Hello everyone,
I would like to perform a purification (fractionation) of tubulin from cell lines in order to study the effect of some tubulin binding agents.
I found a protocol explaining how to perform the ultracentrifugation part, but there is no explanation on how to load the fractions on the gel for a silver staining. Do I load the whole fraction or do I dose the proteins first?
Also, I wanted some advice on when to treat the cells. When I performed the experiment, I treated my cells for 24 h before the extraction, but since there are depolymerization and polymerization steps during the purification I wonder if I have to add the drugs at that moment too?
I’m a little lost, so any advice you can give me will be very helpful!! Thank you.
Hello,
we have extensively done tubulin fractionations in the motor neuron cell line NSC34. You can download the methods from ResearchGate, see link below : Schäfer et al, J Neurosci 2007 and Bellouze et al HMG 2014. In our experience it is preferable to analyze equal volumetric fractions on WB.
As a first, less quantitative approach, you can also use IF by comparing cells extracted (and not) for soluble tubulins.
Hope this helps. Otherwise come back to me.
Good luck
GH
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