Luminol HRP Western Blot Substrate From Boston BioProducts

Although there is no shortage of choices when it comes to horseradish peroxidase (HRP)-based chemiluminescent detection kits for Western blots, Boston BioProducts (Worcester, MA) has recently introduced a product that is well worth looking into. Their kit consists of two reagents, a luminol solution and an oxidizing solution. As with most other commercial chemiluminescence kits, the working reagent is prepared by mixing together equal volumes of the luminol and oxidizer solutions. The working solution is then applied to the immunoblot following incubation with primary and secondary antibodies. The blot is then used to expose X-ray film.

I recently compared Boston BioProducts’ Luminol HRP substrate to SuperSignal West Femto (Pierce, Rockford, IL). The results are shown in Figure 1. As expected, the SuperSignal gave a very strong signal with a 1:50,000 dilution of the HRP-conjugated secondary antibody and a 30 sec exposure. At this same dilution, Boston BioProducts’ Luminol gave a respectable signal with a 5 min exposure time. Increasing the secondary antibody concentration permitted shorter exposure times. There was essentially no background staining with the Luminol even with a 30 min exposure.

Although SuperSignal West Femto is very impressive when it comes to sensitivity, there are a couple of pertinent issues to keep in mind. For one thing, we have found that such sensitivity is only rarely required and frequently is an annoyance. Most commercial chemiluminescent substrates are sufficiently sensitive to detect all but the least abundant of antigens. In fact, ultra-high sensitivity substrates often result in high background or the need to let the signal decay before a clear exposure is obtained. The second issue is cost. While SuperSignal West Femto is probably the most sensitive substrate available, it is also likely the most expensive. A kit containing 50 ml of each reagent (to make a total of 100 ml working solution) costs $310. Pierce’s less sensitive product, SuperSignal West Pico costs $200 for a kit that will make 500 ml working solution. These prices translate to $3.10/ml for the Femto and $0.40/ml for the Pico. In stark contrast, Boston BioProducts’ Luminol is $95 for the 1000 ml working solution kit, or 9.5 cents/ml. That’s over 32 times less expensive than the Femto and about 4 times less expensive than the Pico.

The take-home message is that a very good signal can be obtained using Boston BioProducts’ Luminol solution simply by lengthening the X-ray film exposure time and/or increasing the concentration of the secondary antibody, relative to the conditions used for a good SuperSignal West Femto signal. If you currently use a product less sensitive than SuperSignal West Femto, your usual secondary antibody concentration and exposure time should work well with Luminol. The big plus is the cost. Boston BioProducts’ Luminol is so much less expensive than SuperSignal West Femto it more than makes up for the increased expenditure of secondary antibodies.