Anti-Tubulin monoclonal antibody, clone 656

Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the beta-subunit of the alpha-beta tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy.

Objectives To examine the antiproliferative effect of a rationally designed, novel noscapine analogue, 9-((perfluorophenyl)methylene) aminonoscapine, '9-PAN') on MDA-MB-231 breast cancer cell line, and to elucidate the underlying mechanism of action. Methods The rationally designed Schiff base-containing compound, 9-PAN, was characterized using IR, NMR and mass spectra analysis. The effect of the compound on cell viability was studied using an MTT assay. Cell cycle and cell death analyses were performed using flow cytometry. Binding interactions of 9-PAN with tubulin were studied using spectrofluorometry. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were investigated using the probes, DCFDA and rhodamine-123, respectively. Immunofluorescence imaging was used to visualize cellular microtubules. Key findings 9-PAN inhibited cell proliferation (IC(50)of 20 +/- 0.3 mu m) and colony formation (IC50, 6.2 +/- 0.3 mu m) by arresting the cells at G(2)/M phase of the cell cycle. It bound to tubulin in a concentration-dependent manner without considerably altering the tertiary conformation of the protein or the polymer mass of the microtubulesin vitro. The noscapinoid substantially damaged cellular microtubule network and induced cell death, facilitated by elevated levels of ROS. Conclusions 9-PAN exerts its antiproliferative effect by targeting tubulin and elevating ROS level in the cells.