Andrea Brazdova, Oumsaad Naas, Nicolas Visez, Jean-Pierre Sutra, Hélène Sénéchal et Pascal Poncet
Hôpital d’Enfants Armand Trousseau, Laboratoire de Biochimie, Equipe «Allergie & Environnement», 26 avenue du Dr Arnold Netter,
75012 - Paris.
Université de Lille, Laboratoire de Physico-chimie des Processus de Combustion et de l’Atmosphère, Lille.
Institut Pasteur, département Infection & Epidémiologie, Paris. 2015.

Introduction

Allergy is a hypersensitivity disorder of the immune system. According to epidemiological studies, at present, 20-30% of the population in many countries around the world suffers from allergies, and this percentage is growing trend.

Allergens are proteins with a broad range of molecular weights (5-50 kDa) exhibiting different features of solubility and stability, able to cause IgE-mediated hypersensitivity after contact with the immune system.

The development of new types of allergy treatments needs diverse and well-characterized allergenic source materials. This study describes an effective method for allergen characterization.

Overview

  • Keywords: Allergy, citrus, allergen isolation, citrus homogenization
  • Aim of the study: Identification of grinding method for citrus allergen isolation
  • Application: West blot analysis
  • Sample type: Citrus
  • Sample name: Green lemon, yellow lemon, orange, grapefruit
  • Material: FastPrep-24™ instrument, CoolPrep adapter, 2ml Lysing Matrix A, C & E tubes, IKA grinder
  • Buffer: PBS

Protocols & Parameters

  1. Add citrus peel and pulp with 500µl of PBS in 2 ml Lysing Matrix A, C or E tubes.
  2. Load Lysing Matrix tubes in a CoolPrep Adapter containing dry ice.
  3. Process with the FastPrep-24™ 5G: 40 sec at a speed setting of 6.0 m/s.
  4. Centrifuge the Lysing Matrix tubes 20 min at 18.000 x g, 4°C to pellet debris.
  5. Keep the supernatant at -20°C prior to analyses

Results

Comparison of citrus protein extraction with different lysing matrix


Conclusion

  • Protein extraction from peel and pulp of citrus samples with the FastPrep-24™ 5G showed to be highly effective with the 3 types of lysing matrix tested.
  • The protein yield with IKA grinder method was very low with high concentration of pectins, preventing protein migration on SDS- PAGE gel (data not shown).
  • The effectiveness of the FastPrep® method is quantitative, higher protein yield, and qualitative, wide variety composition of protein extracts pectins free.
  • The FastPrep® system is a powerful tool to get rapidly and with a very high reproducibility protein extracts ready for electrophoresis (SDS-PAGE) analysis.
  • Protein extracted with the FastPrep-24™ 5G instrument have conserved their immunoreactivity.