3.6.1 Protein sample preparation
For protein extraction and Western blot analysis, cells were washed with 1xPBS (Table 3) and afterwards destructed in protein lysis buffer (Table 14) using a cell scraper and thoroughly transferred to a 1.5 mL tube. Liquid nitrogen was used to devastate cell membranes for release of whole protein lysates. Insoluble fragments were removed by centrifugation at 10,000 x g for 15 minutes at 4 °C. The protein-containing supernatant was transferred into a fresh tube and kept on ice for measuring the protein concentration for further experiments.
Table 14. Protein lysis buffer, pH 7.8
Substance Quantity
D-Mannitol 0.25 M
Tris-HCl 0.05 M
EDTA 1 mM
EGTA 1 mM
DTT 100 mM 1:100
Triton® X-100 1:100
PhosSTOP™ (Roche, Penzberg, Germany) 1 tablet cOmplete™, EDTA-free Protease Inhibitor Cocktail
(Roche, Penzberg, Germany)
1 tablet
Bidest. H2O Ad 10 mL
3.6.2 BCA assay
The total protein concentration of the sample was determined using the bicinchoninic acid (BCA)-based Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Darmstadt, Germany).
The underlying biochemical reaction includes the reduction of Cu2+ to Cu+ by peptides in a stoichiometric manner. Based on this reaction, bicinchoninic acid reactis with the cuprous ion and the resulting purple colored solution can be measured with the 560±10 nm bandpass filter of FLUOstar OPTIMA reader (BMG Labtech, Ortenberg, Germany). Protein sample concentration was compared to a protein standard curve of bovine serum albumin (BSA).
3.6.3 Polyacrylamide gel electrophoresis (PAGE)
Gel electrophoresis was conducted to separate proteins based on their molecular weight.
Table 15. 1.5 M Tris-HCl, pH 8.8
Substance Quantity
Tris-HCl 23.6 g
HCl q.s. for pH adjustment
Bidest. H2O Ad 100 mL
Table 16. 0.5 M Tris-HCl, pH 6.8
Substance Quantity
Tris-HCl 7.88 g
HCl q.s. for pH adjustment
Bidest. H2O Ad 100 mL
Table 17. 10 % Sodium dodecyl sulfate (SDS)
Substance Quantity
SDS pellets 1 g
Bidest. H2O Ad 10 mL
Table 18. Stacking gel 3.5 %
Substance Quantity
Acrylamide/bisacrylamide 30 % 0.58 mL Sodium dodecyl sulfate solution 10 % 0.05 mL 0.5 M Tris-HCl buffer, pH 6.8 1.2 mL Ammonium persulfate solution 10 % 30 µL Tetramethylethylenediamine (TEMED) 8 µl
Bidest. H2O Ad 5 mL
Table 19. Running gel 10 % / 12.5 %
Substance 10 % 12.5 %
Acrylamide/bisacrylamide 30 % 3.34 mL 4.17 mL Sodium dodecyl sulfate solution 10 % 0.1 mL 0.1 mL
1.5 M Tris-HCl buffer, pH 8.8 2.5 mL 2.5 mL
Ammonium persulfate solution 10 % 62.5 µL 62.5 µL Tetramethylethylenediamine
(TEMED)
12.5 µL 12.5 µL
Bidest. H2O Ad 10 mL Ad 10 mL
3.6.4 Western blot
After SDS-PAGE, proteins were transferred onto a PVDF membrane (Roche Diagnostics, Mannheim, Germany). Incubation with the primary antibody against the protein of interest was conducted over night at 4 °C and at room temperature on the next day for additional two hours. After washing the membrane with 1xTBST, the secondary antibody was applied for two hours and the chemiluminescent signal was visualized with Chemidoc software (Bio-Rad, Germany). Tubulin was frequently used as a loading control for total protein lysate.
Table 20. 5x SDS sample buffer; 10 mL
Substance Quantity
1.5 MTris-HCl, pH 6.8 2 mL
Glycerol 5 mL
SDS pellets 1 g
β-Mercaptoethanol 2.5 mL
1 % Bromophenol blue 0.5 mL
Table 21. 10x SDS-PAGE buffer (Running buffer)
Substance Quantity
Tris base 30 g
SDS pellets 10 g
Glycine 144 g
Bidest. H2O Ad 1 L
The 10x SDS-PAGE buffer was diluted 1:10 with aqua bidest prior to use.
Table 22. 10x Transfer buffer, pH 8.3
Substance Quantity
Tris base 30 g
Glycine 144 g
HCl q.s. for pH adjustment
Bidest. H2O Ad 1 L
To dilute the 10x transfer buffer, 70 % aqua bidest and 20 % methanol was added.
Table 23. 10x TBS, pH 7.5
Substance Quantity
NaCl 292 g
Tris base 24.2 g
HCl q.s. for pH adjustment
Bidest. H2O Ad 1 l
Table 24. 1x TBST
Substance Quantity
10x TBS 100 mL
Tween 20 0.5 mL
Bidest. H2O Ad 1 L
Table 25. 5% Blocking milk
Substance Quantity
Skim milk powder 5 g
1xTBST 100 mL
3.6.5 Antibodies Table 26. Primary antibodies
Antibody Dilution MW [kDa]
Host Company Purpose
Cofilin1 1:1000 16 rabbit Cell Signaling and Witke-Laboratory [12]
WB
phospho-Cofilin1 (Ser3)
1:1000 16 rabbit Cell Signaling WB
PGC1α 1:1000 92 rabbit Rockland WB
DRP1 1:1000 83 mouse BD Bioscience WB
α-Tubulin 1:10 000 54 mouse Sigma-Aldrich WB
MCU 1:1000 30 rabbit Cell Signaling WB
Fis1 1:500 17 rabbit Enzo Life Science WB
p62 1:1000 62 rabbit Cell Signaling WB
Actin C4 clone 1:2000 42 mouse MP Biomedicals WB
LC3B 1:1000 14-16 rabbit Cell Signaling WB
Mfn2 1:1000 80 rabbit Cell Signaling WB
TFAM 1:1000 28 rabbit Abcam WB
Nrf-1 1:1000 68 rabbit Cell Signaling WB
INF2 1:1000
180-200
rabbit Proteintech WB
GFP 1:500 27 goat Rockland WB
H2AX 1:2000 14 rabbit Novus Biologicals WB
MAP2 1:100 mouse Abcam
Immunocyto-chemistry
GFAP 1:100 mouse Cell Signaling
Immunocyto-chemistry phospho-DRP1
(Ser616)
1:1000 83 rabbit Cell Signaling WB
phospho-DRP1 (Ser637)
1:1000 83 rabbit Cell Signaling WB
Tim23 1:1000 23 mouse BD Transduction
laboratories
WB
Table 27. Secondary antibodies
Antibody Dilution Host Company Purpose
Peroxidase-labeled anti-goat IgG (H+L)
1:2500 horse Vector laboratories WB
Peroxidase-labeled anti-mouse IgG (H+L)
1:3000 horse Vector laboratories WB
Peroxidase-labeled anti-rabbit IgG (H+L)
1:2500 goat Vector laboratories WB
Anti-mouse IgG (H+L) Dylight™
650
1:200 goat Thermo Scientific Immunocytochemistry
Anti-rabbit IgG (H+L) Dylight™
488
1:200 goat Thermo Scientific Immunocytochemistry
3.6.6 Immunoprecipitation
Immunoprecipitation was performed to investigate protein-protein-interactions. To achieve sufficient pulldown quantity of the protein of interest, Dynabeads™ Protein A for Immunoprecipitation (Invitrogen, Karlsruhe, Germany) were used according to the manufacturer’s protocol. In brief, magnetic Dynabeads were coupled to 2.5 µg of the primary antibody diluted in 200 µL 1xPBS/0.1 % Tween20 by incubation on a rotator at room temperature for 30 minutes. Afterwards, the Dynabead-antibody complex was washed once with PBS/Tween20 by restraining the tube on a magnet following resuspension of the magnetic beads in 6 mg Crosslinker BS³ (Thermo Fisher Scientific, Darmstadt, Germany) diluted in 250 mL aqua bidest. 750 µL PBS/Tween20 was added prior to rotation for
30 minutes at room temperature to fix the crosslinked beads with the antibody.
Subsequently, the fixed complex was washed one with PBS/Tween20 and 1 mL of 30 mM Tris-HCl, pH 7.4 solution was added for 15 minutes. Then, 250 µg of protein sample diluted in 600 µL protein lysis buffer (Table 14) was added to the Dynabead-antibody-complex and incubated over night at 4 °C on a rotator. On the next day, the complex was washed three times with PBS and finally the proteins were eluted by boiling the sample with 50 µL of 2.5x SDS sample buffer (Table 20) for 10 minutes at 95 °C. The antigens were visualized by primary and secondary antibody detection after SDS-PAGE and wet blot procedure via chemiluminescence with Chemidoc software (Bio-Rad, Germany).
3.6.7 Mitochondrial isolation
Table 28. Mitochondrial isolation buffer, pH 7.2, 4 °C [147]
Mitochondrial isolation of freshly dissected cortical or hippocampal brain tissue (~50 mg) was performed as previously described [147]. Briefly, the tissue was charged with 2 mL mitochondrial isolation buffer (Table 28) and roughly homogenized with a 20G Neoject needle (Dispomed, Gelnhausen, Germany) and then sieved through a 100 µm nylon cell strainer (Corning Incorporated, Corning, NY, USA). To homogenize the tissue efficiently and extract mitochondria from the cell structure thoroughly, a cell homogenizer (Isobiotec, Heidelberg, Germany) with appropriate 1 mL gas-tight syringes (Supelco, Munich, Germany) were used and restrained into a device to ensure constant rate of 700 µL/min. The cell homogenizer contains a spherical tungsten carbide ball with a clearance of 10 µm to decompose the tissue but simultaneously maintain the integrity of mitochondria. The cell homogenate was transferred into 1.5 mL tubes and centrifuged at 800 x g for 10 min at 4 °C to remove cell debris. Afterwards, the supernatant was transferred into a fresh tube and centrifuged at 10 000 x g, again for 10 min at 4°C (Heraeus™ Fresco™ 17 Mikrozentrifuge;
Thermo Fisher Scientific, Darmstadt, Germany). The resulting pellet consists of a crude
Substance Final concentration Weight
Sucrose 300 mM 102.8 g
TES 5 mM 1.146 g
EGTA 200 µM 76 mg
Bidest. H20 add 1 L
KOH q.s. for pH adjustment
mitochondrial fraction, which was finally resuspended in MSHE-BSA buffer (Table 29). All steps were performed on ice or at 4 °C. Pierce™ BCA Kit was used to determine the protein amount of the mitochondrial fraction.
Table 29. MSHE-BSA buffer, pH 7.2
Substance Final concentration Weight
Sucrose 70 mM 24 g
Mannitol 210 mM 38.26 g
HEPES 5 mM 1.19 g
EGTA 1 mM 0.38 g
BSA (freshly added) 0.5 % (w/v) 5 g
Bidest. H2O ad 1 L
KOH q.s. for pH adjustment ad 1 L H2O
Table 30. 1xMitochondrial Assay Solution (1xMAS), pH 7.2
Substance Final concentration Weight
Sucrose 70 mM 24 g
Mannitol 220 mM 40.08 g
KH2PO4 10 mM 1.36 g
MgCl2 5 mM 0.476 g
HEPES 2 mM 0.476 g
EGTA 1 mM 0.380 g
BSA (freshly added) 0.20 % (w/v) 2 g
Bidest. H2O ad 1 L
KOH q.s. for pH adjustment