The use of spent renal dialysis membranes for the isolation of large numbers of human neutrophils for biochemical studies. Application to purification of the myeloid IgA receptor (Fc alpha R).
Human neutrophils (PMN) can be eluted from spent Cuprophan renal dialysis membranes in large numbers (10(9)-10(10) per dialyser cartridge) and in relatively high purity by washing the membranes with 0.35 M NaCl. This offers the possibility of isolating relatively large amounts (10(-4)-10(-3) g) of minor PMN proteins such as those expressed on the cell surface. Here the technique is applied to the purification of the neutrophil IgA receptor (Fc alpha R). Affinity chromatography on IgA-Sepharose of NP-40 extracts of 125I-labelled PMN isolated from fresh venous blood routinely gave a receptor preparation showing one diffuse band, Mr 50-70 kDa, upon analysis by SDS-PAGE and autoradiography. When the same method was used with larger numbers of unlabelled PMN from fresh venous blood or renal dialysis membranes a preparation was obtained which gave multiple bands upon analysis by SDS-PAGE silver stained gels due to contamination of the receptor with cytoplasmic proteins which bound non-specifically to the IgA-Sepharose. Most of these contaminants could be removed by chromatography of the IgA-Sepharose eluates on wheat germ agglutinin-Sepharose.
Mazengera
R L
RL
Department of Pathology, Ninewells Hospital and Medical School, University of Dundee, U.K.
Kerr
M A
MA
Todd
A S
AS
D015703:Antigens, CD; D002448:Cell Adhesion / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007070:Immunoglobulin A / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000302:isolation & purification; D008567:Membranes, Artificial; D009504:Neutrophils; D011961:Receptors, Fc / Q000302:isolation & purification*; D016622:Silver Staining
D015703:Antigens, CD; C036832:Fc(alpha) receptor; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D011961:Receptors, Fc