ProAct™ Membrane Stain from Amresco

There are numerous commercial products that allow for the visualization of protein that has been transferred to solid membrane supports. Perhaps the most commonly used technique is Western blotting where stains are often used to determine the successful transfer of protein to membrane (usually nitrocellulose or PVDF) and further allow visual estimation of protein loading.

As a lab that performs Western blotting on a routine basis, we most often use ponceau solution. Unfortunately, a major disadvantage of ponceau is its relative ineffectiveness staining proteins that have been transferred to PVDF membranes. In addition, we have recently been interested in more quantitative protein detection techniques and for this purpose have been utilizing the Licor Odyssey near-infrared (NIR) imaging system. However, despite the visual removal of ponceau stain from the membrane, it exhibited significant background signal in the IR 680nm (red) channel to the point where some positive signals could not be clearly identified. In an attempt to resolve this issue, we used the Licor-recommended 1:5 dilution of ponceau. Unfortunately, this also proved to inadequately suppress the background to an acceptable level. We therefore set out to test several commercially available stains based on the following criteria; 1) ability to stain both nitrocellulose and PVDF with an equivalent or superior sensitivity to ponceau and 2) low background in the IR 680nm channel when using the Licor Odyssey. In summary, Amresco’s ProAct™ membrane stain satisfied both the above criteria.

ProAct is supplied as a 1 liter ready-to-use solution. The manufacturer recommends that membrane-bound protein be stained for between 30 seconds and 2 minutes. However, we have found that longer incubation periods (up to 10 min) give superior staining results. The background staining of the membrane is easily removed with several changes of distilled water, leaving the proteins visible as purple bands. In contrast to ponceau, ProAct is not readily removable with common Western wash buffer solutions, e.g. PBST/TBST. Although the manufacturer recommends incubating stained membranes in 0.1N NaOH for complete stain removal we have not observed any interference of the stain in subsequent antigen detection steps if it is not removed. In fact, we have noticed that the strength of the stain decreases significantly following the numerous washing steps utilized in a typical Western blot protocol.

The distinct advantage that ProAct offers over ponceau becomes apparent when blots are analyzed on the Licor Odyssey system. In contrast to the high background staining of membranes observed with ponceau, ProAct exhibits very low non-specific background staining in the IR 680nm channel of the Licor. Due to its low background, ProAct has become our preferred stain when quantifying our blots using Licor.