The aim of a Western blot is to provide information about the presence and expression levels of specific proteins in a complex mixture such as a cell lysate or tissue homogenate. The technique was first developed almost 40 years ago, yet despite being highly popular it is a notoriously time-consuming process.
During a typical Western blot, samples are loaded into a gel, to which an electrical current is then applied. Under denaturing conditions proteins are separated on the basis of size, whereas native conditions allow separation of proteins in their active state based on both size and charge. Following separation, proteins are transferred to a membrane, which is then subjected to a series of treatments, typically: blocking, incubation with primary antibody, washing, incubation with secondary antibody, further washing, and, finally, development.
Researchers are turning to automated Western blot processors, which offer consistency across all steps of the protocol and also provide a substantial time saving.
Each stage requires careful optimization, yet once the experimental conditions have been finalized, the resulting Western blot can vary considerably from day to day, and between different operators. To overcome these issues, researchers are turning to automated Western blot processors, which offer consistency across all steps of the protocol and also provide a substantial time saving. These systems allow the researcher to load the necessary solutions, select a staining protocol, and then walk away.
Although many researchers believe that the success of a Western blot is determined by the nature of the primary antibodies that are chosen, every variable within the assay can influence data quality. According to Russ Yukhananov, CEO at Precision Biosystems, the importance of the washing steps should not be underestimated. “Manual processing can lead to significant variability in results between blots,” says Yukhananov, “whereas automated processing using the Precision Biosystems BlotCycler™ produces excellent reproducibility, in part due to the precise timing and consistency of solution changes.”
Reproducibility of Western blot processing using the BlotCycler™ from Precision Biosystems. Lane 1: HeLa whole cell lysate, lane 2: recombinant human IL-2, lane 3: HeLa whole cell lysate + recombinant human IL-2. Blocking, antibody incubation and washing steps were performed using the BlotCycler. The fluorescent intensity between different blots was subsequently normalized using a molecular weight standard. Automated processing with BlotCycler demonstrated excellent reproducibility.
As well as contributing to the production of more uniform results from one blot to another, the use of an automated Western blot processor can dramatically cut down on the number of trips that the researcher is required to make to the blot rocker platform, allowing precious time to be spent elsewhere.
Primary Antibody Recycling
The GOBlot™ Western Blot Processor from Cytoskeleton was developed following communication with hundreds of research scientists, and is programmed with the four most popular Western blotting routines. Once the machine has been loaded with blocker and antibody solutions, the program is simply left to run. Two membranes can be probed with the same antibody solutions simultaneously, while the modular nature of GOBlot allows multiple machines to be run at different times with different routines and different antibodies, providing excellent flexibility. “A further advantage of GOBlot” says Ashley Davis, vice-president of Cytoskeleton, “is that it enables the primary antibody, often the most expensive reagent within a Western blot, to be recycled.”
Re-use of a primary antibody with the GOBlot from Cytoskeleton. A Western blot was probed with a primary antibody, which was subsequently re-used three times. Automated processing with GOBlot demonstrated successful antibody recycling with negligible loss of signal.
Prasanna Satpute-Krishnan is a strong advocate of the GOBlot™. “GOBlots have been very helpful during the start-up phase of my laboratory, when I was the only person in the lab,” she says. “I could spend 10-15 minutes planning my Western blot and putting together the blocking solution, primary and secondary antibodies, and wash buffer, then focus on other things without disruption while the machines finished off the Western blot. One of my favorite features is the separate collection chamber for used primary antibody solution, which allows re-use. Altogether, the GOBlot not only saves me time, but money as well.”
Amy Emery, research associate at the University of Cambridge, regularly runs Western blots during her work within cancer research and has often been reliant on custom-synthesized antibodies that are available in only limited quantities. “Although I appreciate that automated processors can save time by eliminating the need to manually wash membranes and change antibody solutions,” says Emery, “I’ve been dissuaded from using these in the past since I was under the impression that they would incur substantial antibody loss. Primary antibodies are really expensive, so unnecessary waste isn’t something that I would be willing to accept, but Western blot processors appear to have evolved significantly, and knowing that I could re-use my primary antibody would definitely make it more likely that I would consider giving them a try.”
Diagnostic Use
As well as providing convenient methods of gel staining and Western blot processing, automated systems have utility within diagnostics. The ProfiBlot™ 48 from Tecan processes up to 48 strip-based samples per run, automating reagent dispensing, washing, and incubation steps. It supports a range of Western blot applications, such as the confirmation of infectious diseases and autoimmune diseases, as well as screening for allergies. The system is designed to meet the IVD-directive 98/79/EC for Europe, and processes the entire assay in a disposable tray within an enclosed environment to minimize human exposure to potentially infectious samples.
Gerald Habenbacher, project manager at Tecan, has worked with several labs that are successfully using the ProfiBlot™ for Western blot analysis, and says that “while the reagent saving features of the system minimize dead volumes to keep running costs low, the dispense volume auto-calibration function and the predefined cleaning protocols make it easy to use.”
Strip assay processing with the ProfiBlot™ 48 from Tecan.
Another popular automated system with the capacity for processing strip-based samples is the AutoBlot 3000 from Bio-Rad Laboratories. This instrument can dispense and aspirate up to 20 strips in just 90 seconds, and is provided with pre-programmed protocols as well as the option to input user-defined methods. “This system provides excellent flexibility,” says Greg Stewart, senior product manager, infectious disease at Bio-Rad, “since it allows dispense volumes, incubation times, rock speed, and platform temperature to be programmed in any combination within a protocol. The AutoBlot 3000 has a specially designed tray platform and unique dispense and aspiration systems to enable generation of accurate, highly reproducible results.”
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Although many researchers are content to follow traditional, manual protocols for Western blot processing, all of which have stood the test of time and been extensively literature-cited, there is a growing awareness of the utility and benefits that are offered by automated systems. Laboratories in which multiple researchers routinely run Western blots while simultaneously performing other experiments can benefit from significant time savings, at the same time generating highly reproducible results. Those working within diagnostics have the added peace of mind that exposure to potentially harmful samples is minimized through the enclosed environment provided by the processor.
As automated Western blot processors become ever more sophisticated, the number of membranes that can be developed concurrently will increase, and staining protocols will offer additional steps, resulting in membranes that have been probed for multiple target antigens with minimal hands-on time required by the researcher. The use of these systems to enhance data quality and reproducibility affords yet further technological advancement in scientific research.
Images: Preciscon Biosystems, Cytoskeleton, Inc., and Tecan.