Preparing yeast cell extracts SDS-PAGE gives a snapshot of proteins in an extract Proteins are extracted from cells.

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.