Noelle Changes Her Brain Receptor Research Game with Wes
"One of the best advantages of using Wes is that it saves a lot of time, both in terms of hands-on time and in time to completion of the experiment. We were spending more than 4 hours of hands-on time per Western blot with traditional methods, and we can set up a run on the Wes in about 45 minutes. Our Wes runs are completed in less than 4 hours, as opposed to 4 days with traditional methods."
- Noelle Anastasio, Ph.D., Assistant Professor, Department of Pharmacology and Toxicology, University of Texas Medical Branch
Getting to the bottom of addictive disorders
Noelle is an Assistant Professor in the Department of Pharmacology and Toxicology at the University of Texas Medical Branch, and has a full membership in the UTMB Center for Addiction Research. Her studies use a multi-disciplinary approach (protein biochemistry, cellular and rodent behavioral models) to elucidate the biochemical and pharmacological properties of the N-methyl-D-aspartate receptor in the corticostriatal brain circuit that are altered in vulnerable versus resistant phenotypes in addictive disorders.
Numbers that didn't quite cut it
The lab's research interests lie at the interface of pharmacology, neuroscience, and psychiatry. Over time that's grown into a primary focus on elucidating the molecular, neurochemical and behavioral underpinnings of impulsivity—a trait that has been implicated as an important risk factor and contributor to relapse for addictions, obesity and eating disorders. They were identifying target proteins with traditional Western blotting, but they needed better reproducibility and quantification than they were getting.
Knocking it up a few notches
Wes and his 12-230 and 66-440 kDa Master Kits let Noelle's team streamline their experiments and up their reproducibility. Several traditional Western and co-immunoprecipitation protocols were transferred to the Simple Western format—using a variety of protein preparations and greater than 10 antibodies—with only minor changes to buffer composition, detergent, and denaturing conditions.
Wes let them successfully interrogate protein expression and protein:protein interactions from high and low impulsive rats (Figure 1, Fink et al., 2015; Anastasio et al., 2015). Simple Western assays also let them detect the GluN1, GluN2A, and GluN2B subunits of the NMDAR from rat brain. Now they use Wes for all their immunoblotting assays.
Figure 1. Protein was immunoprecipitated from medial prefrontal cortex (mPFC) of low impulsive (LI) and high impulsive (HI) mice to understand protein:protein interactions of 5-HT2AR and 5-HT2CR. Immunoprecipitation of 5-HT2CR followed by immunoblotting for 5-HT2AR on Wes (A) and the reciprocal experiment (B) demonstrated decreased 5-HT2AR and 5-HT2CR interaction in HI mice.
Saving big in more than one way
Noelle feels Wes gives her team quite a few advantages over traditional Western blot methods. First off, using smaller sample sizes is vital when protein samples are extracted from small brain regions. Wes typically only needs 2-4 µg of protein per well, that's significantly lower than the traditional methods they'd been using which required 30-40 µg per well. As an added plus, Wes uses much less antibody—about 2.5 µL for a 1:100 dilution for a full plate as opposed to 60 µL for one 10-15 well gel in traditional methods—which helps cut experimental costs too.
But the best advantage is Wes just saves the team a lot of time—both hands-on time and the time it takes to complete an experiment. Before Wes, they were spending more than 4 hours-worth of hands- on time per Western blot. With Wes they set up a run in about 45 minutes, and it's done in less than 4 hours—compared to the 4 days it took with traditional methods. This lets them collect more data much more quickly.
According to Noelle, ProteinSimple products were designed with end-users in mind, and she feels the ProteinSimple team is truly supportive of scientists and their research. She's now also worked with another on-campus group to help them set Wes up and start using Simple Western assays in their laboratory.
A few recent publications
Serotonin (5-HT) 5-HT2A receptor (5-HT2AR):5-HT2CR imbalance in medial prefrontal cortex associates with motor impulsivity, NC Anastasio, SJ Stutz, LH Fink, SE Swinford-Jackson, RM Sears, RJ DiLeone, KC Rice, FG Moeller and KA Cunningham, ACS Chem Neurosci., July 2015; 15;6(7):1248-58, doi: 10.1021/acschemneuro.5b00094.
Individual differences in impulsive action reflect variation in the cortical serotonin 5-HT2A receptor system, LH Fink, NC Anastasio, RG Fox, KC Rice, FG Moeller and KA Cunningham, Neuropsychopharmacology, July 2015; 40(8):1957-68, doi:10.1038/npp.2015.46