ny body clarify me lysis buffer(protenase cockail, PBS) are use in yeast cell protein extaction?
The standard extraction buffer published by Horvath and Riezman in the 90s contains 0.06 M Tris-HCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) sodium dodecyl sulphate (SDS), 5% (v/v) 2-mercaptoethanol, 0.0025% (w/v) bromophenol blue. The extracts can be directly separated via 1D-SDS-PAGE.
In the attachment you will find an updated protocol from 2011 that gives better protein yield.
Regards, Christian
Article An improved method for whole protein extraction from yeast S...
Hi there,
In case the purpose of extraction is to recover a clarified lysate containing active soluble proteins, I would suggest to resuspend cells in 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1% NP-40 supplemented with Complete anti-protease cocktail from Roche. And then to submit the resuspension to glass-bead breakage.
Guna,
Dominique and Christian have given you good starting primers.
]Depending on the final application (biochemical assay, mass spectrometry analysis, immunoblotting), there are several techniques as well as buffers. Glass bead-based methods for e.g. should be avoided when dealing with unstable or fragile proteins. You can even lyse cells by grinding in liquid nitrogen or avoid mechanical disruption altogether using a TCA or urea-based buffer. Alternatively, there are also enzyme-based disruption methods should you need particularly gentle lysis methods.
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_yeast/
I have used a fairly complex buffer in the past to extract proteins for MS analysis of phosphorylated residues on my proteins of interest that was phosphate-based rather than Tris-based; I have also used a HEPES-based buffer to extract proteins for binding assays.
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