Protein biochemical methods 84 

5.2.1.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Denatured protein samples were applied to 8%, 10%, 12%, or 15% polyacrylamide gels and separated by electrophoresis as described (Laemmli, 1970). Separating gels were of the above mentioned acrylamide (Acrylamide/Bis Solution, 37.5:1, Serva) concentrations, stacking gels were always 4%. To denature protein samples they were incubated at 65°C for 10 min in HU buffer/ DTT (8 M urea, 5% SDS, 0.2 M Tris pH 6.8, bromophenol blue and 0.1M DTT prior to use). Electrophoresis of mini gels (Hoefer Mini Gel System) was carried out using Laemmli running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS) at 20 mA per gel and depending on application a maximum of 150 V. Approximate protein size was estimated by comparison to a pre-stained protein standard (AllBlue, BioRad).

5.2.1.2 Western blot

After SDS-PAGE proteins were blotted to PVDF membrane (Millipore) using a semi-dry blotting system. The membrane and Whatman filter papers were cut to gel size and soaked in blotting buffer (25 mM Tris, 192 mM glycine, 0.01 % SDS, 20 % methanol). Gels were blotted at constant current

(150 mA per mini gel) and limited to 15 V for 2-3 hours. After blotting the membranes were blocked using 5 % non-fat milk (Merck) in TBS-T (TBS/ 0.02% Tween20) for at least 30 min. For immunological detection of blotted proteins incubation with primary antibody in blocking solution (with 0.065% sodium azide) was carried out overnight at 4°C. After washing with TBS-T (1x5min, 3x10min) the membrane was incubated with horseradish peroxidase coupled secondary antibody for 1-2 h at room temperature. This was again followed by washing with TBS-T (6x10 min) and once with ddH2O. For

chemiluminescent detection the ECL reagent mixture (GE Healthcare) was added to each membrane, spread evenly, and the signal detected using ECL-Hyperfilm (GE Healthcare). Developed films were scanned and processed using Adobe Photoshop (Adobe Systems Inc.) and/ or ImageJ (http://imagej.nih.gov/ij/) software.

5.2.1.3 Yeast whole cell lysates- TCA precipitation

The necessary amount of cells (release/ FACS cell amount in 1 ml of OD600=0.6, otherwise in 1 ml of

OD600=1; OD600=1 is approximately equal to 107 cells/ ml) was harvested by centrifugation and then

resuspended in 1 ml ddH2O. Alternatively the appropriate amount of cells was resuspended in 1 ml

ddH2O directly from plates. To lyse cells, 150 μl 1.85% NaOH/ 7.5 % β-mercaptoethanol were added

and the samples were vortexed. After incubation for 10 min on ice 150 μl 55 % TCA were added to the samples, they were vortexed and again incubated on ice for 10 min. Protein precipitates were then collected by centrifugation (20000 g, 10 min, 4°C), the supernatant was aspirated, and the pellet was centrifuged again to remove the remaining liquid. Pellets were then resuspended in 50 μl HU/ DTT and incubated at 65°C for 10 min. If necessary the pH of the samples was adjusted by addition of 2-4 μl 2 M Tris-base.

5.2.1.4 Immunoprecipitation (IP)

Yeast cultures were grown in YPD to an OD600 of 1, harvested and washed once with cold ddH2O/

1 mM PMSF (1000 g, 4°C, 5 min). Cells were then lysed in IP buffer (50 mM Tris/ HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.1% NP-40, 10% glycerol, 10 mM NaF, 2 mM PMSF, complete protease inhibitor cocktail (Roche)) by addition of zirconia beads (Biospec) and vortexing (6x 30sec). After lysis the NP-40 concentration was increased to 1%, and the extracts were centrifuged at 2,600 g for 5 min, followed by centrifugation at 20,000 g for 25 min. An input sample (10 μl) was taken prior to antibody addition and denatured by adding an equal amount of HU/ DTT and incubation at 65°C for 10 min. The supernatants were incubated with 20 μl pre-coupled HA-antibody (Santa Cruz Biotechnology Inc.), or 4.5 μl myc, or 2 μl Shp1 antibodies and rotation at 4°C over night. Immunocomplexes were then either bound to 20 μl protein A sepharose beads (GE Healthcare) for three hours (4°C) or directly washed if pre-coupled beads were used. Beads were washed four times (600 μl IP buffer/ 1% NP-40 for 10 min, 800 μl IP buffer/ 1%NP-40 8 min, 800 ml IP-buffer 5 min, 1 ml IP buffer) and eluted in 25 μl HU/ DTT, 10 min, 65°C.

5.2.1.5 Denaturing Ni-NTA Pull-down

Denaturing Ni-NTA pull-downs of yeast overexpressing 6xHis epitope tagged ubiquitin were used to detect in vivo ubiquitination of proteins. Yeast strains were either transformed with YE-pCUP-HisUb, or

86

strains in which YI211-pADH-HisUb had been integrated were used. Overnight cultures were grown in selective medium without induction of the copper promoter. 200 ml selective medium were inoculated to OD600 of 0.1 and incubated at 30°C or 25°C. In the case of strains carrying pCUP-HisUb,

overexpression of His_{ubiquitin was induced approximately 3 h before harvesting the cultures by}

addition of 100 µM CuSO4 to the culture. Upon reaching OD600 0.8-1.0, the cultures were harvested by

centrifugation (5500 g, 10 min, 4°C), were resuspended in 10 ml ice-cold sterile ddH2O and were

transferred to 50 ml round-bottom tubes. After centrifugation (3500 g, 10 min, 4°C), the supernatant was discarded and the pellets were frozen in liquid nitrogen. Cells were lysed by resuspending the frozen pellet in 5 ml 1.85 N NaOH/ 7.5 % β-mercaptoethanol and incubation on ice for 15 min. 5 ml 55 % (w/v) TCA were added, the samples were vortexed and incubated on ice for 15 min. Precipitated proteins were collected by centrifugation (3500 g, 10 min, 4°C) and the pellet was washed twice with 5 ml ice cold (-20°C) acetone. The protein pellets were then thoroughly resuspended in 2 ml denaturing buffer A (6 M guanidinium chloride, 0.1 M NaH2PO4, 10 mM Tris, 0.05 % Tween20 pH 8)

and were incubated at RT and 80 rpm for one hour to further increase solubilization of the TCA- precipitate. Remaining insoluble material was removed by centrifugation (23000 g, 20 min, 4°C). The imidazole concentration of the supernatant was increased to 20 mM and a 10 % input sample was taken. The lysate was then incubated with 50 µl magnetic Ni-NTA agarose (Qiagen) at 4°C overnight. Using a magnetic rack (Dynal), the beads were washed three times with 800 µl buffer A/ 20 mM imidazole and then five times with 800 µl buffer C (8 M urea, 0.1 M NaH2PO4, 10 mM Tris pH 6.3 and

0.05% Tween20). To elute bound proteins the beads were incubated with 30 µl 1 % SDS for 10 min at 65°C. The eluate was then concentrated using a speed-vac (25 min, 45°C) and was resuspended in 15 µl HU/ DTT and 10 µl ddH2O. Input samples were subjected to TCA-precipitation (see 5.2.1.3) and

were finally resuspended in 25 µl HU/ DTT. Both samples were incubated at 65°C for 10 min prior to SDS-PAGE. Typically 3 µl input and 7 µl pull-down were loaded on SDS-PAGE gels.

5.2.1.6 Two step purification of phosphorylated Shp1 for mass-spectrometry Strains expressing Shp1myc7His _{were grown to OD}

600 of 1-1.5 and harvested. Lysis and Ni-NTA pull-

down were performed as described above for His_{ubiquitin pull-downs. Most (25 µl) of the SDS eluate}

was directly diluted 1:24 with RIPA*** (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1.25 % Triton X-100) buffer and incubated with 20 μl of pre-coupled anti-myc beads (Santa Cruz Biotechnology Inc.) overnight. The remaining 5 µl were used as a PD eluate/ IP-input control. The beads were then transferred to empty protein columns (BioRad), the flow-through was collected and the beads were then washed six times with 600 µl RIPA* (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1.25 % Triton X-100, 0.1 % SDS) buffer and bound protein was eluted in 50 µl 1 % SDS. The eluate was concentrated using a speed-vac and then resuspended in 7.5 µl HU/ DTT and 12.5 µl ddH2O.

For mass-spectrometry the sample was analyzed on a pre-cast 10% Gel and run with MOPS buffer (Invitrogen). The strong band at approximately 50 kD was excised and analyzed using an OrbiTrap mass spectrometer and peptides were identified using Mascot Daemon software (all performed by Frank Siedler MPI of Biochemistry, Martinsried).