We often find that the most crucial reagents in a given experiment, such as antibodies, are also the most limiting. The molecular characterization of protein interactions frequently requires considerable amounts of antibody. In extreme cases, when the primary Ab titer recovered from immunized animals may be so low that standard detection protocols demand copious amounts of primary and secondary antibodies, the reagents are used up long before the project is even completed. Under these circumstances, detection reagents that require a smaller quantity of primary and secondary antibodies, while maintaining high signal detection with low background, are critical. Our lab has tested several kits and protocols for chemiluminescent horseradish peroxidase (HRP)-conjugated antibody detection. In terms of sensitivity, cost and overall ease of use, the Supersignal West Dura HRP Detection Kit from Pierce is the only one still stocked on our shelves.
We have been quite successful using 15-20 ng/ml of primary antibody in conjunction with at least 40 ng/ml of the supplied secondary antibody (Pierce stock concentration is 0.01 mg/ml). For high concentrations of a specific antibody (at least 0.5 mg/ml), a dilution of 1:40,000 of the primary antibody can be used with 1 ng/ml of the provided HRP-secondary antibody. Of course, the appropriate dilutions for both primary and secondary antibodies must be determined empirically for each situation. Our experience has found that the dilution ranges we use allow us the flexibility to conserve valuable materials (either primary or secondary antibody) in the laboratory.
The kit comes with two detection reagents: Luminol/Enhancer (Reagent A) and Stable Peroxide Solution (Reagent B). These two reagents can also be used nominally, extending the number of samples that can be processed with a single kit. For example, the minimum volume required to cover a miniblot (7 x 8.5 cm) membrane is 1.5 ml, which would require only 0.75 mls of Reagents A and B. By using a smaller volume, we can increase the number of miniblots that can be analyzed by approximately three-fold over the manufacturer’s recommendations. Depending on the concentration of sample antibodies, chemiluminescent detection can take as little as 5-10 seconds. One extra bonus is that the reagents can be stored at room temperature, saving valuable 4C shelf space. The advantages of using the Supersignal West Dura HRP Detection Kit, make it a highly recommended product for cost-conscious laboratories.
Rhoel Dinglasan, Ph.D., M.P.H.
Postdoctoral Fellow
Johns Hopkins Bloomberg School of Public Health