Exceptional Researchers and LI-COR Imaging Systems Driving Innovation.
Dr Patrick Kiely is a Principal Investigator and Senior Scientist in Professor Rosemary O'Connor's laboratory, in the Department of Biochemistry. The lab is located in the BioSciences Institute at the University College Cork in Ireland.
Dr Kiely studies cell migration, a process that involves a network of tightly regulated signalling pathways. Breakdowns in this regulation contribute to immune disorders, and are fundamental to the invasiveness of many cancers. Dr Kiely is investigating the molecular mechanisms of tumor cell migration and invasiveness.
Using arrays of immobilized target peptides, Dr Kiely studies the protein-protein interactions that occur during cell migration. He identifies proteins that compete with one another for binding to a target protein, and examines their binding preferences.
"The cost savings certainly did merit the purchase of a second instrument within 24 months."- Dr. Patrick Kiely
Equimolar concentrations of test proteins are overlaid onto an array of target peptides. Antibodies are then used to detect bound proteins. Distinct antibody species permit multiplex fluorescent imaging at 680 and 800 nm with an Odyssey® Infrared Imager.
The assay provides key information about what amino acids on the target protein are preferred by the test proteins. Fluorescent imaging indicates whether that region is preferred by test protein 1 (680 nm signal; red) or test protein 2 (800 nm signal; green). If the test proteins compete for binding to the same peptide, a yellow signal is observed (overlap of 680 nm and 800 nm signals).
"We couldn't do this without the Odyssey's ability to do dual detection of two different colors, based on detection of two different proteins. So that's proved to be really cool," says Kiely. "The time that the Odyssey saves us in Western Blot techniques is critical, it cuts down our workload dramatically."
"We've gotten very good at using the Odyssey software to look at quantification of specific signals," says Kiely. "Because the Odyssey is working on intensity of the signal, we can get a lot more specific information about a band. We pick up 5 - 10% differences in protein concentration or protein turnover, and that is something we cannot do without the Odyssey."
Dr Kiely's group uses the In-Cell Western™ assays, a quantitative immunofluorescent method, to optimize siRNA concentrations in 24- and 96-well plates. They are also using the Odyssey to generate quantitative readouts of cytotoxicity assays and 2D electrophoresis gels.
For more information about Dr. Kiely's work, visit the links below:
"Another reason we love the Odyssey, and why we have two of them, is the support that we get," says Kiely. "I pick up the phone and call headquarters, and they get us anything we need as far as specific products for the Odyssey and the antibodies." The Odyssey saves this lab around €20-25,000 in consumables per year. Theirs was the first Odyssey purchased in Ireland; there are now two instruments in that department, and a total of four machines at the BioSciences Institute.
We thank Dr Kiely for his contributions to cancer research, and are proud to consider him an "Odyssey Expert".
Publications resulting from work on the Odyssey:
1. Favre, C., Zhdanov, A., Leahy, M., Papkovsky, D., and O'Connor, R. (2010) Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells. Oncogene 29(27), 3964-3976.
2. McMahon, M., Ayllon, V., Panov, K. I., and O'Connor, R. (2010) Ribosomal 18S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation. J Biol Chem 285(24), 18309-18318.
3. O'Callaghan, K. M., Ayllon, V., O'Keeffe, J., Wang, Y., Cox, O. T., Loughran, G., Forgac, M., and O'Connor, R. (2010) Heme-binding protein HRG-1 is induced by insulin-like growth factor I and associates with the vacuolar H+-ATPase to control endosomal pH and receptor trafficking. J Biol Chem 285(1), 381-391.
4. Kiely, P. A., Baillie, G. S., Barrett, R., Buckley, D. A., Adams, D. R., Houslay, M. D., and O'Connor, R. (2009) Phosphorylation of RACK1 on tyrosine 52 by c-Abl is required for insulin-like growth factor I-mediated regulation of focal adhesion kinase. J Biol Chem 284(30), 20263-20274.
5. Kiely, P. A., Baillie, G. S., Lynch, M. J., Houslay, M. D., and O'Connor, R. (2008) Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and ß1 integrin and for tumor cell proliferation and migration. J Biol Chem 283(34), 22952-22961.