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Need Lysis Buffer for SF9 cell vesicles - secreted protein

Need Lysis Buffer for SF9 cell vesicles - secreted protein - Protein Science

Need Lysis Buffer for SF9 cell vesicles - secreted protein - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 02-16-2011, 04:04 PM
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Default Need Lysis Buffer for SF9 cell vesicles - secreted protein



Working on a secreted protein within sf9 cells and I know that a large majority is not making it out of the cells.
So now I want to go after the pellet and get as much protein as I can out of there but of course it will most likely be trapped in vesicles since its a secreted protein.
So the SF9 cell lysis is easy with just a Tris/NaCl buffer but what can I add to also ensure that the vesicles are also being lysed?
Non-Ionic detergents? If so any suggestions on which ones or concentrations?
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Old 02-17-2011, 07:58 AM
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Default Re: Need Lysis Buffer for SF9 cell vesicles - secreted protein

Hola, This is a commun aspect of secretion systems, you improve the recombinant protein expression but the processing membrane machinery stay at normal level so itīs saturated. Years before, Orbigen had virus booster mixes wich coinfected with your viruses improve secretion, but this company was sold to Gentaur and theese products dissapeared. The best way to liberate the protein, is sonication of the whole cells or of the vesicles if you are able of separate them by gradient or sequential centrifugation,improving protein quality, that you could avoid, if your protein is tagged for purification. But remind that this protein has bounded the signal peptide. One more recommendation, try to infect at low MOI harvesting and changing medium during some days after infection checking the aspect of cells, and lowering the volume each change untill 5 times less. Buena suerte
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