Hi
I am preparing Microtubules polymerized in-vitro and observe them under fluorescent microscopy. In addition to the Microtubules, I see aggregates which are very brights dots ("aster like"). I have read on Cytoskeleton datasheets that an initial centrifugation of the tubulin stock (14krpm, 10", 4'C) would minimize this "aster like" aggregate. I have tried this, but still have this unwanted aggregates in my sample. Here is the protocol I am using.
4.0 mg/ml Tubulin in BRB80 + 10% Glycerol + 1mM GTP
1:4 rhodamine labeled:unlabeled (Cytoskeleton Inc)
--> on ice,
--> Centrifugation at 14krpm, 10", 4C
--> Taking the supernatent
--> 37C, 30"
--> addition of taxotere (final 20uM), final DMSO :15%
--> 37C, 10min
--> Centrifugation at 14krpm, 10", RT (remove free tubulin, needed in my application)
--> re suspension of MTs in warm BRB80 + 10uM Taxotere to final 1mM
--> 1 in 150 ul dilution in warm BRB80 + 10uM Taxotere
Fluorescent microscopy
I hope someone can help me on this issue.
- I am suspicious to DMSO content when adding the drug. As far as I read, DMSO will promote polymerization and have used instead of Glycerol in some protocols.
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