I'm trying to express a protein in Pichia pastoris that ideally should be secreted. However, I would also like to see if the protein is being retained by the yeast. To that end, I want to lyse my cells, collect the proteins, and run them through an SDS-PAGE gel. My concern is preserving the proteins. Is there a good method out there for lysing Pichia cells without destroying the proteins?
This paper reports an alkaline method of lysis. http://onlinelibrary.wiley.com/store/10.1002/yea.1905/asset/yea1905.pdf;jsessionid=DE6C63CB8FD37E69C210B8FA700DF47D.f02t03?v=1&t=ivvn0nfk&s=c3d3790eb7057d7a7ffc057eabefeb337ab8e720&systemMessage=Wiley+Online+Library+will+be+unavailable+on+Saturday+26th+November+2016+from+07%3A00-11%3A00+GMT+%2F+02%3A00-06%3A00+EST+%2F+15%3A00-19%3A00+SGT+for+essential+maintenance.++Apologies+for+the+inconvenience.
Are you running a native gel? Your proteins will be denatured by SDS anyway.
Oh, I see. I'm running a non-native gel, but maybe I can try native and see what results I get. Thank you!
Here is a summary of methods for breaking yeast cells.
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_yeast/
Hi Aster,
a colleague of mine with lots of working experience in yeast just sent me this link to a research gate thread asking a similar question:
https://www.researchgate.net/post/Pichia_Pastoris_lysis
Hopefully you can find something there useful.
- Badges
- Science method