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Proteins, Expression, Isolation and Analysis> | … The primary antibody, either anti-NuMA (Invitrogen, GT3511) or anti-Eg5 (custom, Genscript), was then incubated on the coverslip at 5 μg/ml for 45 min, washed with PBS-N, then the … | Get A Quote |
Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is co... More