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Preparing yeast cell extracts
Proteins are extracted
from cells
SDS-PAGE gives a snapshot
of proteins in an extract
There are major challenges in analyzing yeast cell proteins!
How can proteins be efficiently extracted without being degraded?
Tough cell wall surrounds
the plasma membrane
Vacuoles contain multiple
proteases
Christopher Bruser, www.celllibrary.org
used with permission
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 and reducing agents convert proteins to random coils
surrounded by negative charge
2-mercaptoethanol (β-Me)
reduces disulfide bonds
between cysteine residues
SDS binds and solubilizes unfolded proteins:
1 gram protein binds 1.4 grams SDS
Corresponds to ~1 SDS molecule for every 2
amino acids
SDS binding imparts a uniform charge to mass ratios to all proteins in a sample!
“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 SDSPAGE.