Separation and Analysis of Honeybee Venom Components.ppt

Separation and Analysis of Honeybee Venom Components Levi Blazer Liz Denning Laura Rhodes Juniata College Research Advisors: Dr. Lorraine Mulfinger and Dr. Michael Boyle Honeybee Venom Components Melittin: Our Primary Interest Comprises 50% of raw honeybee venom Has antibacterial and lytic properties Melittin tetramer (4 protein chains) Gel Filtration Chromatography SEPHADEX? G-50 (MW 30,000 – 1,500) Stationary phase consists of porous beads Beads composed of cross linked dextran (Sephadex) Degree of crosslinking determines the size of the pores of the beads Our column optimized for melittin Separation According to Size Small molecules enter the pores of the beads and flow through the column more slowly Large molecules will not be able to enter the beads and will flow through more quickly Sephedex Gel Chromatography Honeybee Venom Sample: 20mg/mL Lyophilization-Freeze Drying Process Lyophilization Process Lowering the temperature and pressure draws out solvent vapor leaving behind frozen faction sample Solvent removed via sublimation Solid phase Gas phase Analysis of Column Fractions Gel Electrophoresis SELDI-TOF Mass Spectrometry Purpose of Gel Electrophoresis Determine purity of column separation Compare with whole bee venom Identify protein components of whole venom Polyacrylamide Gel Electrophoresis Samples placed in 20% sucrose solution Bands separated by charge Stained in Rapid Reagent Polyacrylamide Gel Results Acknowledgements Dr. Lorraine MulfingerAssistant professor, Juniata College Dept. of Chemistry Dr. Marielena McGuire Field Scientist, Mid-Atlantic Region, Ciphergen Biosystems, Inc. Dr. Michael Boyle Von Lebig chair in Biomedical Sciences, Juniata College Dept. of Biology Dr. Tom Lyons Fisher Professor of Chemistry Juniata College Dept. of Chemistry References Altmann F, Kubelka V, Staudacher E, Uhl K, Marz L. 1991. Characterization of the isoforms of Phospholipase A2 from honeybee venom. Insect Biochem 21(5) 467-7