Protein-biochemistry methods

Removed mice tissues were weighed and homogenized in five volumes of ice-cold tissue homogenization buffer (Table 3.4). After douncing for 20 strokes using a 1 ml dounce homogenizer, 1 % (v/v) Triton X-100 was added to the homogenate and incubated for 1 h on ice. For a separation into soluble and insoluble Triton X-100 fractions, the mixture was centrifuged at 15.000 x g for 15 min at 4 °C using a refrigerated centrifuge. After centrifugation, the insoluble fraction (pellet) was discarded and the soluble fraction (supernatant) was transferred into a new 1.5 ml reaction tube for protein concentration determination or stored at -20 °C.

3.2.2.2 Protein concentration determination

Based on Lowry assay protocol (Lowry et al. 1951), the colorimetric determination of protein concentration was followed using detergent compatible Bio-Rad DC^TM protein assay.

According to the manufacturer’s manual, 20 µl of reagent S was added to 1 ml reagent A to prepare reagent A’. 5 µl of the protein sample and seven BSA standard dilutions, namely 2.0000, 1.0000, 0.5000, 0.2500, 0.1250, 0.0625, 0.0312 and 0.0000 µg/µl were loaded in triplicates into a 96-well microplate. 25 µl of working reagentA’ and 200 µl of reagent B were added successively to the already loaded samples. The mixture was incubated for 15 min at RT and the absorption was measured at 750 nm wavelength using Tecan GENios microplate reader. The protein concentration of the sample was calculated with the help of the BSA standard.

3.2.2.3 Tritosome enrichment

Tritosomes are lysosomes isolated from mice liver using a sucrose gradient-based technique (Wattiaux et al. 1963) after an injection of Triton WR1339 solution (Tyloxapol) (Leighton et al. 1968). Their content corresponds to that of the lysosomes and thus are expected to show comparable catalytic reactions (Gersten et al. 1974).

Enrichment of tritosomes was performed as previously described (Markmann et al. 2017). Four days ahead of sacrifice, control and starved mice were intraperitoneally (IP) injected with 17 % (w/v) Triton WR1339 solution in 0.9 % (v/v) NaCl. 4 µl/g of bodyweight. In the case of starved mice, chow depletion was introduced prior to the experimental day in accordance with the time of starvation.

On the experimental day, mice were sacrificed by a cervical dislocation and livers were removed. Mice livers were homogenized in five volumes of 250 mM ice-cold sucrose buffer. After douncing for three strokes using 5 ml dounce homogenizer, homogenates were centrifuged at 1000 x g for 10 min at 4 °C. The supernatant was transferred to a new 15 ml conical plastic tube, while the pellet was re-homogenized in 3.5 ml of 250 mM sucrose buffer followed by another similar centrifugation. Both post-nuclear supernatants (PNS) were pooled and ultra-centrifuged for 7 min at 56.500 x g at 4 °C using Ti-50 fixed angle rotor. After discarding the supernatant and resuspending the pellet in 10 ml of 250 mM sucrose buffer with a glass pestle, another ultra-centrifugation was performed under the same conditions. The resulting pellet, containing mitochondria and lysosomes, represents the M/L layer. In order to separate both organelles, a

Materials and Methods 26

discontinuous sucrose gradient using different sucrose density solutions was carried out (Table 3.4). The M/L pellet was resuspended in 3.5 ml sucrose solution of ρ 1.21 density, forming the first layer of a discontinuous sucrose gradient. Using a 1000 µl micropipette, the following 2.5 ml sucrose solution layers of ρ 1.15, 1.14 and 1.5 ml of ρ 1.06 density were carefully added. The density gradient was ultra-centrifuged at 110000 x g for 150 min at 4 °C, using SW41 swinging-bucket rotor. Subsequently, the resulting tritosome-enriched interphase fraction, located between ρ 1.14 and ρ 1.06 layers, was carefully collected and further prepared for western blot analysis and tandem mass tag (TMT) labeling or stored at -20 °C.

3.2.2.4 Mouse serum preparation

6-month-old male wild-type mice were sacrificed, and the thorax was opened.

After collecting 400-1000 µl of whole blood samples from the chest region using a 1000 µl micropipette, coagulation at RT was performed for 30 min. Samples were centrifuged at 18000 x g for 10 min at 4 °C and the supernatant was transferred to a new 1.5 ml reaction tube. 50 µl of serum samples were sent to Dr. Eberhard & Partner medical care center (Dortmund, Germany) for the determination of amino acid and acylcarnitine concentrations by mass spectrometry.

3.2.2.5 Measurement of blood glucose levels

Accu-Chek^® Guide instrument was used to test the blood glucose levels in control, 6 and 24 hours starved mice. To prevent blood clotting, samples were collected quickly and directly after the sacrifice. According to the manufacturer’s manual, the test strip was inserted into the instrument and a fresh whole blood drop was carefully added to the edge of the strip. The test results were obtained in mg/dl and the mean out of three individual measurements was calculated.

3.2.2.6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐

PAGE)

In a discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis, negatively charged proteins are separated based on their molecular mass while migrating through a SDS polyacrylamide gel to the anode (Laemmli 1970).

Either 12.5 % or 7.5 % running gel was prepared, followed by a 5 % stacking gel (Table 3.4) and both mixtures were cast using a BioRad Mini-PROTEAN Tetra Cell Stand. The desired number and size of pockets formed in the stacking gel was decided using the appropriate comb. After polymerization, the gel was placed into an electrophoresis chamber filled with 1x SDS-running buffer (Table 3.4).

Ahead of samples loading, 10-20 µg of protein samples were mixed with 1x laemmli buffer (Table 3.4) and incubated for 5 min at 95 °C. During this process, proteins will be denatured and negatively charged. In addition, 3 µl of Pageruler^TM prestained protein ladder or 7.5 µl of Spectra^TM multicolor high range protein ladder were loaded into the gel pocket. Gel electrophoresis was carried out at a constant 90 V for 2 h.

3.2.2.7 Western blot (WB) analysis

Western blot is an analytical method used for transferring previously separated SDS-PAGE proteins onto a protein-binding membrane. The protein of interest is then detected using specific antibodies. Antibodies are usually coupled to enzymes or chemical compounds to visualize the signal (Towbin et al. 1979).

For the immune detection, separated proteins were transferred onto a nitrocellulose (NC) or a methanol activated polyvinylidene fluoride (PVDF) membrane using Trans‐Blot^® Cell electrotransfer system. First, three thin Whatman papers were soaked in blotting buffer (Table 3.4). Subsequently, the membrane and the gel were submerged shortly into the blotting buffer and placed on top of it, followed by three additional presoaked Whatman papers. The semi-dry electrotransfer was carried out for 45 min at 300 mA.

After the electrotransfer, the membrane was blocked for 1 h by using 5 % (w/v) milk powder or 5 % (w/v) BSA blocking buffer (Table 3.4) to avoid unspecific binding of antibodies. Afterwards, the membrane was washed three times with a washing buffer (Table 3.4) and incubated with the diluted primary antibody in blocking buffer overnight at 4 °C. The appropriate horseradish peroxidase (HRP) conjugate secondary antibody was applied for 1 h at RT.

For a specific protein signal detection, additional three washes using the same washing buffer were completed. An equal amount of the enhanced chemiluminescence (ECL) substrates was mixed in a 1:1 ratio and poured over

Materials and Methods 28

the whole surface of the membrane. After 1 min, visualization was accomplished using Fusion Solo imaging system.

3.2.2.8 Western blot stripping

To investigate more than one protein on the same WB membrane, primary and secondary antibodies need to be removed before the detection with other antibodies. Therefore, a mild stripping protocol was used.

The dry membrane was equilibrated in 1x TBST for 5 min. Following this, the membrane was agitated thrice with an acidic stripping buffer (100 mM glycine, pH 2.0), while alternating with three washes of 1x TBS (Table 3.4) for 10 min each. Subsequently, the membrane was blocked again, if necessary, prior to the addition of primary and secondary antibodies.

3.2.2.9 Western blot quantification

Enhanced chemiluminescence (ECL) was developed and visualized protein signals were quantified using FusionCapt Advance Solo 4. The software displays the emitted light from each detected band in the form of a gray scaled signal. By choosing the appropriate exposure time, the detected protein signals below the saturation limit were further used for quantification purposes. After defining the boundary and subtracting the background intensities from the signal of interest, the quantity of the detected protein was determined by the sum of the pixel intensities in the selected area. For normalization, housekeeping proteins were utilized as a control. Band intensities originating from the protein of interest were divided by the corresponding housekeeping (control) protein band intensities, for example: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for whole lysates or tripeptidyl-peptidase 1 (TPP1) for lysosomal fractions. Subsequently, the amounts of the normalized signals were compared to the control sample intensities which was set to 1. Values greater or less than 1 represent an increase or a decrease in the amount of protein in the treated sample compared to the control sample, respectively.

For a statistical analysis of the selected normalized signals, a two samples t-test (two-tailed distribution, unpaired) was performed, with a p-value <0.05 was defined as significant, a p-value <0.01 as highly significant and a p-value >0.05

as not significant. The standard deviation was determined using the n-1 method and the standard error of the mean was calculated using the following formula:

Standard error = standard deviation/square root of total number of samples