separation and analysis of honeybee venom components l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Separation and Analysis of Honeybee Venom Components PowerPoint Presentation
Download Presentation
Separation and Analysis of Honeybee Venom Components

Loading in 2 Seconds...

play fullscreen
1 / 15

Separation and Analysis of Honeybee Venom Components - PowerPoint PPT Presentation


  • 245 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Separation and Analysis of Honeybee Venom Components' - Audrey


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
separation and analysis of honeybee venom components

Separation and Analysis of Honeybee Venom Components

Levi Blazer

Liz Denning

Laura Rhodes

Juniata College

Research Advisors:

Dr. Lorraine Mulfinger and Dr. Michael Boyle

melittin our primary interest
Melittin: Our Primary Interest
  • Comprises 50% of raw honeybee venom
  • Has antibacterial and lytic properties
  • Melittin tetramer (4 protein chains)
gel filtration chromatography
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
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
Sephedex Gel Chromatography

FRACTIONS

UV MONITOR

COLUMN

honeybee venom sample 20mg ml
Honeybee Venom Sample: 20mg/mL

Melittin

Phospholipase

???????

Hyaluronidase

lyophilization freeze drying process
Lyophilization-Freeze Drying Process
  • Purpose:ability to reconstitute peptide into varied solvents as necessary for certain experimentation
lyophilization 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
Analysis of Column Fractions
  • Gel Electrophoresis
  • SELDI-TOF Mass Spectrometry
purpose of gel electrophoresis
Purpose of Gel Electrophoresis
  • Determine purity of column separation
  • Compare with whole bee venom
  • Identify protein components of whole venom

Figure 1: Shows electrophoresis components

polyacrylamide gel electrophoresis
Polyacrylamide Gel Electrophoresis
  • Samples placed in 20% sucrose solution
  • Bands separated by charge
  • Stained in Rapid Reagent

Figure: Shows PAGE gel results. (In lane 6: Whole Bee Venom, in lane 5: Melittin Standard, and in lane 1-4: Melittin Fractions)

polyacrylamide gel results
Polyacrylamide Gel Results

1 2 3 4 5 6

  • Top Gel:
    • Lane 1: Whole Bee Venom
    • Lane 2: Melittin Standard
    • Lane 3-6: Melittin Fractions
  • Bottom Gel:
    • Lane 1: Whole Bee Venom
    • Lane 2: Melittin Standard
    • Lane 3-6: Phospholipase A Fractions

Melittin Fractions

1 2 3 4 5 6

Phospholipase A Fractions

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

Acknowledgements
references
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) 467-72.

Kemeny DM, Dalton N, Lawrence AJ, Pearce FL, Vernon CA. 1984. The purification and characterization of hyaluronidase from the venom of the honey bee, Apis Mellifera. Eur J Biochem 139(2) 217-23

Mulfinger LS. 1989. Synergestic activity of honey bee venom with antibiotics. The Pennsylvania State University.

Staay FJ, Fanelli RJ, Blokland A, Schmidt BH. 1999. Behavioral effects of apamin, selective inhibitor of the SKCA channel in mice and rats. Neurosci. Biobehav. Rev. 23 1087-1110