Dear all,
I have experiment to check the cellular localization of my protein of interest. Therefore, I decide to use transient expression analysis with protoplast system.
However, every time I check in the confocal microscope, I always observed big vacuole in my protoplast. This big vacuole is hindering my observation in the confocal, as I want to observed the localization of protein in cytoplasm and in the chloroplast envelope.
I wonder if somebody also experience this kind of thing, and maybe have some solution for it?
For protoplast isolation and transformation I use the Tape-Arabidopsis sandwich method (Wu et al., 2009). I use ~3 weeks old Arabidopsis grown at LD. I also incubate my protoplast after transformation for ~12hours in the dark.
Interestingly, before transformation, my protoplast has normal vacuole as chloroplast is spreading inside the protoplast. However, after incubation, I observed big vacuole and chloroplast is clustered together.
I really appreciate if somebody has solution for this problem.
Thank you
Hi Hendry,
I'm affraid that the single big vacuole phenotype is the result of the stress induced by your transformation protocol and you can't avoid it.
As Hayat said try to plasmolyse your protos. I used to do this with 6-800mM sorbitol and it works well. You can also use 50% glycerol.
Oliv
Hi,
Thank you for the answer, after several trial, I actually found a simple way to prevent the big vacuole. Basically I am just using the 6-well culture plate (with 500ul W5 buffer) and incubated for ~12hours. I think the big vacuole occur because of hypoxia or stress from dense population of protoplast culture.
Try to do the incubation with a very
low agitation and in a large erlenmyer for aeration
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