Although traditional Western blotting requires significant optimization for satisfactory results, one advantage it affords is assay flexibility. For example, the incubation times for the antibody steps are flexible, allowing for overnight incubation to either improve the signal or for convenience. Also, traditional chemiluminescent Western blotting allows for blots to be reused.
After completing a Western blot, the detection antibodies can be removed with stripping buffers and the blot re-probed for another antigen. Both of these features were tested using the fast Western blot protocol. A fast Western blot was first performed using a phospho-S6 ribosomal antibody. The resulting blot was stripped and then successfully reprobed for a second target (Figure 2, Panel A).
The fast Western protocol also allows overnight primary incubations (Figure 2, Panel B). Because preblocking the membrane is not necessary, the membrane was incubated in primary antibody overnight, and then completed in approximately 30 minutes the next day.
Western blotting has become and will remain a mainstay technique in laboratories for many years. Its popularity and wide scientific acceptance stems from its ability to combine a straightforward protocol with high sensitivity. The Western blot readout is visual with specific protein identification and detection with protein size information. There is no doubt the method will continue to evolve.
Recent significant advances include products like the Pierce Fast Transfer System and Pierce Fast Western Blot Kits, which allow scientists to obtain results more rapidly than ever before. In addition, these new methods have helped to make Western blotting more environmentally friendly by reducing waste streams and eliminating the need for hazardous chemicals.