Comparison of extraction methods for Poaceae pollen allergens
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While pollen sampling has been standardised in the last decades, allergen extraction from aerobiological samples still needs standardisation. With the aim to identify the most practical, efficient and reproducible method, in this study, protein extraction protocols from Poaceae pollen, i.e. Lolium perenne, Phleum pratense and Dactylis glomerata, were evaluated. Three extraction protocols were selected and compared in terms of protein yield and allergen extraction. The methods were based on the use of: (1) lysis buffer; (2) pollen hydration in PBS buffer followed by sonication; and (3) pollen hydration in PBS buffer. After extraction, protein concentration was measured. Results indicated that the different extraction protocols could extract significantly different protein amounts, being the lysis buffer-based protocol the most efficient in terms of total protein extraction. However, when allergen extraction was compared, no significant differences were detectable among the different protocols. Results afforded the selection of a suitable extraction protocol for Poaceae pollen allergens. In particular, results suggested a PBS extraction followed by sonication; as it allowed the highest allergen extraction, it might be the most reproducible protocol minimising any possible interference with proteomics and immunological studies when compared to lysis buffer method.
KeywordsComparison of extraction protocol Poaceae pollen Poaceae allergens Protein yield
We are very thankful to Juan Asturias for providing pollen samples and primary antibodies. This study was partially supported by AERORAIN project (Ministry of Economy and Competitiveness, Grant CGL2014-52556-R, co-financed with FEDER funds).
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