GE Healthcare Life Sciences recently launched the Amersham WB system for quantitative SDS-PAGE and Western blotting of proteins using fluorescence detection. The system was standardized for the entire Western blotting process, including electrophoresis, transfer, probing, scanning, and image analysis.
According to Scott Ripley, general manager for commercial research and applied markets, traditional Western blotting requires a complex combination of analytical knowledge, extensive manual handling, dexterity, and strict adherence to protocols in order to achieve reliable results. User-to-user variation can be as high as 35–45%, he explained.
“We set out to bring to market an all-new system that addresses a number of key issues that customers were seeing, namely quantitation, reproducibility, and ease of use,” said Ripley. The Amersham WB system relies on little manual handling and uses optimized and standardized reagents, gels, membranes, and detection antibodies. The only things that the researcher needs to input are the protein samples and the primary antibodies, he noted.
“The whole process is fully monitored and alerts are given when intervention is needed. In addition, the system provides a novel and efficient normalization method for highly reproducible quantitative data of target protein levels,” Ripley continued, adding that this is achieved through built-in quantitative Western blot protocols, which use multiplexing of Cy™3 and Cy5 signals on the same blot. The system supports three applications based on Western blotting and one based on SDS-PAGE: Easy Western, Western with endogenous protein normalization, Western with total protein normalization, and Easy SDS-PAGE.
The system software allows easy visualization of each process, keeping track of blots and data, pointed out Ripley. The system then carries out automated data analysis at the end of a run. All running parameters, such as current, voltage, and temperature, are also logged for each experiment. Fluorescence detection was designed into the system to enable multiplex analysis and normalization using either total protein signal (acquired from fluorescent prelabelling using Cy5 dye reagent) or a housekeeping protein.
“Multiplexing, using different antibodies, can identify different proteins within a single sample, which again can be important in helping understand the cellular mechanisms in disease,” said Ripley, explaining that fluorescent detection is highly beneficial for multiplexed experiments as it enables discrimination of signals not only on size but also on fluorescent wavelength. “The Amersham WB system is useful for validation and antibody testing due to its ease of use and the possibility of unattended operation,” he added. “More samples can be run quicker and in a more reproducible way.”