tag:blogger.com,1999:blog-7356964387844331141.post6820566460375341924..comments2024-03-14T03:40:44.524-06:00Comments on Steve Koch Science: A PLoS ONE Success Story--Taxol Crystals Masquerading as MicrotubulesSteve Kochhttp://www.blogger.com/profile/09076719001132389463noreply@blogger.comBlogger6125tag:blogger.com,1999:blog-7356964387844331141.post-73822863816042560092023-08-31T17:03:33.865-06:002023-08-31T17:03:33.865-06:00https://saglamproxy.com
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I am not in the field anymore, but I am still suspicious that a lot of people haven't picked up on Foss et al.'s excellent work. I'm glad you did, and wish you the best in your research!Steve Kochhttps://www.blogger.com/profile/09076719001132389463noreply@blogger.comtag:blogger.com,1999:blog-7356964387844331141.post-72874108906669221612014-01-02T13:41:55.643-07:002014-01-02T13:41:55.643-07:00Fantastic blog. I completely agree that the power ...Fantastic blog. I completely agree that the power of journals to allow comments post publication, has given the meaning of publication and discussion a whole new twist. <br />Indeed we also struggled to understand what was going on in our negative controls with taxol-microtubule mixtures with BRB80, till we saw the report by Foss et al. I have worked with microtubules before but in Xenopus extracts, so there was no question of adding taxol. Now in an in vitro system it isn't that simple. <br />Thanks.<br />-Chaitanyamastishkvigyanhttps://www.blogger.com/profile/03708666507223917100noreply@blogger.comtag:blogger.com,1999:blog-7356964387844331141.post-17503611280417309152009-12-11T12:22:28.593-07:002009-12-11T12:22:28.593-07:00Thanks for blogging about our paper, and for advoc...Thanks for blogging about our paper, and for advocating for open access. Clearly there's still a lot to be learned about Taxol/tubulin interactions. Since this is not the focus of Dahong's lab, we're no longer working on this project. But I'm very pleased that other people are taking an interest.<br /><br />I see you’ve referenced the 1994 Caplow paper for the 10 nM binding constant. I’m curious as to whether their experiments may also have been affected by the presence of Taxol crystals.<br /><br />For example, in Table I, footnote c, the authors write, "Microtubules assembled with 1 mM GTP and 50 uM tubulin were made 50 uM in taxol and incubated for 10 min. This mixture was diluted 10-fold into 5 uM taxol and incubated an additional 10 min. Aliquots were then added to [3H]taxol contained in either a 5% volume or a 9-fold greater volume; after 90 s microtubules were isolated with an Airfuge. Radioactivity and protein were measured in the resultant pellet."<br /><br />My feeling is that some Taxol crystals would likely have been present in the 50 uM Taxol before the 50 uM tubulin mixture was added. It's unclear to me whether all such crystals would have remained intact after the addition of tubulin or whether there would have been some dissociation, freeing up additional Taxol to stabilize the MTs. Since Taxol is so insoluble in water, I suspect at least some crystals would remain even after the 10-fold dilution. If so, it's possible that some tritiated Taxol could get incorporated into the Taxol crystals rather than stabilizing the MTs. I also suspect that any good-sized Taxol crystals would pellet along with the stabilized MTs. So when the authors measure the tritium vs protein in the pellet, I'm not certain that these numbers reflect only Taxol that's MT-associated. And that's ignoring the complication that free tubulin heterodimers could nucleate Taxol crystals, according to the Castro 2009 reference. In addition, some tritiated Taxol may be associated with small Taxol crystals that are too light to pellet, perhaps accounting for the 15% of tritated Taxol that didn't pellet in fig. 2.<br /><br />Anyway, I've made a lot of assumptions above, and they may not all be true, or the effects may not be significant enough to affect the outcome. And I haven't gone through the whole paper to look for other possible discrepancies. But it may be worthwhile for you to consider these issues before fully accepting the 10 nM figure.<br /><br />Again, good luck with your experiments!<br /><br />-MargitMargit Fossnoreply@blogger.com