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Allergy pp 223-237 | Cite as

Preparation of Samples for a Mass Spectrometry-Based Method to Identify Allergenic Proteins

  • Mary BrianEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2020)

Abstract

Western blotting is an immunological technique that has been combined with mass spectrometry analysis, to create a high-throughput method for protein identification. Western blotting using serum allows us to identify a protein within an allergenic extract that specifically binds to a serum antibody, immunoglobulin E. This specific IgE binding protein can then be detected with a highly sensitive chemiluminescence detection substrate.

Proteins detected by western blotting can be analyzed by mass spectrometry following an in-gel digestion protocol. The protein band of interest is excised from the gel and digested with trypsin to form peptides. Mass spectrometry will almost certainly have a pre-chromatographic step in which these peptides are separated before becoming ionized and entering a mass analyzer. It is in the mass analyzer that peptides are identified according to their mass-to-charge ratio, compiled into a mass spectrum which is compared to mass spectra held within online protein databases.

Key words

Allergen Mass spectrometry Gel electrophoresis Western blotting 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Occupational and Environmental MedicineNHLI at Imperial CollegeLondonUK

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