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Isolation of Canine Neutrophil Plasma Membranes
Author(s) -
O'Donnell Robert T.,
Andersen Burton R.
Publication year - 1985
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.38.3.353
Subject(s) - percoll , vesicle , biology , membrane , concanavalin a , differential centrifugation , affinity chromatography , sepharose , chromatography , organelle , biochemistry , centrifugation , chemistry , enzyme , in vitro
This paper describes the isolation of plasma membrane vesicles formed by nitrogen cavitation of canine neutrophils. Plasma membranes from disrupted cells were separated from other membranes and organelles by Percoll‐density gradient centrifugation. Transmission electron microscopic examination of membrane preparations chromatographed on either Sephacryl S‐1000 or Sepharose 4B revealed that two populations of plasma membrane vesicles were formed: large (176 ± 22nm), and small (119 ± 11 nm). Purified large vesicles were separated from Percoll and contaminating cytosol by Sephacryl S‐1000 chromatography. Small vesicles were obtained free of Percoll by recavitating purified large vesicles. Problems encountered due to the presence of a soluble 5′‐nucleotidase inhibitor also are discussed. Large and small membrane vesicles were separated into adherent and non‐adherent populations by affinity chromatography on either concanavalin A‐Sepharose or lentil lectin‐Sepharose columns.