by Catherine Shaffer
Western blotting is a foundation technique for protein chemistry. The basic technique—transferring size-separated protein bands from a gel onto a membrane and visualizing with the use of antibodies—has been in existece for decades, and the protocols have changed very little. However, incremental improvements in materials, equipment and reagents have made the technique continually more reliable and more quantitative over the years. In addition, technology advances are making the Western blotting procedure faster than ever, shaving time off the preparation, run and analysis stages of the process. Solutions for faster Westerns include faster reagents, faster transfer equipment and even whole systems built from the ground up for a faster overall procedure, from sample prep to results.
Automated Western blotting
Performing a Western blot can be a time-consuming and labor-intensive process, with many preparative steps and time intervals to manage. Protein Simple has automated the entire work flow with its Simple Western™platform. According to Trent Basarsky, Vice President of Corporate Development for Protein Simple "We have reinvented the entire Western blot process." Running a Simple Western requires minimal prep time at the beginning, to mix samples and reagents. "Then you load the reagents and samples into this instrument, press ‘go,’ and a few hours later you come back to a completed experiment, including fully analyzed data."
Because the operator does not need to perform tasks at intervals during the procedure, as with a standard Western blot, there is a large savings in terms of uninterrupted time that can be used for other tasks as well as higher reproducibility by virtue of removing manual processes. According to Basarsky, "The response to this product has been really strong."
Within the system, the nanoliter protein separation is carried out in a capillary filled with a solution matrix. The proteins do not get transferred, and there is no membrane. The results are shown in a variety of formats, including a digital image that "looks a like a regular Western," says Basarsky.
Gel electrophoresis system
A standard Western protocol can run anywhere from a full day to five days. Because standard protocols require multiple manual manipulations, the elimination of any of these steps makes the work flow shorter and less cumbersome.
GE Healthcare Life Sciences has launched the Amersham™ ECL™ Gel electrophoresis system with precast gels and easy-to-use CCD imagers. "By [doing] this, we are excluding manual extra steps and manually induced errors," explained Karola Winkel, Marketing Manager for GE Healthcare Life Sciences.
The company also offers Amersham ECL Prime and Amersham ECL Select detection reagents, which offer improved sensitivity and increased signal intensity for better protein detection. According to Winkel, the fact that GE offers the whole Western blotting work flow (including instruments, reagents, gels, membranes, detection reagents and analysis software) enables the company to optimize its protocols for quality results.
Streamlined Western blot procedure
The V3 Western Workflow™ from Bio-Rad combines a set of Bio-Rad products to produce a more efficient process with optimum results. Key products include TGX™ Stain-Free™ precast Gels, Trans-Blot® Turbo™ transfer system and ChemiDoc MP Imager system. The work flow speeds the total time to results by shortening the electrophoresis, transfer and quantification steps. TGX Stain-Free gels require a 15-minute run time versus 45 min for traditional mini gels. The Trans-Blot Turbo completes a transfer in 7 minutes compared with 1hr or more for traditional tank transfer, with comparable transfer efficiency.
Researchers using the stain-free detection option of the ChemiDoc MP Imager system can quantify total protein on the membrane in less than 5 minutes without needing to normalize data with housekeeping proteins, which often requires stripping and probing the membrane a second time, or probing with multiple antibodies simultaneously.
"Other methods used to speed results often come at the expense of data quality," says Ryan Short, a Bio-Rad marketing manager in the Lab Separations Division. The V3 Western Workflow can be used in most life science protein research. "A great example is the study of cellular signaling pathways, where researchers are not just identifying the presence or absence of a protein but want to know the relative level of expression change," he continued.
Fur-Chi Chen, Ph.D., a Tennessee State University professor in the School of Agriculture and Consumer Sciences, is using the V3 Western Workflow to help identify proteins that might correspond with tender, juicier meat. Dr. Chen is able to check the work flow throughout the process. He particularly appreciates how easy it is now to quantify proteins with the V3 Western Workflow in contrast to normalization using housekeeping proteins. “Before the V3, I quantified using housekeeping proteins,” said Dr. Chen. “Probing with the first antibody, then stripping and reprobing with the housekeeping proteins, took a half-day. In addition, it was difficult to eliminate signal from the first antibody. Now, with the stain-free imaging technology of the ChemiDoc MP, I can normalize using total proteins in minutes and save those five to six hours.”
Reagent-based Western blotting
New instruments and systems for Western blotting are attractive, but some users would like a faster Western solution that does not involve the expense of a major investment in technology or the inconvenience of switching to a new method. A cost-effective, reagent-based system that works without vacuum pumps or lines, the Thermo Scientific Pierce Fast Semi-Dry Blotter and Pierce Fast Western blotting kits shorten the time required to perform a typical chemiluminescent Western blot to about 1 hr. Priya Rangaraj, Ph.D., Market Segment Manager for Thermo Scientific Pierce Protein Detection Products, said, "This time savings increases a researcher's output four-fold." The Fast Western Kits contain optimized reagents, including a special proprietary diluent for accelerated detection. The Fast Antibody Diluent is optimized for faster blocking of the membrane. It also contains agents to unravel more epitopes on the target, which results in faster and more efficient binding of the primary antibody to the target.
According to Rangaraj, the Fast Western Blotting Reagents work without a loss in sensitivity and are compatible with nitrocellulose or PVDF membranes. "Some instrument-based systems require the use of transfer stacks that can be used once and then need to be purchased again, providing an additional expense for the user."
Solutions are available to make Western blotting faster and quantitative. Options include purchasing a comprehensive system to replace an entire work flow, or optimizing the existing protocol with new components and reagents.
The image at the top of this page is from Thermo Scientific Pierce Protein Research Product's Fast Transfer System.