An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.
Western blotting is an established method that detects antigens on a transferred membrane and is commonly used in biomedical research, but it takes hours or days to complete the entire procedure. Using the combination of immuno-enhancing technology with cyclic draining and replenishing, once you've got a block membrane, the entire procedure can be accomplished in as little as 20 minutes without sacrificing sensitivity. Because Western blotting is common to many different research areas, it has broad applicability to speed up research.
The successful use of this protocol relies on the optimization of antibody dilution with immunoreaction enhancing reagent and incubation time. Begin by incubating the PVDF membranes in appropriate blocking buffers for one hour with agitation at room temperature. While the membranes are incubating, prepare 10%immunoreaction enhancing agent one solution or IRE1 with distilled water and vortex it well, then dilute the primary antibody with 10%IRE1, mixing it gently and thoroughly.
Pick up the PVDF membrane with tweezers and briefly drain the blocking solution. Insert the membrane into a conical tube with primary antibody, ensuring that the entire membrane adheres to the wall of the tube and that the side of the membrane that was originally in contact with the gel is facing inward. Cap the tube tightly.
Insert the tube into a glass bottle for the hybridization oven and turn on the oven. Incubate the membrane with six RPM rotation for at least five minutes. Stop the rotation and carefully remove the PVDF membrane from the tube with tweezers.
Place the membrane into a container with 50 milliliters of PBST and rinse it, then rinse it in a container with distilled water until no bubbles appear, which will remove the majority of the antibody. Transfer the membrane to a salad spinner that contains 250 milliliters of PBST. Place the strainer basket in the spinner and ensure that the lid has been put on securely, then activate the spinner and run it for 20 to 30 seconds.
Discard the solution and briefly rinse the inside of the spinner with distilled water. Add 250 milliliters of PBST into the spinner and repeat the spin, then incubate the membrane with the secondary antibodies and wash it again as described in the text manuscript. Place a piece of semi-transparent flexible film on a flat surface.
Mix two components of the chemiluminescent substrate in a five milliliter tube and immediately transfer 1.5 milliliters of the mixed substrate to the semi-transparent film. Place the PVDF membrane on the solution and transfer the rest of the substrate solution onto the membrane. Incubate the PVDF membrane with the mixed substrate for one minute.
Pick up the PVDF membrane with tweezers and drain the substrate solution briefly. Sandwich the membrane between a pair of transparency films and image it under chemiluminescent mode. The quantities of antigen and antibody respectively necessary for chemiluminescent detection of Western blot are shown here.
In both static and CDR incubations, usage of IRE solutions increased the sensitivity. Similarly, the minimum concentration of anti-6s His tag antibody was lowered from 100 to 50 nanograms per milliliter under static conditions and from 100 to 12.5 nanograms per milliliter under CDR conditions. CDR incubation for either 5 or 10 minutes dramatically lowered the detection limit compared to static incubation.
Furthermore, the combination of CDR with IRE revealed superior sensitivity on the Western blot, even within the extremely short incubation period. Successful application of this method to a Western blot with fluorescent detection is demonstrated here. Fluorescent detection in contrast to chemiluminescent detection has the unique ability to detect multiple targets on the same blot at the same time without stripping and reprobing antibodies.
His-tagged alkaline phosphatase and beta-actin in the cell lysates were simultaneously detected using a two-color fluorophore. CDR incubation with IRE not only accelerated the detection, but also increased the sensitivity. During the incubation, the membrane should stay attached to the wall of the tube.
The tube should be rotated horizontally so that solution washes continually over the membrane surface. This protocol significantly accelerated our research productivity, which strongly depends on Western blotting procedures.
