Opisthorchis viverrini metacercariae were collected from naturally infected cyprinid fish from a water reservoir in Amphur Muang, Khon Kaen Province, Thailand. The fish were purchased from a local fish market in Khon Kaen, Thailand. Following collection, O. viverrini metacercariae were examined under a dissecting microscope and identified as described (Sripa et al. 2010 (link)) and used for infection of laboratory hamsters. The experimental infection of hamsters were performed as described (Intapan and Maleewong 2006 (link); Sadaow et al. 2019 (link)). Adult O. viverrini worms were recovered from the livers and bile ducts of the hamsters three months later. Adult somatic antigen was obtained as described (Intapan and Maleewong 2006 (link)). Briefly, the adult worms were homogenized with tissue grinder in a small volume of 0.1 M phosphate-buffered saline, pH 7.4, containing proteinase inhibitors (cOmpleteTM ULTRA Tablets, Mini EASYpack Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). The suspension was sonicated with an ultrasonic disintegrator and centrifuged at 10,000 x g for 30 min at 4°C. The protein concentration of the antigen was determined (Bradford 1976 (link)). The supernatant as the antigen was aliquoted and stored at −80°C until used.
Mini easypack protease inhibitor cocktail tablets
Manufactured by Roche
Sourced in Switzerland
The Mini EASYpack Protease Inhibitor Cocktail Tablets are a laboratory product designed to inhibit protease activity. The tablets contain a combination of protease inhibitors that can be used to prevent the degradation of proteins during sample preparation and analysis.
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4 protocols using mini easypack protease inhibitor cocktail tablets
1
Extraction and Preparation of Opisthorchis viverrini Antigen
2
Recombinant SsIR Protein Expression
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The synthesized gene (rSsIR; GenBank no. AF035657.1) at position 1–471 bp was optimized in Escherichia coli expression system and constructed into pET43.1a (+) vector from the company (GenScript, Piscataway, NJ, USA). The rSsIR-plasmid was transformed into an E. coli JM109 (Novagen, Darmstadt, Germany) cloning host and an E. coli Rosetta-gami 2 (DE3) expression host (Novagen). Following this, the sequencing identified a recombinant plasmid, which yielded the in-frame sequence. Expression of N- and C-terminal-fused His-tag rSsIR was induced with 1 mM Isopropyl 1-thio-β-d -galactopyranoside (IPTG) at 33 °C for 24 h. The soluble rSsIR antigen was purified using Ni-NTA His Bind Resin (Novagen) and dialyzed against distilled water containing proteinase inhibitor (cOmpleteTM ULTRA Tablets, Mini EASYpack Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). The protein concentration of the purified rSsIR protein was determined by Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and stored at −70 °C before use.
3
Preparation of S. japonicum Antigen
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Frozen S. japonicum males and females (Philippine strain) were obtained from the Applied Malacology Center, Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Thailand. Briefly, the adult worms (200 mg wet weight) were homogenized with a tissue grinder in a small volume (500 mL) of distilled water containing proteinase inhibitors (Complete ULTRA Tablets, Mini EASYpack Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). The extract was then sonicated with an ultrasonic disintegrator and centrifuged at 10,000 Â g for 30 min at 4 C. The protein concentration of the resultant supernatant was estimated (1.17 mg/mL) using the standard method (Lowry et al., 1951 ) and stored at À70 C until use as the antigen.
4
Trichinella spiralis Larval Antigen Extraction
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Antigen was prepared from the T. spiralis strain ISS62. This strain, originally from a domestic pig and now maintained in mice, caused an outbreak of human trichinellosis in Thailand's Mae Hong Son Province in 1986 ( Pozio and Khamboonruang, 1989 ) . The muscles of mice, one month after oral inoculation with Trichinella larvae, were digested with pepsin-HCl. Trichinella spiralis larvae were harvested using a modified Baermann technique ( Justus and Morakote, 1981 ) (link) and were washed several times in distilled water. The extraction of somatic antigen from these larvae was previously described ( Intapan et al., 2006 ) (link). Briefly, the T. spiralis larvae (500 mg wet weight) were homogenized with a tissue grinder in a small volume (10 0 0 μL) of distilled water containing proteinase inhibitors (Complete ULTRA Tablets, Mini EASYpack Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland). The extract was then sonicated with an ultrasonic disintegrator and centrifuged at 10,0 0 0 × g for 30 min at 4 °C. The protein concentration in the supernatant was assayed using the QuickStart Bradford Protein Assay (Bio-Rad Laboratories Inc., CA). The supernatant was kept as the source of antigen and stored at -70 °C until used.
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