Western Blot

2.2.15.1. Protein sample preparations-total cell lysates

For Western Blot analysis, cells were seeded in 6 well plates and treated as described. Supernatant and trypsinized cells were pooled and centrifuged (600 x g, 5min, 4°C). After washing the cell pellet with cold PBS, cells were lysed in the lysis buffer for 30 min at 4°C. Cellular debris was removed by centrifugation at 10,000 x g, 10 minutes, 4°C. Superna- tants were transferred to new tubes.

Table 2.14: RIPA and triton protein lysis buffer

RIPA Buffer Triton lysis buffer

Tris/HCl 0.79 g Tris/HCl, pH 7.5 30 mM

NaCl 0.87 g NaCl 150 mM

Nonidet NP40 1.0 ml EDTA 2 mM

Deoxycholic acid 0.25 g Triton X-100 1 %

Tris/HCl 0.10 g Complete® 1:25 Complete® 1:25 H2O PMSF 1.0 mM Na3VO4 1.0 mM NaF 1.0 mM H2O up to 250ml

Table 2.15: Sample buffer.

5x SDS sample buffer 3 x Laemmli buffer

Tris/HCl 3.125 M 100 µl Tris/HCl 187.5 mM SDS 20% 250 µl SDS 6 % Glycerol 500 µl Glycerol 30 % DTT 16% 125 µl Bromophenolblue 0.025 % Pyronin Y 5% 5 µl β-Mercaptoethanol 12.5 % H2O H2O 2.2.15.2. Protein quantification

Protein samples were quantified according to Bradford et al.63 The standard calibration curve was generated using samples which contain defined concentrations of BSA sus- pended in H2O (50 µg/ml-500 µg/ml). 190 µl of the Bradford reagent (Bradford reagent

stock was diluted 1:5 in H2O) was added to each 10 µl aliquot of the diluted protein sam-

ples (diluted 1:10 in H2O) and calibration samples in a 96-well flat bottom plate. All meas-

urements were performed in triplicates. Probes were incubated for 5 minutes and absorb- ance was measured using the SpectraFluor PlusTM (Tecan, Männedorf, Austria).

2.2.15.3. SDS-PAGE

The SDS-PAGE was performed according to Laemmli et al.67. Here the Mini Protean III system from Bio-Rad (Munich, Germany) was used. Prior to loading the samples, the ap- paratus was assembled as described by the producer, and the chamber was filled with ice-cold electrophoresis buffer. Before loading, samples were boiled at 95°C for 5

minutes. Equal protein amount/per sample was loaded onto the SDS-gel. 2 µl of the Fer- mentas page rulerTM prestained protein ladder was loaded on each gel to estimate the molecular weights of the separated proteins. Proteins were separated in a discontinuous electrophoresis: Proteins were focused by running the probes through the stacking gel, pH 6.8 (100 V, 20 minutes) and then separated in the separating gel, pH 8.8 (200 V, 35- 45 minutes).

Table 2.16: Electrophoresis buffer Electrophoresis buffer

Tris base 3.0 g

Glycine 14.4 g

SDS 1.0 g

H2O up to 1 l

Table 2.17: Preparation of SDS-PAGE

Stacking gel Seperating gel

30 % PAA solution 1.28 ml 30 % PAA solution 5.0 ml 1.25 M Tril HCl pH 6.8 0.75 ml 1.25 M Tril HCl pH 6.8 3.75 ml 10 % SDS 75 µl 10 % SDS 150 µl H2O 5.25 ml H2O 6.1 ml APS 75 µl APS 75 µl TEMED 20 µl TEMED 20 l 2.2.15.4. Tank blotting

After separating the samples in the SDS-PAGE, proteins were transferred to a nitrocellu- lose membrane (Hybond-ECL TM, Amersham Bioscience, Freiburg, Germany) via tank blotting. A blotting sandwich was prepared in a box filled with 1 x Tank Buffer as follows: cathode-pad, blotting paper, separating gel (from SDS-PAGE)-nitrocellulose membrane, blotting paper-pad-anode. Pads, papers, and the membrane were equilibrated with 1 x Tank buffer 15 minutes prior to running the tank blot. Sandwiches were mounted on the Mini Trans-Blot® system (Bio-Rad, Munich, Germany), and the system was filled up with ice cold 1 x Tank buffer. Transfers were carried out at 4°C at 23 V overnight.

Table 2.18: Tank buffer

5 x Tank buffer 1 x Tank buffer

Tris base 15.2 g 5x Tank buffer 200 ml

Glycine 79.2 g Methanol 200 ml

H2O up to 1 l H2O up to 1 l

2.2.15.5. Detection

After the transfer, sandwiches were disassembled and gels were stained with coomassie blue (see below). Membranes were incubated in 5 % blotto for 1 hour, RT to block unspe- cific binding reactions. After one washing step with 1x TBS-T, membranes were incubated with the primary antibody dilution overnight at 4°C . The next day, membranes were washed three times 10 min with 1x TBS-T and then incubated with the secondary anti- body for either 1 hour (IR-dye, light protection) or 2 hours (HRP-dye) at room temperature. 2.2.15.6.1. Detection method-LICOR

Secondary antibodies coupled to IRDyeTM 800 and Alexa Fluor® 680 with emission max- ima at 800 and 700 nm were used. Membranes were incubated for one hour, RT in ap- propriate dilutions. After the incubation period, membranes were washed three times for 10 minutes with TBS-T. Membranes were scanned and analyzed using the Odyssey im- aging system (LICOR Biosciences, Lincoln, NE). Thus, the intensity of each protein band could be determined.

2.2.15.7.2. Enhanced Chemiluminescence

Proteins coupled to peroxidase-conjugated antibodies were detected with ECL solution. After incubation with the secondary antibody, membranes were washed three times for 10 minutes with TBS-T. Then the membrane was incubated protected from light in ECL Plus TM Western Blotting detection reagent (Amersham Bioscience) for 1 minute, and lay- ered between two plastic sheets. Chemiluminescence was detected by exposing the membranes to a X-ray film (Super RX, Fuji, Düsseldorf, Germany) for the appropriate time period in a darkroom. X-ray films were developed in a Curix 60 developing system (Agfa- Gevaert AG, Cologne, Germany)

Table 2.19: Primary antibodies for Western Blot analysis

Antigen Isotype Dilution Provider

Actin mouse 1:1000 Santa Cruz

pAkt (S473) mouse 1:1000 Cell signaling

Ductin (ATP6L) rabbit 1:500 Millipore

EGF-R rabbit 1:500 Cell signaling

pEGF-R rabbit 1:1000 Cell signaling

ERK (tot) rabbit 1:1000 Cell signaling

pERK mouse 1:1000 Cell signaling

Rab5A rabbit 1:1000 Santa Cruz

Rac1 mouse 1:1000 Upstate

Table 2.20: Secondary antibodies for Western Blot

Antigen Dilution Provider

AlexaFluor ® 680 Goat anti-mouse 1:10.000 Molecular Probes IRDyeTM 800CW Goat anti-mouse 1:10.000 Li-COR Biosciences AlexaFluor ® 680 Goat-anti rabbit 1:10.000 Molecular Probes IRDyeTM 800CW Goat-anti rabbit 1:10.000 Invitrogen

Goat anti-mouse IgG1: HRP 1:5000 Biozol

Goat anti-mouse IgG2b: HRP 1:5000 Biozol

Goat-anti rabbit IgG: HRP 1:5000 Dianova

To verify a homogenous loading, polyacrylamid gels were stained for 30 minutes with coomassie-blue, and destained with coomassie-destaining solution. After protein detec- tion, membranes were stained with Ponceau S (0.2% Ponceau S in 5% acetic acid) for 5 minutes and destained with distilled water. Membrane was scanned (Canon, Krefeld, Germany).

Table 2.21: Coomassie staining solutions

Coomassie staining solution Coomassie destaining solution

Coomassie blue 3.0 g Glacial acetic acid 100 ml

Glacial acetic acid 100 ml Ethanol 333 ml

Ethanol 450 ml H2O up to 1 l

H2O up to 1 l