Marine-derived fungal strains were cultured on PDA at 25 °C for 7 d. Ten agar plugs from the PDA plates were removed using a cork borer (10 mm in diameter) and inoculated into liquid basal media [32 (link)] containing 1% (w/v) colloidal chitin. Following a 14 d incubation at 25 °C, the supernatant was separated from fungal tissue using a sterile Miracloth (Merck, New Jersey, USA) and sterilized using syringe filters with a 0.2 μm pore size membrane (Corning, Corning, NY, USA). Extracellular crude enzymes were concentrated using Amicon Ultra 3000 nominal molecular weight limit filters (Merck) and were buffer-exchanged with 20 mM sodium phosphate buffer (pH 6.0). Protein quantification in the supernatant was conducted using a Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA).
0.2 μm pore size membrane
Manufactured by Corning
Sourced in United States
The 0.2 μm pore size membrane is a laboratory filtration device designed for the removal of particles, bacteria, and other contaminants from aqueous solutions. The membrane has a pore size of 0.2 micrometers, which allows the passage of small molecules and ions while retaining larger particles and microorganisms. This membrane is suitable for a variety of laboratory applications, including sample preparation, sterilization, and purification.
Automatically generated - may contain errors
2 protocols using 0.2 μm pore size membrane
1
Extraction of Fungal Extracellular Enzymes
2
Preparation of Toxoplasma gondii Antigen
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Toxoplasma gondii tachyzoites (RH strain) were maintained in BALB/c mice by intraperitoneal passages. Peritoneal exudates from 40 mice were harvested and washed twice (720 × g, 10 min, 4°C) in PBS supplemented with a protease cocktail inhibitor (10 mg/ml aprotinin, 50 g/ml leupeptin, and 1.6 mmol/L phenylmethylsulfonyl fluoride). To prepare soluble T. gondii antigen (STAg), the parasite suspension was lysed by five sonication cycles (60 Hz for 1 min each) on ice. After centrifugation (10,000 g, 2 h, 4°C) supernatants were collected and sterilized by filtration through a 0.2 μm-pore size membrane (Corning Costar Corp., Cambridge, MA). The protein concentration was determined by Bradford (Quick Start™ Protein Assay, Bio-Rad laboratories, Hercules, CA) and aliquots were stored at −80°C until use.
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