Protein Transfer and Detection
Protein transfer and detection are areas where reducing assay time and simplifying the process can result in substantial time savings. Currently, protein transfer can take one hour to overnight with a traditional tank unit. Also, tank transfers typically require prechilling of the buffer and ice packs or setup in a cold room. The introduction of semi-dry transfer devices reduced the transfer process to 45 minutes without prechilling the buffer.
More recent advances have resulted in even more significant reductions in transfer times. The Thermo Scientific Pierce Fast Transfer System, composed of an optimized methanol-free transfer buffer and semi-dry transfer unit, enables rapid and efficient transfer of proteins in 7–10 minutes for most gel types. This new buffer system also eliminates the methanol additive, removing the need for hazardous waste disposal.
The traditional Western blot immunoassay protocol is time consuming with multiple hands-on steps and extended incubation periods. Without proper optimization of blocking buffer, primary and secondary antibody concentrations, and adequate washing, Western blot results can be non-reproducible and problematic. Thermo Fisher Scientific has developed a reagent-based assay system that eliminates the need for extensive optimization of these immunoassay variables.
The Pierce® Fast Western Blotting Kits reduce immunoassay time to approximately one hour while eliminating many of the hands-on steps of the traditional protocol (Figure 1, Panel A). This method has completely eliminated the traditional blocking and reduced the procedure to three simple steps: primary antibody incubation, HRP-conjugated secondary reagent incubation, and washing. Optimized diluents and wash reagents enable reduced processing time for each step. Without the need for additional equipment or expensive and wasteful disposables, the streamlined protocol and optimized reagents provide a cost-effective means to reduce immunoblotting time without sacrificing performance or throughput.
Combining the Pierce Fast Transfer System with the Pierce Fast Western Blotting Kits streamlines the protein transfer and Western blot immunoassay to less than 1.5 hours. This significant reduction of the traditional immunoblotting protocol (~4 hours) is accomplished without loss in assay sensitivity (Figure 1, Panel B). This protocol has been validated using a wide variety of primary antibodies and cell lysates.
For example, we separated cell lysate by gel electrophoresis and used the Pierce Fast Transfer System to complete a 10 minute transfer to nitrocellulose and PVDF membranes. A traditional Western blot protocol was performed in parallel. Importantly, the significantly shortened fast Western protocol produced comparable results to the classical protocol (Figure 2, Panel B). These results highlight the significant advancement to the Western blotting procedure.
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.