Summer Research
* Student(s): Timothy Weaver , Thomas Heacock
Advisor: Herman Lehman
Title: Characterization of Octopamine Antibodies - Western Blot Analysis
Abstract:
Octopamine (OA), a biogenic amine, serves as an important neuromodulator in most invertebrates (David and Coulon, 1985). In locusts, it has been shown that OA is essential in long lasting and energetically demanding motor behaviors, such as migratory flight and fighting (Libersat and Pflueger, 2004). Additionally, research has shown that it plays an important role in associative learning and memory processes in Apis mellifera (honey bees), which directly induces foraging versus non-foraging behavior (Farooqui et al., 2004).
Octopamine is the final product in the biosynthetic pathway beginning with the amino acid tyrosine. Tyrosine is converted into tyramine by way of the enzyme tyrosine decarboxylase. With the binding of copper and ascorbic acid to the enzyme tyramine beta hydroxylase (TBH), tyramine is then converted into octopamine.
This study focused on the measurement of TBH as a direct assessment of octopamine production. The nucleotide sequence of TBH in honeybees was derived from data compiled by the Honey Bee Genome Project. Two regions (23-36, 478-491) of this protein were synthesized and bound to a carrier protein, keyhole limpet hemocyanin (KLH), and then injected into rabbits. The antisera were obtained from the rabbit blood and stored at -80 degrees Celsius.
We used the Western Blot technique with 10% SDS-PAGE gels to test the efficacy of the antibodies to bind to TBH protein on brains isolated from Apis mellifera and Manduca sexta. Proteins were separated by size, transferred to PVDF membranes and then incubated in antisera. By preabsorbing the antisera with a protein conjugate or KLH, the specificity of the antibody to TBH was verified.
Based on the molecular weight of the amino acid sequence, it was expected that the Apis TBH protein would be 64 kDa and the Manduca protein around 75 kDa. One of four Manduca sexta trials incubated in the TBH 478 antisera yielded bands around 75 kDa. Apis brains incubated in TBH 23 antisera produced similar results, showing bands near 64 kDa. With no preabsorption, there was a doublet band approximately at 64 kDa. When the antisera was preabsorbed in the protein conjugate, the band was still present although the lower molecular weight band was diminished in intensity. Furthermore, preabsorption in the protein conjugate yielded fewer distinct bands as compared to isolated antisera. Preabsorption in KLH yielded no change in intensity at 64 kDa as compared to incubation in antibody only but some higher molecular weight bands were diminished.
In summary, this study showed the specificity and effectiveness of the TBH antibodies. By not binding to the preabsorbed KLH, we showed that in fact the antibodies were recognizing and binding to the 13 amino acid sequence (23-36) and not the carrier protein KLH. Lastly, we can deduce that the amino acid sequence of the Manduca is very much conserved to the Apis since the Apis TBH 478 antisera produced bands in the protein gel of the Manduca TAG.
Research by TW supported by the Ralph E. Hansmann Science Student Support Fund. Reseach by TH supported by a grant from the National Science Foundation.