Webinar Highlights: Automated Western Assays to Study the Role of Serotonin Receptors in Cocaine Use Disorder
Validating commercial antibodies and exploring receptor expression
29 Jul 2015
Dr. Noelle Anastasio, Assistant Professor, Department of Pharmacology and Toxicology, University of Texas Medical Branch
Dr. Noelle Anastasio, Assistant Professor, and Sarah E. Swinford-Jackson, Graduate Assistant, both at the Center for Addiction Research, University of Texas Medical Branch, discussed their research into the neurobiological underpinnings of cocaine use disorder in this exclusive webinar, which you can watch on-demand here.
Learn how they optimized and implemented a quantifiable and reproducible automated Simple Western assay in Wes™ to examine serotonin and glutamate receptor expression, as well as protein:protein interactions from rodent brain samples. Read on for highlights from the webinar Q&A session.
Is the Wes Western blotting system compatible with most sample prep buffers or did you have to change your sample preparation to work with this system?
We've actually done several different protein preparations. We also altered our buffer composition slightly to reduce salt concentration, and we used a slightly stronger non-ionic detergent, using NP-40 instead of dodecyl maltoside. Furthermore, previously we denatured our samples at 70°C for 20 minutes, and now we denature at 95°C for five minutes. However, barring these slight modifications, our current buffer systems worked very well with this system.
How much sample did you have to load?
It's all dependent on what target we're looking at. For serotonin 5-HT2A and 5-HT2C receptors, we load 3-4 micrograms of protein per capillary, whereas for NMDA receptor antibodies, we load 1 microgram of protein per capillary. In both cases, this is a 10-30-fold improvement over traditional Western blotting.
Sarah Swinford-Jackson, Graduate Assistant, Center for Addiction Research, University of Texas Medical Branch
Were your experiments performed on fresh samples, and did you do any experiments on samples that had been frozen for a particularly long period of time?
Some of our samples, for example, the brain samples, are flash frozen in liquid nitrogen upon dissection then stored at -80°C until we extract the protein for analysis. Some proteins in these brain samples have been frozen for up to a year and we were able to run them on the Wes system without any problems.
Has any progress been made on ascertaining whether the orbital frontal cortex and the anterior cingulate gyrus are in fact the frontal cortical areas most frequently implicated in addiction to cocaine?
The complexity of the cortical circuitry that's involved in cocaine addiction is still under investigation by a number of laboratories. Interestingly, in humans, the anterior cingulate gyrus is functionally homologous to the medial prefrontal cortex in our rodents. It's for this reason that we've chosen to initiate our studies looking at the prefrontal cortex. However, we are keenly aware that the orbital frontal cortex and additional regions within the brain are also going to be involved, and we're currently investigating those and their relationship between impulsivity and cue reactivity.
How could the information in this webinar help to cure cocaine addiction?
We've focused on understanding how different receptors, for example those for serotonin or NMDA are expressed and function differently at vulnerability periods, such as high cue reactivity and high impulsivity. This gives us an idea about which systems are putative pharmacotherapeutic targets. Understanding a little more about how these receptor expression levels can change will help us target medication that is appropriately suited to reduce cue reactivity and impulsivity through these systems.