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 30⁰C with shaking or rotation.

• The next day, inoculate 70 mL of YP sucrose (2%) raffinose (1%) with 3 µL of the overnight pre-culture and incubate 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 1622 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, harvest 17.6 mL of cells; if the OD600 is 0.950, harvest 13.4 mL of cells (i.e., 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 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 1M β-mercaptoethanol. 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 benchtop. 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 store at -20⁰ C. This is the 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 store at -20⁰ C. This is the insoluble protein fraction.

• Store all samples at -20⁰ C to -80⁰ C.

Buffer recipes

Stock solutions

• 200mM DTT
• 100mM PMSF
• 4M NaCl
• 1M Tris-HCl, pH 8.5
• 20% SDS (w/v)

Soluble Protein Buffer, pH 8.5 (SPB)

(50mM Tris-HCl, 500mM NaCl, 1:100 protease inhibitors)

Start by making 50 mL SPB stock solution:

• 2.5 mL 1M Tris-HCl, pH 8.5
• 6.25 mL 4M NaCl
• 41.25 mL protease-free H₂O

Then mix 9.8 mL of SPB stock solution with 100 µL 100mM PMSF and 100uL protease inhibitors to make 10mL SPB.

For protease inhibitors, we use:

• AEBSF, HCl, MW=239.5, targets serine proteases, 100 mM
• E-64, MW=357.4, targets cysteine proteases, 1.5 mM
• Pepstatin A, MW=685.9, targets aspartic proteases, 2 mM
• o-Phenanthroline, MW=198.2, targets metalloproteases, 500 mM

This mixture is commercially available as Calbiochem protease inhibitor cocktail set IV, or BioVision EZBlock protease inhibitor cocktail IV.

Wash Buffer (WB)

(50mM Tris-HCl, 150mM NaCl, 1:100 protease inhibitors) Same as SPB, but with reduced salt to limit pellet loss.

Start by making 50 mL WB stock solution:

• 2.5 mL 1M Tris-HCl, pH 8.5
• 1.875 mL 4M NaCl
• 45.625 mL protease-free H₂O

Then mix 9.8 mL of WB stock solution with 100 µL 100mM PMSF and 100uL protease inhibitors to make 10mL WB.

Insoluble Protein Buffer, pH 8.5 (IPB)

(50mM Tris-HCl, 150mM NaCl, 8M urea, 2% SDS, 1mM PMSF, 2mM DTT, 1:1000 protease inhibitors) Make fresh before each use. Note that at 8M, urea occupies ~72% of the solution volume, so that 4.8g urea occupies 7.2mL of a 10mL solution. Do not use 500mM NaCl in this solution, as it loosens pellets and causes sample loss.

• 8M urea
• 2% SDS
• 50mM Tris pH 8.5
• 150mM NaCl
• 1mM PMSF
• 20 μL 200mM DTT
• 1:1000 protease inhibitors (see above. higher concentrations may crash out.)

Use 500μL IPB per sample, and note that foaming will make ~25% of the solution unusable.

2mL IPB (~2 samples)	13mL IPB (~10 samples)
0.96g urea 683 μL H2O 200 μL 20% SDS (w/v) 100 μL 1M Tris pH 8.5 75 μL 4M NaCl 20 μL 100mM PMSF 20 μL 200mM DTT 2 μL protease inhibitors	6.24g urea 5.58 mL H2O 1.3 mL 20% SDS (w/v) 650 μL 1M Tris pH 8.5 488 μL 4M NaCl 130 μL 100mM PMSF 130 μL 200mM DTT 13 μL protease inhibitors

6x Soluble Protein Loading Buffer, pH 6.8 (SPLB)

(50 mM Tris-HCl pH 8.5, 10% SDS, 0.05% bromophenol blue, 30% glycerol, 5% β-mercaptoethanol [βME])

Start by making 9 mL 6x SPLB Stock Solution:

• 0.5 mL 1M Tris-HCl, pH 8.5
• 1 g SDS
• 5 mg bromophenol blue
• 3 mL glycerol
• 5.5 mL protease-free water

Then mix 712.5 µL of 6x SPLB stock solution with 37.5 µL of 14.3M βME immediately before use.

6x Insoluble Protein Loading Buffer, pH 6.8 (IPLB)

(50 mM Tris-HCl, 0.05% bromophenol blue, 30% glycerol, 5% βME)

Start by making 9 mL 6x SPLB Stock Solution:

• 0.5 mL 1M Tris-HCl, pH 6.8
• 5 mg bromophenol blue
• 3 mL glycerol
• 5.5 mL protease-free water

Then mix 475 µL of 6x SPLB stock solution with 25 µL 14.3M βME immediately before use.

Buffer Z for Zymolyase digestion

(50mM Tris-HCl pH 7.4, 1M sorbitol)

Mix the following to obtain 50 mL Buffer Z:

• 10 mL 5M sorbitol
• 2.5 mL 1M Tris-HCl, pH 7.4
• 37.5 mL sterile water