Identification of Rad21 protein in crude extract of S. pombe with anti-Rad21 antiserum. WT cells and rad21-9xPK cells grown in YS5S to 1x107, collected, washed and broken by glass beads. Twenty ug proteins per lane were run on SDS-PAGE. First antibody at indicated dilutions in PBST plus 2.5% skim milk was incubated at 4ºC overnight. Second antibody at 1/10,000 of anti-rabbit HRP in PBST was incubated at room temperature for 1 h. Detected by ECL (X-ray film). Rad21 protein migrated at the position of ~110 kDa in SDS-PAGE although the molecular mass is 67.8 kDa. Multiple bands of Rad21 reflect multiple phosphorylation states.