Priority Research Area Asthma and Allergy

Clinical and Molecular Allergology

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Techniques

GENETIC METHODS

  • Cloning and recombinant expression of allergens
    • E. coli, "Clear coli"
    • Pichia pastoris
  • RNA-silencing of specific genes
  • (real-time) PCR

ALLERGEN/PROTEIN PURIFICATION

  • Aqueous and organic extraction methods
  • Chromatography
    • Affinity chromatography
    • Ion exchange
    • Hydrophobic interaction
    • Reversed phase
  • (Preparative) gel electrophoresis
  • purification of lipophilic allergens

ALLERGEN/PROTEIN ANALYSIS

  • Electrophoresis
    • Isoelectric focusing (IEF)
    • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
    • 2D-SDS-PAGE
    • Difference gel electrophoresis (DIGE)
  • Immunoblotting
    • Detection of IgE, IgG und IgG subclasses
    • Colorimetric readout
    • Readout by chemiluminescence
  • ELISA Techniques and ELISA establishment
  • N-terminal sequencing
  • Zymography
  • Enzyme kinetics
  • Epitope mapping of allergens

DESIGN OF PEPTIDE ARRAYS

MAPPING OF B CELL EPITOPES: IgE / IgG

ALLERGEN/PROTEIN ENGINEERING

  • Protein fragmentation by protease digestion
    • simulation of human digestion (Enzynorm/Kreon)  
  • Protein analysis: reduction / native analysis
  • Controlled deglycosylation of proteins
    • Enzymatic: Glycosidases
    • Chemical: Periodate

CELL ANALYSIS / CELL CULTUE

  • Cultivation of human / mammalian cells
  • Cell analysis by flow cytometry (FCM, FACS)
  • Analysis of cell activation by allergens (BAT, basophile activation test)
  • Fluorescence microscopy, FRET techniques

FURTHER TECHNIQUES

  • Generation of monoclonal antibodies and antisera (MAB technique)
  • Immunohistochemistry (IHC)
  • In silico protein-ligand docking studies (Autodock)