Because our lab performs western blot analysis a lot, we have had the opportunity to use the Roche BM Chemiluminescence Western Blotting Kit on an almost daily basis. Overall, we have found this kit to be an efficient, effective, and safe product for our western blot experiments. This kit relies on a horseradish peroxidase (HRP or POD) conjugated secondary antibody (against mouse or rabbit IgG). This HRP, in the presence of hydrogen peroxide, catalyzes the oxidation of a substrate (luminol) to create an activated, intermediate product. As this intermediate product decays, light is emitted which can be detected by film (or phosphor-imaging screens). In our hands, this system has been extremely sensitive, allowing us to detect antigen on a blot with very small amounts of antibody. And with most of our antibodies, the film exposure time for detecting our protein bands has been very short. Also, we have not noticed any problems with detection (no fading) due to reagent storage.
The kit itself comes with a number of components, including: Luminescence substrate solution A (2 x 125 ml bottles), Starting solution B (2 x 2ml tubes), blocking reagent (10 % stock solution) (1 x 100 ml bottle), and anti-mouse IgG-POD/anti-rabbit IgG-POD lyophilizate (1 x 50 U bottle). The reagents that are not included in the kit (but are required) are: Tween 20 and Tris-buffered saline (TBS), pH 7.5 (which can be easily made in the laboratory and should be stored at 4C when not in use). The POD-labeled secondary antibody is lyophilized, so it must be reconstituted in 100 microliters of distilled water - the stock solution is good for about a year when stored at 4 C. Also, TBS-Tween 20 (1 ml of Tween 20 diluted in 1l of TBS), 1% blocking solution (10 ml of blocking reagent diluted in 90 ml TBS), and 0.5% blocking solution (5 ml of blocking reagent in 95 ml of TBS) should be made and allowed to equilibrate to 15-25C before use.
Using this kit is not difficult – in fact the electrophoresis and blotting process is the same as for standard western blot protocols. We generally use PVDF membranes and block in 10ml of the 1% blocking solution (with shaking) for about 1 hour at room temperature (or overnight at 4C without shaking). We incubate the blot with our primary antibody that has been diluted in 0.5% blocking solution. The time of incubation can vary depending on how good your antibody is, but we generally incubate the blot with primary antibody for 1 hour (with shaking). Also, we have found that a freshly prepared primary antibody solution works best. The membrane washing steps are standard (2 x 20ml of TBS-Tween Solution, 10 minutes each followed by 2 x 20ml 0.5% blocking solution, 10 minutes each) and the secondary antibody incubation is performed with conditions similar to the primary antibody incubation (freshly prepared, diluted in 0.5% blocking solution, 1 hour, room temperature with shaking). We have found that 40 mU/ml of secondary antibody is sufficient for detection. Also, performing an overnight incubation with the secondary antibody is strongly not recommended as this tends to produce very high background. After incubation with the secondary antibody, the membrane is then washed four times with about 20 ml of TBS-Tween 20 solution at 15 min each. Usually, right after the second wash the detection reagent is prepared. The detection reagent is prepared by mixing substrate solution A with starting solution B at a ratio of 100:1. About 10 ml of the detection reagent should be prepared for each membrane blot. This detection reagent should be equilibrated for about 30 minutes before use for best results. After the wash, the membrane is incubated with the detection reagent for about one minute with gentle shaking and then placed onto an X-ray film (after wrapping the blot in saranwrap). Usually the protein band can be visualized after a one or two minute exposure. If the intensity of the signal is weak, then the film can be exposed for about an hour, but after one hour the signal intensity generally decreases.
The Roche BM Chemiluminescence Western Blotting Kit has become an integral part of our laboratory. This kit has proven to be very versatile in that the concentrations of primary and secondary antibodies, the incubation times, the washing times, etc. can all be modulated to produce the best results. As a side note, to conserve the blocking solution we tried using nonfat milk for blocking and for diluting antibodies, but we ended up with background problems, so we do not recommend trying this. Also, it is important that allthe reagents to be used are equilibrated to room temperature for optimal results. Overall, we are very satisfied with this product, and we highly recommend this product.
Hee Chul Lee
Graduate Student
Dept. of Biochemistry
NYU School of Medicine