Biochemical Methods

2.2.2.1 SDS-Polyacrylamide Gel Electrophoresis and Western blotting

Protein mixtures were separated by discontinuous SDS-PAGE (Laemmli, 1970) and stained with Coomassie Brillant Blue R 250. Alternatively proteins were visualized by semi-dry Western blotting following a modified Towbin protocol (Towbin et al., 1992). Blots were developed after BSA blocking in NCP buffer (10 mM Tris/HCl, 0.15 M NaCl, 0.05%

Tween 20, 2% NaN3) with alkaline phosphatase conjugated secondary antibodies and BCIP.

2.2.2.2 GST-tagged protein expression

Constructs in pGEX vectors were transformed into Rosetta, BL21 RIL or ArcticExpress RIL cells. Cultures were inoculated in LB (10 g bacto-tryptone, 5 g yeast extract, 86 mM NaCl) or LB rich medium (20 g bacto-tryptone, 10 g yeast extract, 86 mM NaCl) (Sambrook and Gething, 1989) and grown overnight at 37°C. Cells were diluted tenfold and grown at 21°C to an OD600 of 0.4 - 0.8. Expression was induced with usually 0.5 mM IPTG and cells were grown overnight at 21°C. After harvesting and washing following routine procedures, cells were resuspended in lysis buffer (1 x PBS (70 mM Na2HPO4, 30 mM KH2PO4, 150 mM NaCl, 0.1% NaN3, pH 6.5), 1 mM DTT, 1 mM EDTA, 5 mM benzamidine, 1 mM PMSF, protease inhibitor cocktail (Sigma)), opened by sonication in the presence of 0.5 mg/ml lysozyme. Lysates were centrifuged at 10,000 g for 45-60 min at 4°C and the supernatants were coupled to glutathione-sepharose 4B (Sigma) by recycling for 3-4 h. The matrix was washed with 10-20 column volumes of lysis buffer and the bound proteins were eluted with lysis buffer containing 20 mM reduced glutathione (pH 7.2). The presence of protein in different fractions was tested by Bradford´s method (Bradford, 1976). Protein containing fractions were analysed on SDS-PAGE. The appropriate fractions were pooled and dialysed against PBS.

If pGEX 6P-1 vector were used for expression, the GST tag could be removed by PreScission protease. Following elution of the GST fusion protein from glutathione sepharose, the eluate was dialysed extensively against PBS containing 1 mM EDTA and 1 mM DTT in order to remove reduced glutathione and protease inhibitors from the sample. 10 µg of enzyme were used to cleave 1 mg of GST fusion protein. Cleavage was carried out at 4°C overnight on a rotatory shaker. Once digestion was complete, the sample was passed through a washed and equilibrated glutathione sepharose to remove free GST and the PreScission protease from the protein of interest.

Wild type cells were harvested and lysed in degassed TEDABP-Buffer (10 mM Tris/HCl, 1 mM EGTA, 1 mM DTT, 0.02% NaN3, 1 mM benzamidine, 0.5 mM PMSF, pH 8.0) by freeze thaw and passed through a filter. The total lysate was centrifuged and used for size exclusion gel chromatography. The supernatant was applied to a Superose 12 10/300 GL

column (GE) that was controlled by the Äkta. 500 µl fractions were collected and blotted against the appropriate antibodies.

Polyclonal antibodies for DstA and Krs2 were generated in haemophilic New Zealand rabbits. Protein solutions mixed (1:1) with adjuvant were injected into the rabbits. After the primary booster polyclonal serum was for the antibody titer by western blot. In both cases a dilution of 1:10 000 of the original serum was sufficient for western blot use.

2.2.2.3 Immunprecipitation and Nanotrap

For immunoprecipitations the D. discoideum cells were harvested by centrifugation at 1,200 g for 3 min, washed twice with phosphate buffer and resuspended at a density of 1x108 cells/ml in cold 25 mM HEPES buffer pH 7.4. One millilitre of the cell suspension was sedimented for 2 min at 2,000 g and the pellets incubated with IP-lysis buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM EGTA, 5 mM benzamidine, and 1 mM DTT, 5% glycerol, 1% N-octylpolyoxyethylene (Bachem), 1 mM PMSF and protease inhibitor cocktail (Sigma)). The crude lysates were spun for 10 min at 15,000 g and the clear lysates were each supplemented with 100 µg of polyclonal antibodies together with 150 µl of protein A-Sepharose CL-4B slurry (Sigma) equilibrated in IP-lysis buffer. After 2 h of incubation at 4°C, the beads were sedimented, washed seven times with 1 ml IP-lysis buffer, and bound proteins eluted with SDS sample buffer (Faix et al., 2001). Nanotrap was performed as described (Rothbauer et al., 2008). After immunoprecipitation kinase assays were performed essentially as described (Eichinger et al., 1998). The signals were visualized on autoradiography films (Hyperfilm, GE Healthcare) and developed in CURIX 60 (Agfa).

2.2.2.4 Two-dimensional gel electrophoresis

Aliquots containing less than 100 pg of protein were adjusted by the addition of 2 ml of a solution containing 7.0 M urea, 2.0 M thio urea, 4% CHAPS (Sigma), 4% servalyt, 0.4 g/ml ampholytes (Pharmacia Biotech Inc.), 0.2 M DTT and protease inhibitors. Samples were then applied to the top of the naked IPG strips and incubated over night at room temperature covered with mineral oil. The swollen strips were subjected to electrophoresis for 1 h at 300 V and 16 h at 3500 V. The gel reservoir was filled with mineral oil. Following electrophoresis, IPG strips were extruded and equilibrated for 15 min into equilibration buffer 1 (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS and

0.1 M DTT) and 15 min in equilibration buffer 2 (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS and 0.15 M IAA). Second dimension gels were freshly cast in the Ettan DALTsix gel caster. Slab gels consisted of 12% acrylamide-bisacrylamide, 0.07% w/v SDS, 37mM Tris-HCl, pH 8.8, 0.05% w/v ammonium persulfate, and 0.025% v/v TEMED. The first dimension IPG strips were secured on top of the slab gels. The proteins were subject to electrophoresis overnight at 10 mA/gel in gel running buffer consisting of 0.1% SDS, 25 mM Tris/HCl, and 185 mM glycine. Following electrophoresis gels were stained with Coomassie blue solution.