preparing yeast cell extracts sds-page gives a snapshot of proteins in an extract proteins are...
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Preparing yeast cell extracts
SDS-PAGE gives a snapshot of proteins in an extract
Proteins are extracted from cells
There are major challenges in analyzing yeast cell proteins!
How can proteins be efficiently extracted without being degraded?
Christopher Bruser, www.celllibrary.orgused with permission
Tough cell wall surrounds the plasma membrane
Vacuoles contain multiple proteases
Our procedure depends on the rapid and efficient extraction of yeast proteins
Proteins are denatured during the process
What happens during denaturation?
Proteins are stabilized by thousands of bonds
Vast majority are non-covalent:ionic, polar, hydrogen, van der Waals
Covalent disulfide bonds link cysteines
Proteins are extracted under denaturing conditions: • heat• denaturing detergent (SDS) • sulfhydryl reagent (2- aka β-mercaptoethanol)
Sodium dodecyl sulfate is a denaturing detergent with multiple roles in extract preparation
Detergents are amphipathic molecules that render hydrophobic molecules soluble in aqueous solutions
Long side chain binds hydrophobic regions in proteins and cell
membranes
Hydrophilic head group binds water
molecules
SDS binding imparts a uniform charge to mass ratios to all proteins in a sample!
SDS binds and solubilizes unfolded proteins: 1 gram protein binds 1.4 grams SDS
Corresponds to ~1 SDS molecule for every 2 amino acids
2-mercaptoethanol (β-Me) reduces disulfide bonds between cysteine residues
SDS and reducing agents convert proteins to random coils surrounded by negative charge
“Quick and dirty” protein extraction from yeast
1. Collect cells by centrifugation
2. Wash cells with deionized water
3. Treat cells with 0.2 N NaOH for 5 minutes (weakens membrane without lysing the cells)
4. Resuspend the cells in 2X SDS-PAGE sample buffer and boil IMMMEDIATELY for 3 minutes. (Note: do NOT centrifuge the samples.)
5. Store the samples in the freezer until used for SDS-PAGE.