The "Curto Method" Of Western Blotting Seems To Work

Ernie Curto, working in the Neuro Lab here, brainstormed a method for doing western blots that allows more samples to be tested in less time, using fewer antibodies and other resources. Andrew and Taito did a run of western blotting on some of the fish brain lysates we've collected this winter along with past samples and positive controls, and were able to run three replicates of each samples on (I think) 13 individual fish. Basically, it runs dot samples of each replicate on the membrane that are defined by holes punched in tin foil, allowing the samples to be lined up closely but still separately. Westerns are a form of antibody-antibody application, after which your protein sample (NMDA for us) is more heavily attached to antibodies the more concentrated it is, and then these antibodies hold onto a stain that allows different concentrations to be visualized in a scanner.

Our results were consistent with what we've found before, i.e., male scarlet shiners have lots of NMDA in their brains even in winter (it's for learning and memory channels in brain neurons), while telescope and silverstripe shiners, male and female, have less. We also included a male tennessee shiner that we captured at Estill Fork. This is a very sexually dimorphic species, maybe more than scarlets even, and the male we tested had an even higher NMDA level than the male scarlets. This reinforces our working hypothesis that species with more pronounced sexual dimorphism (like breeding colors and size) should have even more extreme levels of NMDA in males. Taito and Asuka are going to rerun our samples in a more traditional western blotting to confirm the Curto method results. If the results hold up we may sample fish on a monthly basis this breeding season to monitor how NMDA levels vary, since our costs and required effort just dropped dramatically.