Protein chemical techniques

2.15.1 Isolation of total protein from cell cultures

For isolation of total protein from cell cultures the medium was first removed and then the cells were washed with ice cold PBS. Depending on the confluency of the cells and the used culture flask/plate, the appropriate amount of protein lysis buffer (modified RIPA) was added to the

30x

adherent cells; for example 150 μl lysis buffer were used for cells with a 90 % confluency in a 6-well plate. The cells were incubated for 5 min on ice before they were scraped off the bottom with either a pipette tip or a cell scraper and transferred to a reaction tube. To pellet the cell debris the cells were centrifuged at 13000 rpm at 4°C for 10 min. The supernatant, which consists of the isolated protein, was transferred to a new reaction tube and the protein concentration was measured. For short-term storage the protein was kept at -20°C, for long-term storage at -80°C.

2.15.2 Isolation of protein from mouse tissue

Proteins from mouse prostate or prostate tumor tissue was obtained via homogenization of the tissue using the TissueLyser LT (Quiagen, Hilden, Germany). A small sample of the tissue was transferred to a 2 ml reaction tube. A stainless steel bead (Ø 5mm, Quiagen) was added as well as 500 μl modified RIPA lysis buffer. The tissue was homogenized for 5 min at 50 1/s oscillation in the TissueLyser LT. Thereafter the cups were kept for 5 min on ice before centrifuging them for 10 min at 13000 rpm and 4°C. The supernatant was transferred to a new 1.5 ml reaction tube and the protein concentration was measured. For shortterm storage the protein was kept at -20°C and for long-term storage at -80°C.

2.15.3 Determination of protein concentration

The protein concentration was measured via Bradford protein assay (Bradford 1976). Roti®- Nanoquant (Carl Roth GmbH, Karlsruhe, Germany) was used, which consists of the Coomassie Brilliant blue dye. In acidic solutions, this dye binds unspecific to cationic and hydrophobic side chains of proteins. Once bound, the absorption maximum of the dye is shifted from 495 nm to 595 nm since the binding reaction stabilizes the dye in its non-protonated and anionic form. A series of Roth Albumin Fraction V dilution was used for calibration according to the manufacturer’s recommendation. The protein concentration was determined by extrapolating to this standard curve.

5x Roti®- Nanoquant dye was diluted with ddH2O to a final 1x concentration and stored at 4°C.

Protein samples were diluted 1:100 with ddH2O. 50μl of the sample were added to a 96-well plate in triplicates. ddH2O was used as a control. 200μl of 1x Roti®- Nanoquant were added to samples and incubated for 5 min at RT. The protein concentration was measured using the SynergyMx plate reader spectrophotometer (BioTek, Friedrichshall, Germany) and calculated by inherent Gene5 software.

2.15.4 Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE gel electrophoresis (Western blot) is a useful biochemical method to separate proteins of a sample according to their molecular weight. The NuPage® Pre-Cast Gel System from Life Technologies (Darmstadt, Germany) was used which is based on the SDS-PAGE gel chemistry by Laemmli (Laemmli 1970). This polyacrylamide gel system, designed for high performance gel electrophoresis, consists of pre-cast gels and buffers with an operating pH of 7.0, which increases stability in both proteins and gels. The samples consisted of 20-30 μg protein, 0.25 vol sample buffer (LDS Sample Buffer (4x), NuPAGE®, Life Technologies, Darmstadt, Germany) and 10 % 1M DTT (reducing agent). The samples were denatured at 70°C for 10 min and afterwards kept on ice for 5 min. Before the samples were loaded onto the gradient gel (NuPAGE® 4-12% Bis-Tris Gel, Life Technologies, Darmstadt, Germany), they were centrifuged briefly. 7 μl of the pre-stained molecular weight standard (See Blue® Plus2, Life Technologies, Darmstadt, Germany) were also loaded onto the gel to determine the size of the separated proteins. Gel electrophoresis was performed at 160 V and 160 mA in 1x MES buffer (Life Technologies, Darmstadt, Germany). Depending on the molecular weight of the protein of interest the gel was run for 1-3 hours.

2.15.5 Transfer of proteins onto a PVDF membrane

After SDS-PAGE gel electrophoresis the proteins were transferred onto a PVDF membrane (GE Healthcare, Freiburg, Germany) using the semi-dry blotting procedure. Thereby, the PVDF membrane (7 cm x 8 cm or 7 cm x 13 cm) was first activated for 10 sec in 100% methanol before it was equilibrated for 10 min in transfer buffer IIa. Six sheets of Whatman GB003 filter paper (Schleicher & Schull, Dassel, Germany) having the same size as the gel and the PVDF membrane, were also soaked in transfer buffer IIa. The electro-blotter (Biometra, Göttingen, Germany) was loaded in a sandwich system in the following order:

Bottom of the blotter, Anode (+): Three sheets of filter paper PVDF membrane

Gel

Lid of the blotter, Cathode (-): Three sheets of filter paper

Before the blotter was closed, remaining air bubbles were removed from the sandwich system.

The transfer was carried out at 25 V and 220 mA for 30 min to 1 hour, depending on the molecular weight of the proteins.

2.15.6 Incubation of protein-bound membranes with antibodies

The PVDF membrane was incubated in the western blot blocking buffer for 1 hour at RT to block unspecific binding sites. Thereafter it was thoroughly washed with western blot washing buffer.

The membrane was incubated with the primary antibody overnight at the recommended dilution in TBS-T at 4°C. The next day, unbound antibody was removed by washing twice for 10 min with the western blot washing buffer. The membrane was then incubated for at least 2 hours with the secondary alkaline phosphatase conjugated antibody, diluted accordingly to the manufacturer’s recommendation in western blot blocking buffer. It was washed three times for 15 min in western blot washing buffer and 5 min in TBS-T to remove the remaining milk. ECL Prime Detection solution (GE Healthcare, Freiburg, Germany) was used to detect chemi-luminescent signals. Therefore, the membrane was placed on a plastic film and the detection solution was pipetted onto the membrane according to the manufacturer’s instruction and incubated for 5 min. The signals were captured using the western blot detection system (FlourChem® Q Alpha Innotech, Logan, USA) and evaluated using the inherent AlphaView Software for FluorChem® systems (FlourChem® Q Alpha Innotech, Logan, USA).