Supernatant/pellet fractionation of budding yeast cells by cryogenic lysis

23 July 2015

For studies of protein aggregation, few techniques are simpler than two-phase fractionation by centrifugation: big stuff (aggregates) pellets, small stuff (unaggregated proteins) stays in the supernatant. Breaking open cells (lysis) can alter or even cause protein aggregation. For yeast cells, which have a tough cell wall, lysis requires a lot of force. We and others find that cryogenic milling does a good job in lysing most cells without causing aggregation.

With minor adaptations, we have applied the same lysis/fractionation to E. coli, cyanobacteria, mammalian cell culture, squid axoplasm, and alternative yeasts. Appearance of membrane proteins in the pellet and cytoplasmic proteins the sup is a good quality control for unstressed samples.

Based on Mike Dion and Allan Drummond’s 2011 protocols, with extensive modifications by Edward Wallace with help from Quincey Justman.

Protocol

Prepare

  • soluble protein buffer (SPB; 20mM HEPES-KOH pH7.4, 120mM KCl, 2mM EDTA, 0.2mM DTT, 1:100 PMSF, 1:100 protease inhibitors cocktail IV). Make stock of salt and buffer, add DTT and inhibitors shortly before use, and chill.
  • total protein buffer (TPB; 20mM HEPES-NaOH pH7.4, 150mM NaCl, 5mM EDTA, 3% SDS, 1:100 PMSF, 2mM DTT, 1:1000 protease inhibitors IV).

  • insoluble protein buffer (IPB; 8M Urea, 20mM HEPES-NaOH pH7.4, 150mM NaCl, 2% SDS, 2mM EDTA, 2mM DTT, 1:100 PMSF, 1:1000 protease inhibitors IV.). Mix fresh daily the urea, DTT, and inhibitors (use a stock solution of other ingredients) and keep at room temperature. Use 250 μL IPB per sample, and note that Urea is slow to dissolve, and foaming will make ~25% of the solution unusable. IPB solidifies at 4°C due to urea and SDS; also, don’t substitute KCl as it precipitates SDS even at RT.

    2 mL (~4 samples) 13 mL (~20 samples)  
    950 μL 6.2 mL H2O
    200 μL 1.3 mL 20% SDS (w/v)
    40 μL 260 μL 1M HEPES-NaOH pH7.4
    8 μL 52 μL 0.5M EDTA
    75 μL 390 μL 5M NaCl
    20 μL 130 μL 100mM PMSF
    4 μL 26 μL 1M DTT
    2 μL 13 μL protease inhibitors IV
    0.96g 6.24g urea
  • For heat treatment, pre-warm water bath to 46°C or pre-warm media as required.
  • Appropriate numbers of safe-lok tubes loaded with 7mm steel balls, racked in liquid nitrogen (LN2).
  • Pre-chill a centrifuge, also label and lay out all tubes and equipment in advance as the protocol moves very quickly once started.

  • Protocol can be paused and samples stored at -80°C when cells have been flash-frozen (step 8), and after cells have been ground in mixer mill (step 11).

    Sample Growth

  1. For each strain, inoculate a 3 mL pre-culture of SC-complete medium with a single yeast colony and incubate overnight at 30with shaking or rotation.

  2. Inoculate overnight culture to OD600 ~ 0.01 in SC-complete medium in a baffled flask, grow to 4×106 cells/ mL (OD600 ~ 0.45) at 30°C with 200rpm shaking.

    Sample Harvesting and Lysis

  3. Transfer 2×108 cells to a 50 mL conical tube (50 mL of a 4×106 cells/ mL culture).

  4. Spin at 2500 g for 30s in a swinging bucket rotor at RT. The end of this spin marks the start of the timed treatment duration. Gently decant and discard supernatant. For heat shock treatment, hold tube containing pellet in waterbath at desired temperature for desired time; alternatively, transfer to pre-warmed media for desired time and end by spinning as described.

  5. Resuspend pellet in 1 mL ice-cold SPB, on ice, and transfer to pre-chilled 1.5 mL tube.

  6. Spin at 5,000 g, 4°C, for 30 seconds.

  7. Resuspend new pellet in 150 μL lysis buffer.

  8. Drip 100 μL of resuspended pellet onto upper wall of tube containing steel ball, still racked in LN2. Goal is to get a nugget of frozen material on the wall, and to avoid dripping the material around the ball and thus freezing the ball to the bottom of the tube; having some LN2 remaining in the tube helps. Place the remaining 100 μL of resuspended pellet in a tube for total protein extraction: process, or freeze, immediately.

  9. Place tubes at -80°C; when all LN2 has boiled out of tube (listen – if any popping or hissing, keep waiting), snap tube closed caref μLly, away from other tubes. Keep at -80°C or lower. (Any remaining LN2 in tube will cause tube to explode open and fire the stainless steel ball into your iPad, brain, colleague, or other important equipment.)

  10. Rack the tube into the PTFE 2 mL tube adaptor for the Retsch Mixer Mill MM400 (Retsch #22.008.0005) and submerge the entire assembly in LN2. Agitate for 4x90 seconds at 30 Hz in a Retsch Mixer Mill MM400, returning sample holder to LN2 between sessions. Complete lysis produces fine snowy powder in the tube.

  11. Remove sample tubes from LN2, tap on bench to release powder from lid, and pop the caps to relieve pressure.

  12. Add 400 μL SPB to each tube, thaw on ice with occasional vortexing, and as soon as possible extract ball with a magnet. (We rinse balls in methanol and store in 50% ethanol.)

    Supernatant fraction extraction

  13. Spin at 3000 g for 30 seconds (clarification step) to remove whole cells and very large aggregates.

  14. Decant clarified liquid into a 1.5 mL microcentrifuge tube. If troubleshooting, keep the 3,000g pellet and process it alongside the insoluble fraction; this end product is the unclarified fraction.

  15. Spin at 100,000 g for 20 minutes (fixed-angle TLA-55 rotor at 40,309 rpm, 4, in a Beckman Coulter Optimax tabletop ultracentrifuge).

  16. Decant supernatant into a 1.5 mL microcentrifuge tube: this is the Supernatant fraction.

  17. Take 10 μL aliquot of soluble fraction and mix with 2X Laemmli buffer; use this to run a protein gel and assess protein integrity.

    Pellet fraction extraction

  18. Violently snap pellet to clear remaining liquid.

  19. Add 500 μL soluble protein buffer (SPB) and vortex. (The pellet may not resuspend; that’s fine.)

  20. Spin at 100,000 g for 20 minutes.

  21. Discard supernatant, clear residual liquid with a hard snap.

  22. Add 250 μL insoluble protein buffer (IPB); note this means insoluble sample is relatively 2X concentrated to others. Process samples in IPB at room temperature to maintain solubility of the Urea.

  23. Dislodge the pellet with a pipet tip, Vortex until pellet dissolves, 10-15 minutes for clarified samples.

  24. Spin at 20,000 g, RT, for 5 minutes.

  25. Decant supernatant into a 1.5 mL microcentrifuge tube: this is the Pellet fraction.

    Total protein extraction

  26. Add 400 μL TPB to each total protein tube. Incubate at 95°C with 500rpm mixing for 20 min.

  27. Vortex vigorously for 15 min.

  28. Spin at 6000 g, RT, 1 min. Take supernatant; this is the Total protein fraction.

    Measure protein

  29. Run a 4-15% SDS-page gel; load roughly 5-10 μL of total and soluble fractions. Make aliquots for further analysis.

  30. For more information, western blot against known stress-aggregating proteins (e.g. Pab1).

  31. Chloroform-methanol extract protein from each fraction for mass spectrometry analysis. We use reductive dimethylation following tryptic digestion for post-sample-collection isotopic labeling.