Ac. Estalote et al., AN IMPROVED METHOD FOR THE ISOLATION OF ERYTHROCYTE ANTIGEN BAND-3, Brazilian journal of medical and biological research, 28(9), 1995, pp. 945-949
An improved method for isolation of human and Rhesus monkey band-3 sep
arated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (S
DS-PAGE) is described. Purified band-3 was obtained from human hemoglo
bin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution sonication (CE+S). The section of the gel corresponding to the antigen
was cut out, mechanically disrupted and incubated in 1% NaHCO3 contai
ning 1% SDS, for 2 h, with shaking, at room temperature, followed by o
vernight incubation at 4 degrees C. The preparation was subsequently s
onicated and clarified by centrifugation. Supernatants were dialyzed a
gainst distilled water, their protein contents were measured, and the
presence of purified band-3 was demonstrated by SDS-PAGE. A calibratio
n curve was developed for assay of CE+S material using densitometric e
valuation of the protein profile on SDS-PAGE. An amount of 37.5 mg of
Hb-free ghosts gave 3.15 mg of purified band-3 after CE+S, correspondi
ng to an 8.4% yield. Rabbits were immunized with 50 mu g. CE+S antigen
. Sera were collected and assayed by Western blot analysis against its
proteolytic fragments, which were obtained from packed red blood cell
s by treatment with protease type VI from Streptomyces griseus (1 h at
37 degrees C), followed by extensive washing and hypotonic lysis. Spe
cific antibodies recognized band-3 and its proteolytic fragments 60 an
d 63 kDa in human ghosts obtained from different blood donors, confirm
ing the genetic polymorphism. Analogous serum obtained against the Rhe
sus monkey band-3 proteolytic fragment 63 kDa recognized the human ant
igen and its respective fragments. These results indicate the existenc
e of similarities between these two species of band-3, suggesting the
potential use of this technique in taxonomic and phylogenetic studies.
Purification by CE+S is an efficient and rapid method for isolation o
f band-3 and its fragments with satisfactory yields and maintenance of
both their immunogenic and antigenic properties.