Figure 2.

Thr4-P-specific mAb production. (A) Survey of synthetic phospho-peptides used for characterization of CTD-specific mAbs. Peptide CTD-2 was used for immunization of rats and identification of Thr4-P-specific mAbs (6D7 and 1G7). The binding specificity of Thr4-P-specific mAbs and of previously generated mAbs with specificity for Ser2-P (3E10), Ser5-P (3E8), Ser7-P (4E12), and unmodified CTD (1C7) was determined by ELISA using a panel of 19 CTD peptides with different combinations of phosphorylated amino acids. Phosphorylation of amino acids adjacent to the phospho site used for immunization either inhibited (−), did not inhibit (+++), or inhibited binding of mAbs to various degree (++, +). (B) Quantitative ELISA data for CTD peptides 1–19. Reactivity below 0.05 indicates background. (C) Overview of conditions of phospho-CTD recognition by mAbs. Red amino acids indicate full or partial inhibition of mAb binding. Phosphorylated amino acids in black colour (Tyr1, Ser, Thr) did not inhibit mAb binding. Error bars show standard deviation of three experiments.