The pattern of MAP-2 binding on microtubules: visual enhancement of MAP attachment sites by antibody labeling and electron microscopy

We used affinity-purified rabbit antibody to hog brain microtubule-associated protein 2 (MAP-2) to examine the pattern of attachment of MAPs to microtubules purified by cycles of in vitro assembly and disassembly. Microtubules were fixed, deposited on EM grids, and labeled with antibody and protein A-gold colloid followed by negative staining. We observed that: (1) The sites of MAP attachment were greatly enhanced by antibody binding in negatively stained preparations. (2) The axial repeat revealed by antibody (100 ± 5 nm) was greater than the previously reported value of 32 nm based on thin sectioning and negative staining procedures. (3) The antibody was arranged in a broad band and revealed a helical pattern of binding. (4) Microtubules with and without treatment with α-chymotrypsin to remove the projection portion of MAP-2 looked similar, suggesting that the antibody-enhanced pattern may reflect the sites of MAP attachment on microtubules. (5) Microtubules with an increased MAP:tubulin ratio exhibited the same 100-nm periodicity.