We are trying to study microtubules dynamics using TIRF. The labels used for tubulin labeling are Hilyte-488, Alexa-568 or Atto-565. We are seeing very rapid photobleaching within seconds of imaging under TIRF condition. Also the GMPCPP seeds seem to deform/depolymerize within ~20s after imaging starts at that view. We didn't use oxygen scavenger system in our imaging solution. Could that be the reason for the problems? Or are there other possibilities?
Another problem is that we don't think we actually see dynamic tubulin from the seed. All the supposingly dynamic part of the microtubules look very solid and they just seem to move in and out of focus instead of grow and shrink. Anyone has any idea on that?
Hey Ashish,
Yes. Your troubleshooting is right on point. You must add oxygen scavenging system to your sample, especially for your long-term imaging where you want to monitor microtubule dynamics. Microtubules severe in presence of laser. In the standard protocols, there is also methycellulose (0.5-1%) that increases the viscosity a bit and helps tubulin dimers and less microtubule shaking during the growth.
How long have you been imaging the seeds for? What exposure time you have?
Try adding OS components to your assay first and then image them bit longer, say 500 ms for 10 mins.
For rapid bleaching oxygen scavenger is necessary. Add this to your buffer.
Also what is concentration of tubulin are you using for observing the dynamics? Amount of seed also matters in this aspect.
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