Different signal transduction pathways were investigated upon stimulation of cell with different growth factors. Before stimulation, the cells were starved in DMEM without serum for 18-24 hours. Cellular proteins were brought into solution with the detergent Triton X-100. This detergent lysis the cell membrane and leaves the nucleus and other intracellular compartments intact. Membrane and cytoskeletal fragments are pelleted by centrifugation and the supernatant is used for subsequent experiments. The cells were washed once with ice cold PBS and lysed on ice in an appropriate volume lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 % glycerin, 5 mM EDTA pH 8.0, 1% Triton X-100), to which 10.0 µg/ml aprotinin, 1.0 mM PMSF, 100.0 mM sodiumfluorid, 10.0 mM p-nitrophenylphosphat 20.0 mM sodiumpyrophosphate, 2.0 mM sodium- orthovanadate pH 10.0 were freshly added. Alternatively, RIPA (Radio Immuno Protection Assay) buffer eas used in stead (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH7.2, 2 mM EDTA, 50 mM sodium fluoride, and 0.2 mM sodium vanadate along with 100 U/ml aprotinin added freshly) was used. The volume was adjusted to the size of the cell culture plate. After 10 min the cell lysate was collected in an Eppendorff-tube and the insoluble fragments were pelleted by centrifugation (13'000 rpm, 4 °C, 10 min). The supernatant was collected in
an new Eppendorff-tube and used either for immunoprecipitation as described or as whole cell lysate.
2.2.8.2 Pierce BCA Protein Estimation Assay
The Micro BCA Protein Assay Kit from Pierce was used to determine the protein concentration of the crude cell lysates. The kit is based on the Biuret reaction, in which proteins react with Cu2+ in an alkaline medium to produce Cu1+. The Cu1+ ions then react with bicinchonic acid (BCA) yielding a water-soluble, purple complex exhibiting a strong absorbance at 562 nm. The kit is not sensible to detergents (e.g. Triton X-100), which are used to lyse cells. The assay was performed in microtiter plates according to the manufacturer recommendation. A bovine serum albumin concentration series (100 - 2000 µg protein/ml) was used as standard.
2.2.8.3 Immunoprecipitation of Proteins
In immunoprecipitations antibodies, which were coupled to protein A-sepharose, were used for the enrichment of proteins from total cell lysate. Protein A is a membrane protein of the bacteria Staphylococcus aureus, which binds specifically to the Fc- domain of certains immunoglobulins (Chenais et al., 1977). For the immunoprecipitation, the cell lysate was diluted with an equal volume of HNTG-buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 % glycerin, 5 mM EDTA pH 8.0, 0.1 % Triton X-100), 30 µl 50 % protein A-sepharose and 1-5 µg monoclonal antibody or 2-10
µl polyclonal antibody were added. The samples were rotated at 4 °C for at least 4 hours end-over-end. The protein A-sepharose beads were pelleted by centrifugation (13'000 rpm) and washed three times with HNTG-buffer. After addition of 50 µl 2 x Laemmli- buffer, the beads were boiled at 100 °C for 5 min to denature the proteins.
2.2.8.4 In vitro Binding Assay with GST-Fusion Proteins
5 µg GST-fusion protein were incubated with 40 µl glutathion-sepharose in 600 µl HNTG-buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 % glycerin, 5 mM EDTA pH 8.0, 0.1 % Triton X-100) and rotated end-over-end at 4 °C for 1 hour. The sepharose beads were pelleted by centrifugation (13'000 rpm) and washed once with HNTG- buffer. An equal volume of cell lysate and HNTG-buffer were added to the beads and the samples were processed as described in 2.2.11.3.
2.2.8.5 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins
The SDS-polyacrylamide-gel electrophoresis allows the separation of proteins according to their molecular weight (Laemmli, 1970). The detergent sodium-dodecyl- sulfate binds and denatures proteins. The binding of the dodecyl-sulfate, results in a negative, total charge of the protein independent of the specific amino acid composition, but correlated to its molecular weight. The negative charge determines the migration of the protein through the gel. 7.5, 10 or 15 % SDS-polyacrylamide gels or gradient gels with a continuous polyacrylamide gradient from 7.5 to 12.5 % were used, dependent on the molecular weight of the proteins. The stacking gels contained 4 % polyacrylamide. The SDS-gel electrophoresis was performed as described in Current Protocol in Molecular Biology (Ausubel F. M., 1988). After electrophoresis, the gels were either stained as described in 2.6.7 the proteins were blotted onto nitrocellulose as described in 2.6.8. The following proteins were used as molecular weight markers:
Protein Molecular Weight (kDa)
Myosin 200
β-galactosidase 116
Phosphorylase b 97
Bovine serum albumin 66
Ovalbumin 45
Carboanhydrase 29
Trypsin-inhibitor 21
Lysozyme 14
2.2.8.6 Staining and Fixation of Polyacrylamide Gels
The gels were stained for 5 min in Coomassie-solution (1 % Coomassie G250, 10 % acetic acid, 40 % methanol) and destained with 40 % methanol, 10 % acetic acid. The destained gels were placed on 3 MM Whatmann-paper and dried at 80°C under vacuum. If the proteins were radioactively labeled, the gels were exposed to films.
2.2.8.7 Transfer of Proteins onto Nitrocellulose Membrane
For immunodetection, the proteins were blotted onto nitrocellulose membranes. The transfer was performed with transblot-SD-buffer (50.0 mM Tris-HCl pH 7.5, 40.0 mM glycine, 20 % methanol, 3.75 x 10-5 % SDS) at 0.8 mA/cm2 nitrocellulose membrane in a semidry blot aperture for 2 hours. After the transfer, the proteins were stained with Ponceau S (2 g/liter in 2 % TCA), the bands of the molecular weight standard were marked and the membrane was destained and blocked with 1 x NET-gelatine (50.0 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 150 mM NaCl, 0.5 % Triton X-100 0.25 % gelatine).
2.2.8.8 Immunoblotting of Proteins (Western blot analysis)
Proteins, that are immobilized on nitrocellulose membranes can be detected with antibodies. In a first step the immobilised protein (antigen) is recognized by a specific monoclonal or polyclonal antibody (primary antibody). In a second step the primary antibody is recognized by a species specific secondary antibody, to which a horse radish peroxidase is coupled. In alkaline medium luminol (a diacylhydrazide) is oxidized by horse radish peroxidase and hydrogen peroxide. Immediately following oxidation, the luminol is in an excited state which then decays to ground state via a light emitting pathway, which can be detected by exposure to a film. The nitrocellulose membrane was incubated for one hour with three changes of 1 x NET-gelatine (50.0 mM Tris-HCl pH 8.0, 5 mM EDTA pH 8.0, 150 mM NaCl, 0.5 % Triton X-100. 0.25 % gelatine) to block unspecific binding sites of antibodies. The membrane was incubated with the primary antibody (1:500 - 1:10000 dilution in 1 x NET-gelatin) at room temperature for 3 hours or at 4°C overnight. After removal of the primary antibody, the membrane was washed 3 times for 20 min with 1 x NET-gelatine at room temperature and incubated with the secondary antibody for one hour. The membrane was washed 3 times for 20 minutes with 1 x NET-gelatine before detection with a ECL-kit from NEN. The membrane was incubated in a 1:1 mixture of both ECL-reagents for one minute. Afterwards, the membrane was wrapped with plastic film, and the air bubbles between the membrane and the plastic film were removed by gently smoothing with a tissue paper. The membrane was exposed to the film for different time periods. When the membrane should be reprobed with another antibody, the bound antibodies were
removed by stripping in 62.5 mM Tris pH 6.8, 100 mM β-mercaptoethanol, 2% (w/v) SDS at 50 °C for 1 hour.