Brian Cuts Time to Results and Solves His Membrane Protein Challenges with Wes
"We had been trying to study receptor proteins with traditional Westerns but it was frustrating—we just could not get it. A few months with Wes and we've already had way more success than we had in three years."
- Brian Perrino, Ph.D., Associate Professor, Department of Physiology and Cell Biology, University of Nevada School of Medicine
Digesting gastric smooth muscle signaling
Brian, an Associate Professor in the Department of Physiology and Cell Biology at the University of Nevada School of Medicine, studies the role of gastric smooth muscle signaling in gastric motility disorders like gastroparesis and functional dyspepsia. Patients with these disorders suffer from symptoms like nausea and abdominal pain after a meal.
His lab mainly looks at the dephosphorylation of myosin as a signal processor that regulates muscle contraction and relaxation. They also collaborate with other researchers to get a better handle on the signaling pathways controlled by GPCRs, ion channels, and tyrosine kinase receptors found in the muscle layer. Right now there aren't many treatments or drugs for patients that deal with these stomach disorders, so Brian's team is working hard to get to the bottom of the regulatory mechanisms involved to help fasttrack development of new and better treatments.
The never-ending Western blot challenge
Brian's group routinely looks at the phosphorylation state of myosin light chain, the MYPT1 subunit of myosin phosphatase, and CPI-17. To fully understand the signaling pathway, they need to look at total and serine 19 phosphorylation on myosin light chain, total and threonine 38 phosphorylation of CPI 17, total MYPT1, and phosphorylation of MYPT1 on threonine 853 and 696. That's a grand total of seven Western blots. Add all the control and contractile situations to look at plus protein phosphorylation in the presence and absence of inhibitors into the mix and that's a lot of immunoblots!
Ramping up the speed to results
With Wes, the Perrino lab now gets 24 Simple Western data points in just three to four hours compared to the two and a half days it used to take with traditional Western blots. They can now do assay optimization really quickly—in one afternoon, they can determine the optimal lysate concentration and antibody dilution to use for quantitative results. Wes' fast time to results also helps them look at all the proteins and post-translational modifications associated with their proteins of interest more efficiently. The day-to-day reproducibility they get with Wes also means they aren't having to repeat experiments when day-to-day transfer differences cause signal intensities to suddenly drop in half. So, instead of three weeks to collect tissue and run Western blots they can now get it all done in just a week with Wes!
Rocky road studying smooth muscle membrane proteins
For the past three years Brian had also been trying off-and-on to study molecular mechanisms that regulate smooth muscle signaling at the receptor level with little success. Since they were interested in looking at phosphorylation events as well as protein expression, RNA amplification methods just weren't an option. Because of the low abundance of receptor proteins in the plasma membrane that only makes up a small portion of the total protein in a cell, they had to load a ton of protein in their gels—which resulted in a lot of background. And trying to enrich their lysate prep with different, enrichment columns, magnetic beads, differential centrifugation, membrane preps and sucrose gradients didn't help at all.
Changing the membrane protein game
Wes' sensitivity finally gives them great receptor data. In fact, they're able to detect these low abundant proteins with just RIPA-lysed whole cell lysates so they don't have to worry about losing protein during special protein enrichment methods. In fact, they got great results for c-KIT (a receptor tyrosine kinase that's an interstitial cell marker), SK3 (a small conductance potassium channel expressed on fibroblast-like interstitial cells, and TRPV4 receptor in just a couple of weeks (Figure 1). They were able to do more with Wes in just a few weeks than they had off-and-on for the past three years because of method limitations.
Figure 1. Wes detected a titration of the plasma membrane protein TRPV4 with just 1 µg of murine bladder smooth muscle whole cell lysate using 1:50 diluted a-TRPV4.
Opening new doors for receptor protein research
Wes has let Brian's group to do more than ever before and they're only getting started. Because they can get great sensitivity now, they plan to use Wes to look at their receptor of interests in FACS sorted cells from some GFP-tagged mice. This will give them specific cell populations of cellspecific receptor and ion channels from smooth muscle or different types of interstitial cells. By comparing receptor activation in their interstitial cell knockout and wild-type mice, they'll be able to understand what proteins are there and how they change depending on disease state for the first time.
Wes made such a difference in the Department of Physiology and Cell Biology that the Pharmacology Department couldn't help but take notice. They were looking at similar things in vascular and uterine smooth muscle and got their very own Wes after seeing the throughput, reproducibility, and sensitivity Brian's group got. Brian feels it's this kind of collaboration with smart, interesting people from all over the world that will really contribute to the field's knowledge and potentially help clinicians come up with treatments—and that's why he keeps doing what he does!