Specific in vivo labeling of tyrosinated α-tubulin and measurement of microtubule dynamics using a GFP tagged, cytoplasmically expressed recombinant antibody.

ABSTRACT: GFP-tagged proteins are used extensively as biosensors for protein localization and function, but the GFP moiety can interfere with protein properties. An alternative is to indirectly label proteins using intracellular recombinant antibodies (scFvs), but most antibody fragments are insoluble in the reducing environment of the cytosol. From a synthetic hyperstable human scFv library we isolated an anti-tubulin scFv, 2G4, which is soluble in mammalian cells when expressed as a GFP-fusion protein. Here we report the use of this GFP-tagged scFv to label microtubules in fixed and living cells. We found that 2G4-GFP localized uniformly along microtubules and did not disrupt binding of EB1, a protein that binds microtubule ends and serves as a platform for binding by a complex of proteins regulat

SUBMITTER: Cassimeris L 

PROVIDER: S-EPMC3610906 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature