3). Open in a separate window Figure 3. Posttranslational modifications of purified tubulin evaluated by modification-specific antibodies. study, the TOG resin was packed in an open column (Fig. 1A). To examine whether the corresponding TOG1 and TOG2 domains of MICROTUBULE ORGANIZATION1 (MOR1; Whittington et al., 2001), an Arabidopsis homolog of yeast Stu2, can be used to isolate tubulin, we also purified a recombinant GST fusion of the TOG1 and TOG2 domains from MOR1 and used this fusion to prepare a TOGMOR1 column. Open in a separate window Figure 1. Affinity purification of Arabidopsis tubulin using the TOG column. A, A gravity-flow TOG column. The GST-TOG1/2-coupled resin (2 mL) is sandwiched between the upper and lower porous plastic filters (arrows) and packed in a 15-mm-diameter column. B, Purification of tubulin from Arabidopsis MM2d cultured cells. Protein samples taken from different purification steps were separated by SDS-PAGE and analyzed by Coomassie Blue staining (left) and immunoblotting with anti–tubulin antibody (right). M, Molecular mass marker; Lysate, 5 L of crude cell extract containing 27 g of proteins; Flow-through, 5 L of the flow-through fraction that did not bind to the TOG column; Eluate, 10 L of the TOG column eluate; Final, 1 g of the desalted and concentrated tubulin. C, MT sedimentation assay. Purified MM2d tubulin (20 m) was assembled into MTs in the absence (left) or presence of 5 m taxol (right). Coomassie Blue-stained tubulin bands in the pellet (P) and supernatant (S) fractions represent polymerized MTs and tubulin, respectively. Proteins were quantified by densitometric analysis, and ratios (%) of the pellet to supernatant fractions are indicated. We first tested whether 7-d-old Arabidopsis seedlings could be used as a tubulin source for purification, since we were previously able to purify modest amounts of Arabidopsis tubulin from seedlings using the TOG column (Fujita et al., 2013). After crude cell extracts were applied to the TOG or TOGMOR1 column, the resins were washed and the bound proteins were eluted under high-salt conditions (for details, see Materials and Methods). When the TOG column was used, we obtained approximately 18 g of pure tubulin from 15 g fresh weight of Arabidopsis seedlings, with a Indocyanine green tubulin recovery rate from total extracted proteins of 0.024% (Table I). In some of our early purification attempts, purified tubulin was partly degraded, probably due to slow handling of the samples. In contrast to the TOG column, the TOGMOR1 column yielded no detectable amount of tubulin (Supplemental Fig. S1). Apparently, the first two TOG domains of MOR1 are not functionally equivalent to those of yeast Stu2 and do not bind to tubulin with high affinity. In subsequent studies, we used the Stu2-derived Indocyanine green TOG domains to explore improved conditions for obtaining pure plant tubulin at higher quantities. Table I. Purification of plant tubulins by the TOG column = episodes). Catastrophe and rescue frequencies were calculated from the inverse of the mean time spent in growth and shrinkage, respectively (= total time spent in growth or shrinkage). N/A, FAAP24 Not applicable (since few shrinkage or rescue events were observed in our assay). *, No shrinking MTs were observed during our assay period. = 24)0.62 0.16 (= 15)0.84 0.17 (= 10)0.07 0.03 Indocyanine green (= 10)0.11 0.02 (= 10)0.19 0.01 (= 2)?Shrinkage rate (m min?1)11.3 7.15 (= 10)5.51 1.01 (= 3)N/A* (= 0)N/A* (= 0)N/A* (= 0)N/A* (= 0)?Catastrophe frequency (10?3 events s?1)0.49 (= 20,495 s)0.15 (= 18,813 s)N/A (= 13,450 s)N/A (= 17,444 s)N/A (= 14,772 s)N/A (= 2,819 s)?Rescue frequency (events s?1)0.059 (= 169 s)0.038 (= 78 s)N/A* (= 0 s)N/A* (= 0 s)N/A* (= 0 s)N/A* (= 0 s)Arabidopsis?Tubulin concentration (m)7.510157.51015?Growth rate (m min?1)0.76 0.19 (= 43)0.79 0.27 (= 75)0.74 0.21 (= 73)0.36 0.08 (= 24)0.36 0.15 (= 32)0.39 0.10 (= 40)?Shrinkage rate (m min?1)49.6 32.6 (= 40)55.0 44.0 (= 71)56.6 48.3 (= 71)30.1 17.7 (= 23)38.3 21.9 (= 33)31.5 16.4 (= 40)?Catastrophe frequency (10?3 events s?1)5.52 (= 7,247 s)7.79 (= 9,245 s)5.72 (= 12,420 s)3.94 (= 5,839 s)5.04 (= 6,545 s)4.31 (= 9,290 s)?Rescue frequency (events s?1)N/A (= 115 s)N/A (=.