The Visualizer™ Spray & Glow™ Western Blotting Detection System from Upstate is a chemiluminescent system for detection of horseradish peroxidase (HRP) labeled proteins on Western blots. The system gives a chemiluminescent signal that is both strong and long lasting. The novel feature that differentiates this system from other chemiluminescent systems is that the chemiluminescent substrate and enhancer solution come premixed in a spray bottle. The substrate is ready for use without mixing. The spray bottle is used to apply the solution directly to the membrane.
At this point, the Visualizer™ Spray & Glow™ Western Blotting Detection System comes in one format: a 100ml spray bottle. The bottle is stored at 4°C and is stable under these conditions for 1 year. Due to the sensitivity of the system, and as with all chemiluminescent systems, it is necessary to optimize the components of your assay with respect to primary and secondary antibody concentrations. Optimizing these variables will reduce non-specific reactions and give strong specific results with an optimal signal-to-background ratio.
The procedure for the use of the Visualizer™ Spray & Glow™ System is very straight forward. After the final wash (drain excess wash buffer by touching against absorbent paper), place your membrane onto plastic wrap. Spray the Visualizer onto the membrane as many times as is necessary to ensure even coverage. We usually find this requires between 2 and 5 sprays for a regular precut 8x7cm nitrocellulose membrane (Invitrogen). Upstate claims that each 100ml bottle will be enough for 50 membranes. On occasion we have experienced a problem with the spray and it has required a few squeezes to prime, so we usually spray into a receptacle until the spray is even before spraying onto the membrane. The Visualizer is left to incubate on the membrane at room temperature for 2 minutes (in our hands this is optimal but the time required may vary depending upon the protein, protein concentration, antibody being used, etc). Upstate recommends an incubation time of 1 to 5 minutes. At this point, remove the excess Visualizer by touching the membrane against absorbent paper and enclose the membrane in a fresh sheet of plastic wrap, being careful to remove any air bubbles between the membrane and the wrap. Place the wrapped membrane in a film cassette, protein side up and expose to X-ray film for varying amounts of time (or using an alternative imaging system for chemiluminescence). In our hands, we generally expose the membrane for 1, 5 and 15 minutes but this will, of course, depend on all the previously mentioned variables and exposure times will need to be optimized for each situation. We have used the Visualizer with both primary HRP-conjugated and HRP-conjugated secondary antibodies to detect proteins of varying molecular weight (20kD-80kD) and concentration with equal success.
Upstate’s Visualizer™ Spray & Glow™ system is a fast, convenient and sensitive method for detection of proteins in Western blots.
Boyd Scott
Senior Research Scientist
Vitae Pharmaceuticals
Discovery Biology