![HIS-TAN1-GFP binds to soluble tubulin dimers.(A) Agarose beads fused with an anti-GFP antibody were used to pull down HIS-TAN1-GFP or HIS-GFP in the presence of tubulin dimers. A tubulin band is detected after pull-downs in the presence of HIS-TAN1-GFP (1 µM) and not detected in the pull-down with HIS-GFP (1 µM). Coomassie-stained SDS-PAGE results from two independent in vitro pull-down are shown. Band intensity differences reflect differences in the relative amount of immunoprecipitated HIS-TAN1-GFP between experiments. (B) Size exclusion chromatography of one representative replicate of molecular weight standards and tubulin using fast protein liquid chromatography. The A280 (a.u.) for each standard and tubulin was plotted against elution volume (Ve) for the same running conditions to determine the Ve for each protein. (C) Interpolated molecular weights for tubulin were plotted on a semilog Ve/V0 curve determined from molecular weight standards (void volume determined by running blue dextran through column). Two replicates for 5 µM tubulin (91.45 ± 12.32 kD [average ± SD]) and 10 µM tubulin (111.13 ± 14.18 kD) and protein standards were run, indicating that tubulin elutes as a dimer.](https://cdn.rupress.org/rup/content_public/journal/jcb/219/8/10.1083_jcb.201907184/4/jcb_201907184_figs3.png?Expires=1772836829&Signature=YPM4f8aY5V2pRxB6Y2d-3hZiEJUq9Oum9gr7wf-hb1V1PR7Y39RUOQSW8T29fRwmADXHxyP8WvaY2Ij80bLuUI7hH9eKrK8BLuODmH1G-j19nNZ8z2InaREgPfOVLsHnYt1rtdeErD9M0JsPsHOtiN4JqzuEa2~I-mBTmKIIAxZHSrfhkZg4uwxgCprFkDADYA78yiCUYI6l0bOBR~eHG5MBXmVZUeqR~Tr4j-OVeX4SDDSaZw6E~Z8-~9qH500bywMG6wDb~9iOX98QkFqtZwEywQlOyow2zEtEZUqVdG7FAfEPYrgxG69kXaHlEdgfbXZ62tm8~kuJlRk5XWJK1g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
HIS-TAN1-GFP binds to soluble tubulin dimers. (A) Agarose beads fused with an anti-GFP antibody were used to pull down HIS-TAN1-GFP or HIS-GFP in the presence of tubulin dimers. A tubulin band is detected after pull-downs in the presence of HIS-TAN1-GFP (1 µM) and not detected in the pull-down with HIS-GFP (1 µM). Coomassie-stained SDS-PAGE results from two independent in vitro pull-down are shown. Band intensity differences reflect differences in the relative amount of immunoprecipitated HIS-TAN1-GFP between experiments. (B) Size exclusion chromatography of one representative replicate of molecular weight standards and tubulin using fast protein liquid chromatography. The A280 (a.u.) for each standard and tubulin was plotted against elution volume (Ve) for the same running conditions to determine the Ve for each protein. (C) Interpolated molecular weights for tubulin were plotted on a semilog Ve/V0 curve determined from molecular weight standards (void volume determined by running blue dextran through column). Two replicates for 5 µM tubulin (91.45 ± 12.32 kD [average ± SD]) and 10 µM tubulin (111.13 ± 14.18 kD) and protein standards were run, indicating that tubulin elutes as a dimer.
