Anti epo antibody

rhEPO containing culture media was subjected to SDS-PAGE, followed by transferring to a PVDF membrane (Millipore Corp., Bedford, MA). After blocking with 5% BSA in TBS-T (140 mM NaCl, 10 mM Tris-HCl, with 0.05% Tween 20, pH 8.0) for 1 hour at room temperature, the PVDF membrane was incubated with biotinylated lectins (Wheat Germ Agglutinin; WGA, Maackia Amurensis leukoagglutinin I; MAL I) at 4 °C for 16 hours. After rinsing three times for 5 min using TBS-T, the membrane was incubated with ExtrAvidin-peroxidase (Sigma) at room temperature for 1 hour, followed by developing using ECL kit (Thermo Scientific; Rockford, IL). For reblotting, the membrane was stripped at room temperature for 1 hour with stripping buffer (Candor Bioscience GmbH, Weissensberg, Germany). PVDF membrane was rinsed 5 times with T-TBS. The membrane was blocked using 5% BSA in TBS-T, and then incubated with 1:1000 diluted anti-EPO antibody (Santa Cruz, CA). After washing three times with TBS-T for 5 min, the membrane was incubated with 1:5000 diluted HRP-labeled anti-mouse IgG antibody (Santa Cruz) at room temperature for 1 hour. The membrane was developed using ECL solution (Thermo Scientific; Rockford, IL).