SecureSeal Hybridization Chambers
were originally developed to perform hybridizations on specimens affixed to glass slides or to perform microarray incubations with limited volumes (SecureSeal References). The peel- and- stick adhesive chambers can be created in various sizes to accommodate different samples, including partial membrane blots from protein mini-gels (shown here). Recently we have investigated the utility of these chambers for use in western blots.
The efficacy of using our SecureSeal Hybridization Chambers in a Western blot application was explored, leading to a simple method which provides excellent sensitivity with reduced antibody requirements and reagent volume. The expense of primary antibodies is often the greatest contributor to the overall cost of running a western blot. In some cases when antibodies are generated in private laboratories the supply can be extremely limited and the antibody is of immeasurable value. Researchers often try to conserve precious antibodies by using minimal amounts of antibody to see a signal, and also by re-using their antibodies in multiple experiments. An alternate solution is to reduce the volume as well as increase the dilution of the primary antibody as possible without losing signal.
The reaction kinetics achieved in the SecureSeal chamber allows greater dilutions of primary antibody while minimizing total reaction volume and provides superior signal compared to a traditional open-well chamber. To reduce reaction volume, for example, a 25.5 x 75.5 mm SecureSeal (Grace Bio-Labs product # 621507) attached to a microscope slide (adequate for 2-3 full lanes from a mini-format gel) only requires 500 µl of solution for effective mixing on a tube roller. Further reduction in volume is possible for smaller blot sizes coupled with the appropriate SecureSeal chamber size. More importantly, active mixing in a small volume allows for improved reaction kinetics. A ten-fold lower antibody concentration (0.0001 antibody equivalents (AE) using a SecureSeal achieved comparable signal to incubation in an open well with 1 ml (see Figure 1, 2). Signal was even detectable at a dilution of 0.00001 AE with the SecureSeal, further demonstrating the chamber’s ability to improve reaction kinetics. Similar advantages were achieved with thinner reaction chambers, obtainable using our HybriWell products, and allowed for a further reduction in reaction volume requirements (Figure 3).
Figure 1: Representative results obtained with the SA500 SecureSeal (A) and an open well (B) with various primary antibody dilutions, represented as antibody equivalents. Antibody concentrations are reported at antibody equivalents, where 0.001 is equal to the manufacturer’s recommended 1:1000 dilution. The SecureSeal required 250 µl total volume compared to the open well, which required 1 ml of total volume. Membranes were blocked with 0.1 x Super G Blocking Buffer (Grace Bio-Labs Product #105100) for 1 hour and incubations were conducted with a monoclonal anti-GAPDH antibody overnight at 4°C in 1x PBST with 0.25 % BSA. Colorimetric endpoint detection was performed utilizing alkaline phosphatase with BCIP/NBT substrate (Vectastain ABC Systems, Vector Laboratories).
Figure 2: Average signal for replicate blots achieved with the SA500 SecureSeal western chamber.
Figure 3: Results achieved with the HBW2260-FL HybriWell (Right) and open well (Left). The HybriWell chamber required 200 µl total volume compared to the open well, which required 2 ml of total volume. Membranes were blocked with 1 x Super G Blocking Buffer for 1 hour and incubations were conducted with a monoclonal anti-GAPDH antibody for 1 hour at room temperature in 1x PBST. Colorimetric endpoint detection was performed utilizing alkaline phosphatase with BCIP/NBT substrate (Vectastain ABC Systems, Vector Laboratories).
See our video on use of Secure Seal chambers here.
