The H c-C3BP is a new entity as it differs biochemically from oth

The H.c-C3BP is a new entity as it differs biochemically from other known such proteins. The significance of H.c-C3BP is discussed

in relation to host–parasite interaction. Acrylamide, bis-acrylamide, PMSF, diaminobenzidine (DAB), orthophenyl diamine (OPD), CNBr-activated Sepharose 4B and goat anti-human C3 polyclonal antibody were procured from Sigma–Aldrich (Karnataka, India). Lysozyme, protein molecular weight markers, goat anti-rabbit IgG–horse radish peroxidase conjugate, rabbit anti-goat IgG–horse radish peroxidase and isopropyl thio-D-galactopyranoside were purchased from Bangalore Genei (Bangalore, India). Ni-charged resin, nitrocellulose membranes and sodium dodecyl sulphate were purchased from Bio-Rad laboratories this website (Mumbai, India); rabbit anti-human MAC (C5b-9) antibodies were purchased from Calbiochem (La Jolla, CA, USA) and rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase was procured from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). All other chemicals used were of analytical grade. Sheep and goat abomasums (stomach) were procured from local abattoir; the adult worms were picked up manually and washed several times with prewarmed saline. The excretory–secretory products (ES products) were collected

Birinapant nmr by culturing the adult parasites in RPMI 1640 medium without phenol red containing streptomycin 0·1 mg/mL and penicillin 100 IU (~20 worms per mL of the medium) at 37°C for 6–8 h in a candle jar [10, 11]. The ES products and the adult worms recovered after incubation were stored at −40°C. The infective-stage larvae (L3) of H. contortus were

recovered by mild crushing of the adult parasites and layering the extract over a mixture of autoclaved goat faecal matter and powdered charcoal (3 : 2 w/w) kept on a moistened filter paper in a Petri dish. The Petri dish was kept in a bigger Petri dish containing sterilized distilled water. This assembly was covered with a glass jar and kept at room temperature Bay 11-7085 (25–30°C) with provision for aeration. The larvae, which emerged and were collected in the water reservoir after 5–7 days, were concentrated by filtration through a Whatman No.1 filter paper. The adhered larvae were flushed by dipping the paper in small volume of distilled water and stored at −40°C. Complement C3 was purified as described earlier [15] with modifications. Goat blood was collected in citrate saline. About 120 mL of plasma was treated with 14 mL of 40% PEG-8000 drop wise (~4% (w/v) final concentration). The suspension was centrifuged at 10 000 g for 30 min at 4°C. The supernatant was collected, and 30 mL of 40% PEG was added to increase its concentration to 10% (w/v). It was left overnight at 4°C for precipitation of the C3 protein. The precipitates were collected by centrifugation and dissolved in PBS with stirring to break lump pieces. The solution was dialysed against 20 mm sodium phosphate (pH 7·4) containing 5 mm EDTA.

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