Wittrup: Yeast Preps for Western Blotting

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To prepare whole yeast for SDS-PAGE and Western blotting, or if desired, to prepare separate samples of surface-displayed proteins and intracellular proteins.

Contents

Spheroplast buffer

  • 50 mM Tris-HCl, pH 7.5
  • 1.4 M Sorbitol
  • 40 mM beta-mercaptoethanol (add just before use)

Other required reagents

  • TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)
  • Protease Inhibitor Cocktail (eg. "Complete" from Roche)
  • Zymolyase (Zymo Research)
  • 10% SDS solution or SDS sample buffer

Reducing off surface-displayed proteins

  • Spin down 5 OD.ml of yeast culture and wash with PBS.
  • Resuspend in 250 ul PBS containing 25 mM TCEP.
  • Incubate on ice for 30 min.
  • Pellet the yeast (12K xg, 1 min) and set aside the supernatant containing surface-displayed proteins released by reduction.
  • Resuspend the yeast pellet in 250 ul PBS + 25 mM TCEP and incubate on ice for another 30 min.
  • Pellet the yeast and combine this supernatant with the first "extraction".
  • The reduced-off proteins will probably be too dilute for SDS-PAGE-->Western blotting. Either perform slot blotting with the entire sample, or concentrate it down to ~20 ul.

Lysing yeast to release intracellular proteins

  • The section above can be skipped if you don't need to separate surface-displayed from intracellular proteins.
  • Wash the yeast pellet in 0.5 ml spheroplast buffer.
  • Resuspend in 100 ul spheroplast buffer containing protease inhibitors and 2 U Zymolyase.
  • Incubate at 37oC for 15 min.
  • Add 20 ul SDS solution (if performing slot blot) or SDS sample buffer to 1x (if performing SDS-PAGE) and immediately boil for 5 min.
  • Centrifuge 12K xg, 1 min and use the supernatant for slot blotting or SDS-PAGE.
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