Metáfora y metonimia

Proteins were extracted from liver tissue, as well as from primary hepatocytes and cell lines. Tissue and cells were lysed with RIPA lysis buffer containing 1:100 protease inhibitor and phosphatase cocktail II and III. Depending on the source material, slightly different procedures were applied:

a) Snap frozen liver tissue: 1 ml RIPA buffer was added to a piece of tissue on ice. This was homogenised with a pestle and sonicated. After 20 min incubation on ice, the homogenates were centrifuged for 10 min at 4 °C and 13000 rpm. The supernatants were collected in new tubes.

b) Primary cell culture: 6 well plates were placed on ice and medium was aspirated. The cells were carefully rinsed with 1xPBS before 0.3 - 0.5 ml RIPA buffer was added to each well. Cells were scraped and collected in a 1.5 ml tube. After sonification, lysates were incubated for 20 min on ice before they were centrifuged for 10 min at 13000 rpm and 4°C. The supernatants were transferred to new tubes.

c) Cell culture: 6 well plates were placed on ice and medium was aspirated. The cells were carefully rinsed with 1xPBS. Depending on the confluency of the monolayer, 75- 200 µL RIPA buffer was added to the cells on ice. After some minutes on ice, the cells were scraped and transferred to a 1.5 ml tube followed by the sonification step. All protein lysates were stored at -20 °C or -80 °C.

2.2.7.2 Protein quantification

The bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific) was used to quantify the protein content of the lysates. This assay combines the well-known Biuret reaction, which describes the reduction of Cu2+ _{to Cu}+ _{by peptide bonds under alkaline conditions, and the}

sensitive colorimetric detection of Cu+ _{with BCA. Two BCA molecules and one Cu}+ _molecule

form a purple chelate complex with an absorbance at 562 nm that is proportional to the protein concentration (Smith et al. 1985).

Depending on the expected protein concentration, the protein lysates were diluted with 1xPBS before measurement. 5 µL of the (diluted) sample were mixed with 195 µL BCA working reagent, consisting of 49 parts of solution A and 1 part of solution B in a well of a 96 well plate. A standard curve of bovine serum albumin (BSA), ranging from 0 µg/ml to 2000 µg/ml, was prepared the same way. Both the samples and each BSA standard were set up in duplicates. The plate was incubated for 30 min at 37 °C before the absorbance at 562 nm was measured in a plate reader (Infinite M200 Pro, Tecan). Based on the standard curve, the protein concentrations of the samples were calculated.

2.2.7.3 Western blot

2.2.7.3.1 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-Polyacrylamide gel electrophoresis is a widely-used technique for the analysis of proteins. Protein mixtures are separated in a gel in an electric field according to their electrophoretic mobility which is dependent on weight and charge. The anionic detergent sodium dodecyl sulphate (SDS) linearises proteins and covers their charge negatively (Shapiro et al. 1967). Hereby, the electrophoretic mobility is only dependent on the weight, not the charge of the proteins since the charge/weight ratio remains constant.

Gel preparation

The gels used for SDS-PAGE were either freshly prepared or bought (Thermo Fisher Scientific). Gels were cast using the Mini-PROTEAN Tetra electrophoresis system (BioRad) following the manufacturer’s instructions. Two 1.5 mm thick 10% separation gels consisted of 6.4 ml ultrapure water, 5.28 ml acrylamide solution (30% v/v), 4 ml separation buffer, 160 µL 10% (w/v) SDS solution, 6.4 µL TEMED and 160 µL 10% (w/v) APS solution. These compounds were mixed thoroughly and cast between two glass plates within a vertical frame. To avoid oxidation and evaporation, each gel was overlaid with 1 ml of 2-propanol. After the gels were completely polymerised, the 2-propanol was removed and the stacking gels were cast on top. Two stacking gels were prepared using 4.8 ml ultrapure water, 1 ml acrylamide solution (30% v/v), 0.8 ml stacking buffer, 65 µL 10% (w/v) SDS solution, 5 µL TEMED and 100 µL 10% (w/v) APS solution. Directly after casting the stacking gels, combs were added to form loading wells. These gels were stored in a humid bag at 4 °C for a maximum of 1 week. Small-size proteins, like LC3, were analysed with gradient polyacrylamide gels (4 – 12% BisTris) from NUPAGE (Thermo Fisher Scientific).

Gel electrophoresis

Equal amounts of each sample’s protein were mixed with 5 x loading buffer and denaturated at 95 °C for 5 min. The loading buffer comprised dithiothreitol (DTT) to reduce disulphide bonds of the proteins for a better protein separation during electrophoresis. In the meantime, the gels were fit in their chambers and filled with 1 x running buffer. Denaturated protein samples and 4 µL of Precision Plus Protein Dual Colour standard (BioRad) were added to the wells of the gels. The initially applied current for the electrophoresis was 20 mA/gel and was increased up to 40 mA/gel as soon as the samples passed the stacking gel. The electrophoresis was stopped right before the samples reached the end of the gel.

2.2.7.3.2 Transfer

In the next step, proteins were blotted from the gel to a PVDF membrane to immobilise them as well as to make them accessible to antibody detection on the other hand. The transfers were performed electrophoretically with the semidry blot system from Biometra or BioRad. The transfer chamber consisted of two horizontal plates, which were either the anode (lower plate) or the cathode (upper plate). The membrane was activated in methanol and left in anode buffer for several minutes to equilibrate. On the anode plate, twelve Whatman papers

soaked with anode puffer were placed. The activated membrane was laid onto them and covered with the gel. Since bubbles between the membrane and the gel would disturb the transfer, those were carefully removed with a roller. Four Whatman papers, which were pre- equilibrated in cathode buffer, were arranged on top of the gel. The chamber was closed with the cathode plate and connected to the power supply. The transfer was usually performed at 234 mA or 315 mA, depending on the size of the gel (5 mA/cm²), for 40 min. After the transfer, the membrane was washed in ultrapure water and incubated for one hour in 5% BSA in TBS-T to block unspecific binding between the membrane and the antibody.

2.2.7.3.3 Antibody incubation and protein detection

After blocking, the membranes were incubated with specific primary antibodies, appropriately diluted in 5% BSA TBS-T over night at 4 °C and constantly shaken. The next day, the membranes were washed three times at 10 min with TBS-T before incubated with the secondary horseradish peroxidase (HRP) linked antibody for one hour at room temperature. The secondary antibodies were diluted appropriately in 5% BSA TBS-T. Information about the antibodies and their dilutions are listed in Table 2.22. This incubation was followed by three 10 min TBS-T washing steps. The protein detection was performed via chemiluminescence, where the membranes were incubated with 5 ml chemiluminescent solution and 3 µL hydrogen peroxide. The conjugated HRP of the secondary antibody catalysed the oxidation of luminol which was accompanied by light emission at 428 nm. The emission of light was captured by the Blot-Imager Vilber Fusion Fx7 (Vilber Lourmat), which illustrated a specific signal for the protein of interest. After imaging, the membranes were incubated with stripping buffer for 30 min to remove the antibodies and, after the above-mentioned blocking step, the membranes could be used for further protein analysis. If required, the bands of proteins were quantified densitometrically with the software ImageJ.

Table 2.22: Parameters for antibody incubation (Western blotting)

Primary antibody Secondary antibody Dilution Incubation

anti α-tubulin anti mouse 1:1000/1:5000 in BSA O/N 4 °C/ 1 h RT

anti β-actin anti mouse 1:5000/1:10000 in BSA 30 min RT/20 min RT

anti Agxt anti rabbit 1:1000/1:5000 in BSA O/N 4 °C/ 1 h RT

anti LC3 anti rabbit 1:1000/1:5000 in BSA O/N 4 °C/ 1 h RT