PROTEIN AND DNA DETECTION METHODS

3.4.1. Northern Hybridization

RNA isolation and Northern hybridization were performed as described (Cross and Tinkelenberg 1991). Polymerase chain reaction-generated fragments were radio labeled using a labeling kit (Stratagene) and used as hybridization probes.

3.4.2. Preparation of Cell Extracts (Surana et al. 1993)

At the desired time point, 15 mL of culture were harvested at 2500 rpm for 3 minutes and then washed with 2 ml of 10mM Tris pH 7.5. The yielded pellet can be frozen in liquid nitrogen and stored at –80°C in the freezer for further use. Breaking up off cells was achieved by adding 200µl of glass beads (∅0,25mm) and 200µl of freshly prepared “Breaking Buffer” (50mM Tris/HCL, 1mM EDTA, 50mM DTT, 1mM PMSF, 0,5mM TPCK, 0,025mM TLCK and 2µg/ml Epstein). This mixture was vortexted at 4°C for ten minutes to break up cells. 10µl of the obtained suspension were pipetted to an extra tube and centrifuged for 10 min at maximum speed. The rest of the suspension was mixed with 100µL SDS-buffer (0,25M TrisHCl pH 6,8, 15%

mercaptoethanol, 30% glycerol, 7% SDS and 0,3% brominephenolblue) and incubated for 10 min at 95°C. Finally, the denatured protein samples were centrifuged 10 min at maximum speed and stored at –80°C for further use.

3.4.3. Detection of Protein Content (Bradford 1976)

10µl of the crude cell extracts were centrifuged at maximum speed for 10 minutes.

3µl of the supernatant were mixed in a cuvette with 1ml of 1:5 diluted Bradford solution (BIO-RAD, München, D). After incubation for 10 minutes, extinction at 595 nm was measured in an UV/Vis photometer using 1:5 diluted Bradford solution as blank.

3.4.4. SDS-Polyacrylamide Gel Electrophoresis (Laemmli 1970)

Electrophoretic separation of protein mixtures was carried out in 12% SDS vertical gels build up on two different gel types (running gel: 3,5ml water, 2,5 ml “WUG”-buffer (1,5M Tris Base pH 9, 0,4% SDS), 4 ml 30% acryl amide, 50 µl 10%

ammoniumperoxodisulfate, 10 µl N´,N´,N,N-tetra-ethylen-methylen-diamide) stacking gel: 1,8 ml water, 2,5ml “WOG”-buffer (0,25M Tris Base pH 6,8, 0,2%

SDS), 650µl 30% acryl amide, 25 µl 10% ammoniumperoxodisulfate, 5 µl N´,N´,N,N-tetra-ethylen-methylen-diamide). The two Gels were casted and polymerized successively on each other. Depending on the observed protein, 10 to 60 µg of whole protein was loaded on the gel. Running of the gel was carried out for 60

minutes at 200V in electrophoresis buffer (25mM Tris Base, 192 mM glycine, 0,1%

SDS).

3.4.5. Immunoblotting (Towbin et al. 1979)

Preparation of yeast cell extracts and protein immunoblot analysis were performed as described. Transfer of electrophoretically separated protein bands to a nitrocellulose membrane (“Protan”, SCHLEICHER & SCHUELL) was carried out in a “Mini-Trans-Blot-Electrophoretic-Cell” (BIO-RAD, München, D) in transfer buffer containing 25mM Tris Base, 192 mM glycine and 20% methanol overnight at 35 V.

After accomplished protein transfer, free binding sites on the nitrocellulose membrane were blocked by treatment with 3% milk powder in PBS buffer (80mM Na2HPO4, 25mM NaH2PO4, 100 mM NaCl, Tween 20, pH 7,5) for 45 minutes. Then, the first antibody diluted in PBS milk powder was applied for 60 minutes. Residual antibody was washed away by shaking the membrane 10 minutes once in PBS milk powder and twice in PBS containing Tween 20. The secondary peroxidase-labeled antibody was incubated with the membrane for 60 minutes and washed away analogously to the first.

Detection of proteins on the membrane was carried out using the “Enhanced Chemiluminiscence System” (ECL, (Tesfaigzi et al. 1994)) involving two solutions (A: 2,5mM luminol, 400µM para-coumaric acid, 100 mM Tris HCl pH 8,5 and B:

6mM H2O2, 100mM Tris HCl pH8,5), which were put on the membrane for two minutes. Finally, luminescence was detected on Hyperfilm^TM-ECL^TM (AMERSHAM PHARMACIA BIOTECH, Buckinghamshire, GB).

Antibodies were used in 1:1000 (Clb2), 1:2000 (Cdc28), 1:100 (HA, 12CA5) and 1:100 (MYC) dilutions, respectively.

3.4.6. Immunofluorescence Analysis (Pringle et al. 1991)

For indirect immunofluorescence, cells were fixed in 3.7% formaldehyde for at least 40 minutes, washed three times in phosphate-buffer (0,3mM K2HPO4, 0,1mM KH2PO4) and once in SCE-buffer (1 M sorbitol, 0,1M sodium citrate, 0,06M EDTA pH7).

Then the cells were spheroblasted in 200µl SCE-buffer mixed with 2µl zymoylase (TEIKAGU CORPORATION, Tokyo JPN) and 20µl glusulase (PERKIN ELMER MA, USA) for 1 hour at 30°C. Careful centrifugation (2000rpm, 3 min) was followed by washing with SPC-buffer (1,2M sorbitol, 0,3mM K2HPO4, 0,1mM KH2PO4 and 0,1M sodium citrate). To fix spheroblasts on glass eight-well slides (ICN BIOMEDICALS INC, OHIO, USA), the slides were treated with 10µl polylysine for one minute. Polylysine was washed away by water and 12µl of the spheroblasts suspension was put on the air-dried slide, which was the put into a –20°C methanol bath followed by 30 sec incubation in –20°C acetone. After fixation, the first antibody diluted in 1% bovine-serum albumin in PBS was applied and incubated in darkness for one hour. Intermediate washing was carried out using 1% bovine-serum albumin in PBS for three times. The second antibody diluted in 1% bovine-serum albumin in PBS carrying the light-stimulable alexa domain was incubated on the slide again for one hour. After the last three washing steps with 1% bovine-serum albumin in PBS.

Staining with 4,6-diamidino-2-phenylindole (DAPI) and anti-tubulin antibodies were used for visualization of nuclei and spindles, respectively.

3.4.7. FACS Analysis

Cells were fixed in 70% ethanol overnight, centrifuged (13.000 rpm 1 minute) and washed twice with 50mM Tris pH 7,8. 800µl of 50mM Tris pH 7,8 was used to resuspend the cells before 20µl RNase (10mg/ml) were added and incubated for at least 6h at 37°C. Then, 0,5 mL pepsin (5mg/ml in 55mM HCl) were added and incubated for 30 minutes on a shaker at 37°C. Two times washing with 1 ml 200 mM Tris pH 7,5, 200 mM NaCl and 78 mM MgCl2 and collection in the same buffer preceded staining of DNA by addition of 55 µl propidiumiodide (0,5mg/ml). Shortly before FACS measurement, cells were sonificated. 50µl of cell-suspension diluted in 1 ml 50mM Tris pH 7,8 were used for the analysis.

3.4.8. Single-Step Purification of Proteins

Protein Extracts were prepared as described in 3.4.2. All following procedures were carried out at 4°C. STREP-Tactin-Superflow-Columns were equilibrated using 3 ml buffer W (100 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA). Then, 4 ml of the

crude protein extract containing the recombinant STREP-tag fusion protein or the untagged control was applied to one column and ran through by gravity flow.

Unspecific flow-through was collected. To wash residual unspecifically bound protein off the column, 5 ml of buffer W was applied. Afterwards, the fusion proteins were eluted by adding three times 1 ml of buffer E (100 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA, 2,5mM desthiobiotin) and collected in three fractions of 1ml. After the elution step, the column was regenerated using buffer R (100 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA, 1mM HABA). The obtained purified protein was used for Western Blot, 2D gel-electrophoresis or silver staining.