The use of primary antibodies is essential for Western Blotting procedures. These antibodies are sold in small volumes (larger volumes are also available) and can be expensive. The typical starting dilution of primary antibody is 1:1000 unless there is a recommended starting dilution. Initial testing to optimize the antibody concentration could be a series of dilutions as follows: 1:250, 1:500, 1:1000, 1:4000; this could be carried out to 1:10000 or further if necessary. The ideal antibody concentration is dependent on a number of factors that include antibody concentration, specificity and affinity of the antibody, buffer composition, and the temperature of blot processing (RT or cold room). Suboptimal concentration of antibody can results in a weak signal, non-specific bands and increase background. The problem with antibody optimization is the time necessary to determine the ideal concentration. Precision Biosystems has developed a one-step protocol to perform a serial dilution of six different concentrations in a fully automated fashion using the BlotCycler Touch. The BlotCycler has the capacity to run six different primary concentrations simultaneously; each antibody is delivered to a specific membrane. All steps are automated including delivery of the secondary antibody and all washing steps. The good news is, after a single automated run, the proper antibody dilution is established and can be used for subsequent experiments
