Parvalbumin Content and Expression Analysis
The relative content of fish parvalbumin was analyzed through both protein- and transcriptome-based approaches. For the protein approach, fresh fishes including tuna, salmon, halibut, cod, grouper, catfish, grass carp, and tilapia were purchased from local wet market. Genus/species of the fishes were confirmed by DNA barcoding upon DNA extraction with QIAamp DNA mini kit (Qiagen) and PCR with specific primers: Fish-F1: 5′-TCTCAACCAACCATAAAGACATTGG-3′ and Fish-R1: 5′-TATACTTCTGGGTGCCCAAAGAATCA-3′; Fish-F2: 5′-CATCCTACCTGTGGCAATCAC-3′20 . One milligram of raw fish meat was manually homogenized in 10mL ice-cold phosphate-buffered saline (PBS) until a smooth paste was achieved 21 . Protein was then extracted overnight at 4°C with constant stirring. The protein extract was centrifuged and supernatant was filter-sterilized through a 0.22 μm polyethersulfone membrane. The fish protein extracts analyzed fresh upon extraction. Ten microliters of fish extracts were then separated on a 13.5% SDS-PAGE gel according to their molecular weights using a Mini PROTEAN SDS-PAGE system (Bio-Rad). Protein bands were stained with SimplyBlue SafeStain (Thermo Fisher). Densitometry analysis was then performed to determine the relative band intensity of parvalbumin on SDS-PAGE by Image Lab (Bio-Rad) using salmon parvalbumin as the reference band (i.e. band intensity = 1). Four pieces of fish meat were randomly picked for protein extraction and resolved on separate SDS-PAGE to ensure reproducibility of parvalbumin level analysis. While in transcriptome-based approach, we processed publicly available RNA-seq data of fish meat, ensuring quality, and de novo assembly with Trinity22. Aligning transcripts with known allergens from AllergenOnline 23 via blast method identified homologous parvalbumin transcripts.