Affinity isolation of albumin-binding proteins using nitrocellulose-bound albumin.
Albumin immobilized on a nitrocellulose membrane was used as an affinity matrix to purify albumin-binding proteins (ABP) from extracts of lung, heart, thymus, and isolated microvascular endothelial cells. Albumin was immobilized onto nitrocellulose either (i) directly (physically adsorbed), (ii) cross-linked by treatment with 0.25% glutaraldehyde, or (iii) covalently coupled to the matrix using NaIO4 and Na-borohydride. The affinity support was incubated with a membrane-enriched fraction (obtained from tissue homogenates) in the presence of protease inhibitors; specific binding of ABP occurred within 30 min of incubation. The adsorbed proteins were eluted with 0.5% sodium dodecyl sulfate (SDS) and analyzed by SDS-polyacrylamide gel electrophoresis and ligand blotting. Analysis of electrophoretic mobility of eluted proteins showed that they consisted exclusively of the two sets of polypeptides of 31 000 Da and 18 000 Da previously identified as ABP (N. Ghinea et al., J. Cell Biol. 107, 231-239 (1988]. As demonstrated by ligand blotting, the ABP purified on nitrocellulose-bound albumin maintain the ability to interact specifically with albumin. Preliminary experiments showed that the method employed may be of a broader use for the isolation of receptor proteins from tissue extracts by incubating the latter with the cognate ligand immobilized on nitrocellulose membranes.
Moroianu
J
J
Institute of Cellular Biology and Pathology, Bucharest, Romania.
Simionescu
M
M
D000418:Albumins / Q000378:metabolism*; D000818:Animals; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008722:Methods; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D051381:Rats
D000418:Albumins; D008024:Ligands; D011506:Proteins; D003101:Collodion