Zymolyase protocol

[Colin Rasmussen]

fengyang wrote:
> 
> Dear All:
> 
> I use zymolyase to break down yeast cell wall and then do immunostaining.
> For some reason the zymolyase reaction only gave 10-20% dark cells under microscope.
> The conditions are following:
> 10^7 cells of overnight culture (OD596=1)
> spin to remove media
> wash 2x with 0.1M KPO4(pH6.5)/1.2M sorbitol
> suspend cells in  0.5mL 0.1M KPO4(pH6.5)/1.2M sorbitol
> add 5ul mecaptoethanol, 5ul 10mg.mL zymolyase in 0.1M KPO4(pH6.5)/1.2M sorbitol
> 30'/37degree shaking
> 
> Can somebody offer some advices/protocols?


Try more zymolase for the same length of time you are currently using.