American Society of Hematology

Fig. 1.

$Fig. 1. Purification of neutrophil chemotactic activity from serum. / Chemotactic factors isolated from newborn calf serum were first fractionated in a NaCl (0-2 M) gradient (––) by heparin-Sepharose affinity chromatography (A). The protein concentration (■-■) in the fractions was evaluated by the Coomassie blue binding assay. The neutrophil chemotactic activity (▴-▴), expressed as chemotactic indexes (CI), was determined in the Boyden microchamber assay at 1/10 dilution. (B) The final purification step of the neutrophil chemotactic activity by RP-HPLC is shown. Proteins were recovered from the HPLC column in an acetonitrile (0-80%) gradient (––). Neutrophil chemotactic potencies (▨) were determined at dilution 1/50 and represent the mean CI ± SEM of 5 independent experiments. HPLC fractions containing neutrophil chemotactic activity were analyzed by SDS-PAGE (C) under reducing conditions (fractions 32 to 43, 4 μL/lane). The proteins were visualized by silver staining. The left lane shows Mrmarkers (see “Materials and methods”).$

Purification of neutrophil chemotactic activity from serum.

Chemotactic factors isolated from newborn calf serum were first fractionated in a NaCl (0-2 M) gradient (––) by heparin-Sepharose affinity chromatography (A). The protein concentration (■-■) in the fractions was evaluated by the Coomassie blue binding assay. The neutrophil chemotactic activity (▴-▴), expressed as chemotactic indexes (CI), was determined in the Boyden microchamber assay at 1/10 dilution. (B) The final purification step of the neutrophil chemotactic activity by RP-HPLC is shown. Proteins were recovered from the HPLC column in an acetonitrile (0-80%) gradient (––). Neutrophil chemotactic potencies (▨) were determined at dilution 1/50 and represent the mean CI ± SEM of 5 independent experiments. HPLC fractions containing neutrophil chemotactic activity were analyzed by SDS-PAGE (C) under reducing conditions (fractions 32 to 43, 4 μL/lane). The proteins were visualized by silver staining. The left lane shows M_rmarkers (see “Materials and methods”).

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