Drummond:Protein Isolation

Protocol to Isolate Total, Soluble and Insoluble Protein Fractions from Galactose-inducible Strains of S. cerevisiae

• Inoculate a 3 mL pre-culture of YPD with a single yeast colony and incubate overnight at 30⁰ C with shaking or rotation.

• The next day, inoculate 70 mL of YP sucrose (2%) raffinose (1%) with 3 µL of the overnight pre-culture for 18 h at 30⁰ C with shaking in a 500 mL baffled flask.

• The next day, measure the optical density of the 70 mL assay culture to confirm that it is at an OD600 of around 0.400. Transfer 45 mL of the assay culture to a new 500 mL baffled flask and induce with 40 mM galactose (add 1,622 µL of 20% galactose) for 2 h at 30⁰ C with shaking. Continue to incubate the original flask of remaining uninduced cells alongside the flask of galactose-induced cells.

• Harvest aliquots of the assay cultures. The aliquots will be for either the isolation of total protein, or for the sequential isolations of soluble and insoluble protein fractions. There are three aliquots to harvest; two aliquots from the galactose-induced culture flask, and one aliquot from the remaining uninduced culture flask. The volumes to harvest should be equivalent to 15 mL of cells that are at an OD600 of 0.850.
  For example: If the OD600 is 0.725 then harvest 17.6 mL of cells, or if the OD600 is 0.950 then harvest 13.4 mL of cells (ie, 15 mL X 0.850).

• Pellet each aliquot at 3,000 g for 2 min and thoroughly drain the media. Wash the pellet in 1.8 mL of protease-free water and transfer to a 2 mL microcentrifuge tube. Pellet again at 20,000 g for 10 sec ond and remove the wash with a vacuum trap, being careful to not disturb the cell pellet. Resuspend the cells in 1.4 mL of Buffer Z and 14 µL of βME (1 M). Add 33 µL of Zymolyase (10 µg/µL), mix by inversion and incubate at 30⁰C for 30 to 35 min with gentle shaking.

• Pellet the resulting spheroplasts at 3,000 g for 5 min at 4⁰ C. Aspirate the supernatant with a vacuum trap, being careful to not disturb the cell pellet, and gently wash the pellet with 750 µL of ice cold Buffer Z (do not resuspend the pellet, but rather rinse the walls of the tube and the top of the pellet by gentle inversion). Centrifuge again at 3000 g for 3 min at 4⁰ C and remove the wash with a vacuum trap, being careful to not disturb the cell pellet.
  
  
  Note: Perform the following two steps A and B together, side-by-side.

• A: Total protein isolation
  • Lyse a sample of galactose-induced, spheroplasted-cells, and another sample of uninduced spheroplasted-cells in 400 µL of room temperature IPB by vigorously vortexing for 3 min on the bench top. Centrifuge the samples at 20,000 g for 5 min at 4⁰ C. Thoroughly decant the supernatant to a 1.5 mL microcentrifuge tube, add 67 µL of room temperature IPLB, vortex and heat denature at 60⁰ C for 5 min. Chill on ice, and later store at -20⁰ C. This is total protein fraction.

• B: Soluble and insoluble protein isolation
  • Lyse a sample of galactose-induced, spheroplasted cells in 400 µL of ice-cold SPB by vigorously vortexing for 5 min in a cold room. Centrifuge the sample at 20,000 g for 5 min at 4⁰ C. Thoroughly decant the supernatant to a 1.5 mL microcentrifuge tube, add 67 µL of room temperature SPLB, vortex and heat denature at 60⁰ C for 5 min. Chill on ice, and later store at -20⁰ C. This is the soluble protein fraction.
  • Wash the pellet in 400 µL of ice cold SPB by vigorously vortexing for 5 min in a cold room. Centrifuge the sample at 20,000 g for 5 min at 4⁰ C. Thoroughly decant the supernatant to a 1.5 mL microcentrifuge tube, add 67 µL of room temperature SPLB, vortex and heat denature at 60⁰C for 5 min. Chill on ice, and later store at -20⁰ C. Repeat this step twice on the remaining pellet for a total of 3 washes. Vigorously vortex the washed pellet in 400 µL of room temperature IPB for 3 min on the bench top. Centrifuge the sample at 20,000 g for 5 min at 4⁰ C. Thoroughly decant the supernatant to a 1.5 mL microcentrifuge tube, add 67 µL of room temperature IPLB, vortex and heat denature at 60⁰ C for 5 min. Chill on ice, and later store at -20⁰ C. This is the insoluble protein fraction.

• Store all samples at -20⁰ C, or -80⁰ C.

Recipe for Protein Buffers

Soluble Protein Buffer, pH 6.8, (SPB )

(50 mM Tris-HCl, 150mM NaCl, 1:100 Protease Inhibitor Cocktail Set IV)

Start by making SPB Stock Solution:

1. 2.5 mL 1M Tris-HCl, pH 6.8
2. 1.5 mL 5M NaCl
3. 45.4 mL Protease-free water

Then mix: 3465 µL of SPB stock solution with 35 µL of protease inhibitor

Insoluble Protein Buffer, pH 6.8 (IPB)

(50 mM Tris-HCl, 150 mM NaCl , 2% SDS, 2 mM DTT, 1:100 Protease Inhibitor Cocktail Set IV)

Start by making IPB Stock Solution:

1. 2.5 mL 1M Tris-HCl, pH 6.8
2. 1.5 mL 5M NaCl
3. 5 mL 20% SDS
4. 39.5mL Protease-free water

Then mix: 2716 µL of SPB stock solution with 28 µL of protease inhibitor and 56 µL of 0.1M DTT

Soluble Protein Loading Buffer, pH 6.8 (SPLB)

50 mM Tris-HCl, 10% SDS, 0.05% Bromophenol blue, 30% Glycerol, 5% βME

Start by making 6x SPLB Stock Solution:

1. 0.5 mL 1M Tris-HCl, pH 6.8
2. 1 g SDS
3. 5 mg Bromophenol blue
4. 3 mL 100% Glycerol
5. 5.5 mL Protease-free water

Then mix: 712.5 µL of 6X SPLB stock solution with 37.5 µL of 14.3M βME

Insoluble Protein Loading Buffer , pH 6.8 (IPLB)

50 mM Tris-HCl, 0.05% Bromophenol blue, 30% Glycerol, 5% βME

Start by making 6x SPLB Stock Solution:

1. 0.5 mL 1M Tris-HCl, pH 6.8
2. 5 mg Bromophenol blue
3. 3 mL 100% Glycerol
4. 5.5 mL Protease-free water

Then mix: 475 µL of 6X SPLB stock solution with 25 µL 14.3M βME