The Ovarian Blood Follicle: MATERIALS AND METHODS(2)

1 Jan
2013

MATERIALS AND METHODS(2)

lal and a2-M Cationization

led and a2-M were cationized according to a modified protocol from Border et al.. Briefly, an ethylenedi-amine (EDA) solution was adjusted to pH 4.75 and chilled to room temperature. One milligram of either purified human led or a2-M was diluted in distilled water. The EDA solution and l-ethyl-3-[(3-dimethylaminopropyl)-carbodi-imide hydrochloride] (EDC) was added to the diluted proteins. The solutions were allowed to rotate at room temperature for 2 h, and the reaction was terminated by changing the buffer to PBS in Centricon-100 tubes (Ami-con, Inc., Beverly, MA) via centrifugation at 1000 X g. Dot blot analysis demonstrated that the immunoreactivity of cationized led and a2-M was not compromised (data not shown).

IgG Amortization

Purified rabbit IgG was anionized according to a modified protocol from Chen et al.. Initially, succinic anhydride was used to produce proteins of high and low negative IgG charge density, but to yield more consistent data citraconic anhydride (CA) was substituted in the following experiments. Twenty microliters (1-2 |xl every 5 min) of CA was added to 1 mg of purified rabbit IgG in PBS. The reaction was stopped by changing the buffer to PBS using centricon-100 tubes via centrifugation at 1000 X g. The low negative charge density was obtained by adjusting the pH of the reaction mixture to 5.0 and incubating overnight at 40°C. Changing the buffer to PBS and using the same centrifugation technique stopped the reaction. Dot blot analysis demonstrated that anionization of IgG did not compromise immunoreactivity (data not shown). ventolin inhaler

Isoelectrofocusing

Protein charge was determined using the Bio-Rad Mini IEF Cell System (Bio-Rad Labs., Richmond, CA) according to manufacturer’s protocol. Ten micrometers of each protein in PBS was allowed to diffuse into an acrylamide gel and was electrophoresed under constant voltage conditions in a “stepped fashion” for 1.5 h according to the Bio-Rad IEF Cell manual. Focusing was begun at 100 V for 15 min and then increased to 200 V for 15 min and finally to 450 V for 60 min. Gels were fixed and stained according to the manufacturer’s protocol, and the isoelectric points of all proteins were compared to the standards.

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