I am trying microtubule polymerization in-vitro for TIRF analysis. I am using the standard protocols published from different labs.
A) I have an observation that catastrophe is very rare in my hands. I have tried different concentrations of tubulin (7 to 10 uM), different percentage of labelled tubulin (10 to 30%), but this does not seem to have any impact on MT polymerisation, they appear very happy and keep growing with no or very rare hint of catastrophe on kymograph.
B) Another observation is that MT appears to go through a long phase of no-growth (stalled) but they resume again to grow without any catastrophe.
In the above work I am not using any MABs but just tubulin.
I would really appreciate any input from the experts.
Thank you,
If your buffer, temperature and TIRF conditions are ideal; you can double-check the pig/ goat brain tubulin concentration you used for the experiments.
Also, try to do a polymerization-depolymerization cycle of tubulin before you start a new experiment.
Jijumon A.S. thanks for your response.
In between I tried Porcine tubulin from Cytoskeleton Inc., and it worked well.
Try the protocols listed here. They also provide detailed instructions for handling tubulin: https://puresoluble.com/protocols/
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