Tubulin protein: soybean
- Recommended for IC50 & EC50 determinations for plant-specific tubulin ligands.
- Recommended for examining protein interactions with plant-specific tubulin.
Soybean tubulin is isolated from 6 day old soybean germlings of the variety 0127562 (Garst Inc.
Agripro Round-Up Ready) using anion exchange chromatography. Tubulin from soybean may be
used in all situations where other tubulins have been employed, such as compound screening, motility
assays, binding assays and polymerizaton assays. The advantage of using TP005 over animal
tubulins, it is derived solely from plant cells and thus more accurately portrays the tubuin phenotype
of plant cells. TP005 is supplied as a white powder containing 250 μg of soybean tubulin protein
lyophilized in 80 mM PIPES pH 7.0, 1 mM MgCl2, 0.5 mM EGTA, 0.1 mM GTP, 1.0 mM DTT, 5%
Sucrose, and 1% Ficoll400.
Protein purity is determined by running 20ug of protein on a PAGE-SDS gel, (see Figure 1) and performing scanning densitometry of the protein band stained with Coomassie Blue from 4-20% SDS-PAGE. TP005 is determined to be greater than 90% Soybean tubulin (molecular weight of 54 kDa).
Figure 1. TP005 Protein Purity Determination.
A 20 μg sample of TP005 protein was separated by 4-20% SDS-PAGE and stained with coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02).
Biological Activity Assay
The biological activity of TP005 can be assessed by polymerization assays. Tubulin polymerization
into microtubules can be detected by measuring the optical density (OD) at 340 nm or by fluorescence
in the presence of DAPI at excitation and emission wavelengths of 360 nm and 405-450 nm,
||Figure 2. Polymerization of Soybean Tubulin In Response to Trifluran and Paclitaxel. Soybean tubulin was polymerized in the presence 10 μM trifluran (inhibitor) or paclitaxel (enhancer). TP005 was prepared as stated in the methods sectionfor the fluorescence format. Blue diamonds = control, magenta squares = trifluran and yellow triangle paclitaxel. The intensity of fluorescent emissions at 410 nm were measured every 30 s for 40 min.||
|Table 1. Tubulin Ligand Index Values from Studies with Soybean and Neuronal Tubulin.|
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Feizabadi M.S., et al. 2013. Analysis of a single soybean microtubule’s persistence length. Adv. Biosci. Biotechnol. 4, 930-936.
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