March 15, 2010 (Vol. 30, No. 6)
Rachael M. Berry
Priya Rangaraj Ph.D.
Brian Webb Ph.D.
Recent Improvements Seek to Better Time-Consuming and Labor-Intensive Workflow
The Western blot workflow for immuno-based protein detection was introduced over 30 years ago. Since its introduction, Western blotting has become a well-known and widely accepted technique routinely used in many laboratories to detect a specific protein from a complex mixture. Because of the unambiguous results and wide scientific acceptance, Western blotting is often used to confirm results generated from more complex experimental techniques.
Many advancements have improved the convenience and performance of Western blotting. These advancements include the transition from radioactive to chemiluminescent detection, the transition from using homemade polyacrylamide gels to pre-cast gels for electrophoresis, and, most recently, the transition from using bulky and wasteful tank-transfer devices to the more rapid semi-dry transfer devices. Yet, despite these significant advancements, the Western blotting workflow remains a time-consuming and labor-intensive process with many steps that often require optimization.
To perform a typical Western blot, proteins are separated by gel electrophoresis and transferred to either nitrocellulose or PVDF membranes. The nonspecific binding sites on the membrane, where no protein was transferred, are blocked by incubating the membrane in proteinaceous blocking buffer. Because of its low cost, 5% milk is a popular blocking buffer. Protein detection on the membrane begins with primary antibody incubation followed by wash steps to remove nonspecifically bound antibody. Unfortunately, not all primary antibodies have equivalent performance.
Some primary antibodies generate nonspecific bands (i.e., bands other than the protein of interest), and some require an overnight incubation to obtain adequate detection sensitivity. Empirical testing to determine the optimal concentration is often required. Using an insufficient amount of antibody results in poor signal detection and too much antibody can cause high background or a specific signal that is too intense.
After the wash steps are complete, the membrane is incubated with an enzyme-conjugated species-specific secondary antibody, which is followed by more washes and signal detection. A variety of chemiluminescent detection reagents are available for different sensitivity levels.
The concentration of secondary antibody is a critical step in the process and will vary depending on the substrate of choice. A frequent error is the use of the secondary antibody at a concentration that is too high, which can result in detection of nonspecific proteins, high background, or in some cases, inadequate signal from rapid depletion of the chemiluminescent substrate. Extensive washing is required to remove the nonspecifically bound antibodies and can be essential to reduce background and improve the signal-to-noise.
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.
Rachael M. Berry is associate research scientist, Kelli Feather-Henigan and Greg Kilmer are research scientists, Priya Rangaraj, Ph.D. ([email protected]), is market segment manager, and Brian Webb, Ph.D. ([email protected]), is platform manager, cellular tools and detection.