Yeast Protein Purification in 96-Well Format

Heng Zhu, Metin Bilgin & Michael Snyder

Modified by Joe Tasto, Kyle Nordquist & Andy Link

Yeast culture preparation

  • Yeast glycerol stocks stored in 96-well plates at -80°C were inoculated onto a URA- agar plate (VWR; 46600-638) using a 96-pronger (V&P Scientific; VP 408A).
  • The culture was allowed to grow on agar at 30°C for 48 hours, and visible colonies (2 mm diameter) can be observed.
  • To facilitate uniform growth and aeration, a 3mm diameter glass ball (VWR; 26396-508) was added to each well of a 2.2 ml 96-well plate (Marsh Bioproducts; AB-0661) using a flip dispenser (V&P scientific; VP 725EB).
  • Next, 350 l of SC-URA + raffinose liquid medium was added to each well of the 2.2ml plate using an automated plate filling device (Genetix, UK; Qfill2). Finally, a 96-pronger was used to inoculate yeast cells from agar plates to the liquid cultures and the entire plate was covered with an Airport tape sheet (Qiagen; 19571). The cultures were grown at 30°C and 300 rpm.
  • After the culture reached an O.D.595 of around 2.0 (approximately 16-24 hours after inoculation), 15μl of each culture was inoculated into 750μl of SC-URA + raffinose liquid medium in four new plates (one original pre-culture plate yields 4 new plates containing 750μl of medium + 15μl of cells in each well). Again, each well contained a 3mm glass ball to achieve aeration and even growth. The cells are grown at 30°C and 300.
  • After the culture reached O.D.595 of 0.6 to 0.8 (between14-18 hours), the QFill2 was utilized to add 40μl of 40% galactose stock to each well to yield a final concentration of 2% to induce the GAL promoter for 4 hours at 30°C with shaking. Discard the plate if the O.D.s are over 1.0.
  • The cells were harvested by spinning at 3000 rpm for 4 min and the spent medium was simply dumped out. The cell pellets were resuspended in 200μl of cold water by vortexing. Cells of the same strain in each of the 4 wells were combined in a new 96-well plate to eliminate the 3mm glass beads.
  • Cells were pelleted at 3000 rpm for 4 min, the water was dumped out and the cells were resuspended in 500μl of cold lysis buffer without the protease inhibitors. The cells were harvested again by centrifugation and the lysis buffer was discarded. The washed semi-dry culture was immediately stored in -80°C freezer where it can be kept for weeks.

Protein purification in a 96-well fashion

  • The frozen culture in a 96-well box was transferred from -80°C to ice and approximately 225μl of 0.5mm zirconia beads (Biospec; 11079105z) was added to each well with pipet tips secured in a cap mat. The tips were cut to yield the appropriate 225μl volume. While the culture is still frozen, 325μl of lysis buffer containing fresh protease inhibitors was added. A cap mat was used to seal each well (Marsh Bioproducts; AB-0661).
  • After thawing the culture for 15 min on ice, the cells in the 96-well box were vortexed 6 times for 1 minute with 1 minute intervals in between on ice to achieve >90% lysis efficienc. A custom multitube vortexer was used (Lab-tek; New Zealand).
  • Meanwhile, the required amount of glutathione beads (20μl of beads per sample) (Amersham; 17-0756-01) was washed three times with cold lysis buffer containing protease inhibitors and resuspended in 5X of its original volume.
  • After spinning at 3000 rpm for 5 min, the supernatant was collected using Matrix 1250μl tips (VWR; 53503-272) and transferred into a 96-well filter plate (Whatman; 7700-2817) sitting on top of a 96-well, 1.2ml plate (Marsh Bioproducts; AB-0564).
  • The cell lysate was spun through the filter plate into the 1.2ml collection plate for 5 min at 2500 rpm and 4°C to clarify the lysate.
  • 80μl of washed glutathione beads was added to each well and the plate was sealed tightly with a cap mat (Marsh Bioproducts; AB-0566). The beads were incubated with the lysate on a roller drum at 4°C for one hour. To obtain the best mixing, the boxes were rotated 360 degrees on the roller drum by tying the filter plates on with self-locking cable ties (Roadmaster Corp; ISO 9002).
  • The plate was then spun at 3000 rpm for 1 min to pellet the beads. The lysate was removed with a 96-pin aspirator (VP Scientific; VP 177). The pin height was adjusted to remove most of the buffer without losing the beads.
  • 400μl of Wash Buffer I lacking protease inhibitors was added to the beads, the plate was sealed with a cap mat, and the beads were washed by inverting the plate multiple times. The beads were collected with a brief centrifugation spin and the wash buffer removed by aspiration. This was done three times.
  • The beads were then washed three times with 400μl of Wash Buffer II in a similar fashion.
  • Finally, 30μl of Elution Buffer (make sure pH ~7.5) was added to each well. The plate was sealed with a cap mat then vortexed for 45 minutes at 4°C on a multiplate vortexer (VWR; 57019-600) then vortexed for 15 min at RT on the Lab-tek vortexer.
  • The entire slurry of beads and elution buffer was transferred to a filter plate (Whatman; 7700-1818) that was on top of a 96-well PCR plate (VWR; 10011-228) using wide-open genomic tips (VWR; 53503-612). The eluate was collected in the 96-well PCR plate by spinning through the filter plate for 5 min at 2500 rpm at 4°C. The plate was then immediately stored at -80°C.

Preparation of protein microarray slides

  • Slides were printed by Dr. Shawn Levy in the Vanderbilt Microarray Shared Resource (VMSR) center using a Biorobotics arrayer. Slides were stored in a desicator at 4°C containing a saturated NaCl solution to maintain approximately 75% humidity. Various slide chemistries were tested from Xenopore and Telechem. These included surface coatings of Glutathione, Nickel, Aldehyde, Amide, Epoxy, Silanated, and Telechem’s Zetagrip.

Detection of spotted proteins on coated glass slides

  • In a 4.5 x 3.0 inch plastic container, the printed slides were placed protein side up in TBST and incubated at RT on a belly dancer for 15 min (If background signal is too high, the following steps may be done at 4°C).
  • The protein arrays were blocked with 5% Carnation Instant Milk in TBS for 30-60 min at RT on the belly dancer.
  • The block solution was dumped off and the slide was placed in a Hybridization Chamber (Corning; 26101000). The coverslip was placed over the spotted proteins.
  • 50μl of 5% Carnation Instant Milk in TBS containing 1μl of Rabbit anti-GST antibody (Molecular Probes; A-5800) was added at the base of the coverslip. Capillary action pulls the solution over the slide surface underneath. This primary antibody was incubated with the protein array for 1 hour at RT on a low setting on the belly dancer.
  • The slide was then washed 4-6 times with TBST in the 4.5 x 3.0 inch plastic container with at least two washes done on the belly dancer for 5 min at RT.
  • The slide was placed back in the hybridization chamber and a clean coverslip was placed over the spotted area. 50μl of 5% Carnation Instant Milk in TBS containing 0.5μl of Goat anti-Rabbit AlexaFluor 647 conjugated secondary antibody (Molecular Probes; A-21244) was incubated with the protein arrays for 30-60 mins at RT at a low setting on the belly dancer. The chamber was wrapped in aluminum foil to protect the chromofluor from light---VERY IMPORTANT!
  • The slide was then washed 4-6 times with TBST in the 4.5 x 3.0 inch plastic container with at least two washes done on the belly dancer for 5 min at RT. Be sure to cover the container with aluminum foil to continue protection from light.
  • The slide was rinsed in TBS three times to remove the Tween, transported to the VMSR lab, dried in the centrifuge by spinning for 5 min at 50 rcf and immediately scanned utilizing GenePix Pro 5.0 software.
  • Please be conscientious about keeping the slide protected from light once the chromofluor has been added.

Reagents
Lysis Buffer:

50 mM / Tris-HCl pH 7.5
100 mM / NaCl
1 mM / EGTA
0.1% / TritonX-100
0.1% / beta-mercaptoethanol (BME)
Protease Inhibitors / Benz/PMSF/Complete Tablets (see below)

Protease inhibitor tablets containing EDTA [(Roche; 1836145); 1 complete tablet/50ml buffer] BME, PMSF [(175mg/10ml EtOH); add 0.5ml/50ml buffer],Benzamidine [(400mg/10ml EtOH); add 128μl/50ml buffer] and inhibitor tablets are added immediately prior to use.

Wash Buffer I:

Exactly the same as Lysis Buffer except with 500 mM NaCl.

Wash Buffer II:

50 mM / HEPES pH 7.5
100 mM / NaCl
10% / Glycerol

Elution Buffer:

50 mM / HEPES pH 7.5
100 mM / NaCl
40% / Glycerol
10 mM / Glutathione (Reduced form)
0.5 M / Betaine (Sigma; B-2754)

Make sure the pH is around 7.5!

Betaine is hygroscopic and keeps proteins hydrated on the slide (does not interfere with elution).