Many use enzymes like lyticase to get spheroplasts and subsequently, perform mechanical disruption to disrupt the yeasts cells. The isolation is usually followed by the centrifugation. Apparently, I get to know about the glass bead beater which could bring the required disruption to yeast cells. I am glad to know, during the cell breakage, how far this could affect the integrity of the organelles present in the yeast. Is there any optimized protocols, perhaps, could help me to isolate them without making any undesirable damage.
Hi Michal,
I am glad, to know you and, for sharing the experience of isolating PGVs using glass bead beater. I have gone through the papers in relation to use of sec mutants for isolation. I am gonna use this opportunity to clarify some more doubts with you. sec 6-4 mutants are grown at 25 degree, usually before the phase of accumulation at 37 degree. But unknowingly, my lab members had grown the sec mutants at 30 degree, instead of 25C, continuously for many generations. I would like to know that is there any adverse effects, like revert in the mutant, as a result of the growth at 30 degree. Becoz, we are so afraid that the strain might had lost its ability to accumulate vesicles. If you have ever come across this situation anywhere, i will be glad to know it.
To isolate the PGVs from the rest, what kind of the centrifugation (either differential or gradient) you could recommend me?.
Thanking You
Hi
I also had this problem. But the hot phenol method could take a good result. I suggest you use this method.You can get it from the Internet Protocol. A method is low cost and high performance.
Collart, M.A. and S. Oliviero.Current Protocols in Molecular Biology,1993 by John Wiley & Sons, Inc.
Good luck
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