 |
© 1996 Springer-Verlag. Reproduced with permission.
(from Durso et al. 1996, Protoplasma 190: 141-150)
 a The antiserum (S) from an immunized rabbit, the corresponding preimmune serum (P), and a commercial monoclonal anti-alpha-tubulin antibody (T; Amersham) were used on immunoblots of tubulin binding proteins (TBP; ~4 µg), total soluble carrot proteins (Total; ~10 µg), or purified carrot tubulin (from taxol-stabilized microtubules, cMT; ~0.5 µg). Antibody dilutions: S at 1:10,000 on TBP and Total, 1:1000 on cMT; T and P at 1:1000. The major signal is at 50 kDa. b Affinity purified anti-pp50 antibodies (1:100) were used on immunoblots of pp50-enriched protein (lane 1; ~0.5 µg), purified carrot tubulin (lane 2; 0.5 µg), and total soluble carrot proteins (lane 3; ~15 µg). The major signal is at 50 kDa. c Affinity purification controls. Non-pp50 ligands were used in the affinity purification protocol using anti-pp50 antiserum. The eluates were either off random non-pp50 polypeptides (on a strip excised diagonally from the same membrane on which pp50 was immobilized, but excluding pp50; lanes 1 & 2) or off carrot tubulin (lanes 3 & 4), and were used, each at 1:10, on immunoblots of pp50-enriched protein (lanes 1 & 3; ~0.5 µg;) or purified carrot tubulin (lanes 2 & 4; ~0.5 µg). Note that anti-pp50 antiserum and affinity purified anti-pp50 antibodies are monospecific for pp50 and do not contain anti-tubulin contaminants or other components that react with pp50 on immunoblots. |
