

A harsher method, which will desorb proteins from polyvinylidene difluoride (PVDF) or nitrocellulose membranes, is outlined in an article by Montelaro (Protein antigen purification by preparative protein blotting, Electrophoresis 8, 432-438, 1987). In this method, 40% acetonitrile or pyridine is used to desorb protein from PVDF.


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