The Ovarian Blood Follicle: MATERIALS AND METHODS(1)

31 Dec
2012

Animals

Immature female ICR mice (20 days old) were purchased from Harlan-Sprague Dawley (Indianapolis, IN) and housed in micro-isolator cages, exposed to a 12L:12D cycle, and fed standard chow and water.

Materials

Equine CG was purchased from Professional Compounding Centers of America Inc. (Houston, TX). Human CG, affinity-purified rabbit anti-human a2-macroglobulin (a2-M) immunoglobulin G (IgG), affinity-purified rabbit anti-BSA IgG, a2-M from human plasma, and purified rabbit IgG were purchased from Sigma Chemical Company (St. Louis, MO). Fluorescein isothiocyanate (FITC)-conju-gated goat anti-rabbit IgG was purchased from Cappel (Malvern, PA). Human serum was obtained from Hoxworth Blood Center (University of Cincinnati). Affinity-purified rabbit anti-human lal IgG was purchased from Dako (Car-pinteria, CA). BSA (fraction V) was obtained from Fisher Scientific (Pittsburgh, PA). cialis professional 20 mg

Purification of Human lal Protein

lal was purified from human serum according to the Chen et al. protocol. Briefly, human serum was brought to 0.3 saturation with ammonium sulfate and centrifuged at 25 000 X g. The supernatant was retained and brought to 0.45 saturation with ammonium sulfate and centrifuged. The protein pellet was resuspended in PBS and subjected to HPLC using a SW3000 gel filtration column. Immuno-reactive fractions were pooled, and a final purification was performed by loading the pooled fractions onto an HPLC DEAE column and eluting the pure protein with a linear gradient of NaCl from 0 to 0.5 M (pH 7.4). The protein concentration was calibrated using a total protein determination kit (Sigma), and the protein purity was determined using SDS-PAGE and Coomassie blue staining.

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