|Xingyu Jiang and colleagues created a microfluidic device out of a plastic called PDMS to assay a Western blot with 10 different antibodies at once. Image courtesy of Sha He and Wenying Pan, Jiang Laboratory.
Capillary electrophoresis meets protein immunoblotting
Capillary electrophoresis is one way investigators are overhauling the Western blot. The method separates molecules by their size-to-charge ratio inside a narrow electrolyte-filled tube and was the workhorse with which the Human Genome Project was completed (1). The advantage of CE is that the sieving matrix for separations can be automatically pumped in and out, because it contains entangled polymers rather than the typical crosslinked polymers of gels, explains Kennedy. He adds, “It’s what made the big difference in the Human Genome Project. It didn’t sound like much, but if you’re talking about running many samples over and over again, that simple automation step made life so much easier.”
CE also requires less sample than gel electrophoresis and has a better resolution of protein size. Kennedy’s laboratory has now swapped the gel electrophoresis step for CE. Proteins travel down a capillary and separate according to size. As the individual proteins emerge at its mouth, they drop onto a blotting membrane moving steadily across the capillary opening. In this way, the researchers drop the time-consuming gel-to-membrane transfer step of conventional immunoblotting and develop the blot as usual. The researchers have shown they can separate classic protein standards like carbonic anhydrase and lysozyme within an hour using only a few nanoliters of sample (2).
In September, the company ProteinSimple released the SimpleWestern technology in an instrument called Simon. The technology is based on CE and, according to the company, is a “gel-free, blot-free and handsfree solution to the entire Western blotting process.” Mixtures of proteins in nanoliter aliquots are taken into 12 capillaries filled with a sieving matrix and separated by size. The separated proteins are immobilized at the capillary walls in their positions by exposing the capillary to a ultraviolet-light source that activates proprietary chemistry. The separation matrix gets removed, and the reagents for a standard immunoassay flow into the capillary. The primary antibody enters first, followed by a horseradish peroxidase-conjugated secondary antibody, which generates a chemiluminescent readout. Including the time for sample preparation, the totally automated process takes three to five hours, say company representatives.
The instrument allows for quantitative protein measurements. “The biggest challenge with a traditional Western blot performed today is that you separate your proteins in a polymerized acrylamide matrix and transfer it to a solid membrane surface,” says Peter Fung, ProteinSimple’s Simon product manager. “You have no idea how much of your protein that you loaded into that gel is actually transferring to a solid membrane surface. With our technology, we know the proteins that are separated will be captured on the walls of the capillary.”
Trent Basarsky, ProteinSimple’s vice president of corporate development, and Fung both say that their approach gives more reproducible data. No matter “who is in front of the machine, it’s going to give the same answer,” says Basarsky. Although Basarsky declined to reveal the price of the instrument, he and Fung say that the cost of each run in Simon is comparable to that of traditional Western blotting.
Downsizing to microfluidics
The other approach to changing Western blotting is microfluidics, a technology by which small volumes of fluids and molecules move through microscale channels. Kennedy’s group is looking into using microfluidics to further reduce the amount of sample needed for their method and reduce the size of their setup by swapping the centimeter long capillaries for micrometer long microfluidic channels.
Xingyu Jiang and colleagues at the National Center for NanoScience and Technology in Beijing recently incorporated microfluidics for the immunoblotting step. They designed a microfluidic system with channels that allowed 10 different primary antibodies to probe the membrane. Once the incubation step with the primary antibodies was completed, Jiang’s team incubated the whole membrane in a secondary antibody solution. They were able to analyze the expression and molecular weights of 10 proteins, not just a single protein, from a single sample (3). Jiang explains that, because multiple proteins are detected simultaneously, “researchers can save [themselves] the labor of preparing multiple samples.”