An affinity-purified anti-TK1 antibody (pAb1) raised against a synthetic
peptide (
amino acids K211PGEAVAARKLFAPQ225) corresponding to part of the C-terminus of human cytosolic
thymidine kinase (TK1) was produced and characterized by
enzyme-linked
immunosorbent assay, Western immunoblotting and immunoprecipitation as well as by immunostaining of intact cells. pAb1 recognized a single 25 kDa TK1
polypeptide in extracts of human and rodent cells. The
protein was localized to the cytoplasm, as studied by immunohistochemistry and there was no staining in G1/G0 cells or mutant cells lacking TK1 activity, while it was high in S-phase and G2 cells. When series of
peptides were tested for antibody binding in which
alanine was replacing each of the other
amino acids one by one, lysines 211 and 220,
proline 212 and
glutamic acid 214 were found to be important for antibody reactivity. These results indicate that
amino acids 211-214, which may form a turn region, constitute a major recognition site for pAb1, and this structure may also be involved in the cell cycle-dependent modification of TK1, pAb1 is a very useful tool for studies of the cell cycle regulation of TK1, and it may be used to identify and quantify rapidly proliferating cells such as
tumor cells.