Study of four protein-extraction methods of the Caspian monkey Goby (Neogobius fluviatilis pallasi Pallas, 1814) epidermal mucus for bioassay-guided screening

Fish epidermal mucus protein extraction for bioassay-guided screening is challenging and variable. In this study, four different protein extraction methods, including acetone, trichloroacetic acid (TCA)-acetone, methanol, and solid-phase extraction (SPE), were investigated to study their efficiency in ultrafiltration and to obtain the appropriate peaks in HPLC to measure biological activities. The presented data shows that the solid phase extraction method is better than the other three tested methods in quantity (total protein yield) and quality (protein profile observed in SDS-PAGE) of proteins and peptides from the epidermal mucus of Caspian monkey goby. On the other hand, due to the epidermal mucus hydrophobic nature, all three extraction methods including acetone, (TCA)-acetone and methanol led to the irreversible proteins aggregation, makes ultrafiltration, HPLC, and consequently bioactivity screening impossible. In this study, the highest amount of protein (4.676 μg/ml) (P<0.01) and the best protein profile (13 bands) were related to solid phase extraction. Also, this method simultaneously leads to the extraction, concentration, and initial purification of the samples and therefore improves the efficiency ultrafiltration and HPLC. Thus, we introduce this method for the isolation of active proteins and peptides from various complex biological samples, including fish epidermal mucus.