MATERIAL AND METHODS
54 washing buffer B. When the radioactivity signal became undetectable again, the membrane was wrapped in plastic wrap and exposed and developed using a phosphoimager setup (Fujix, BAS 2000, Raytest Isotopenmeßgeräte).
3.3.21 Long-range-PCR
Long-range PCR was established to confirm the correct insertion of the targeting vector at the 3’
or at the 5’ end of the homology arms of the targeted genes. The volumes of the ingredients are summarized in Table 14. A reaction volume of 25 µL was used for each reaction.
Table 14: Reaction mixture for a long-range PCR.
ingredients volume in µL
10 x Buffer 10
10 mM dNTPs 2
Primer A 2
Primer B 2
Template-DNA 8
H2O 75,5
Taq polymerase 0,5
The following primer combinations (for sequence information see Table 8) were used for the long-range PCR:
Table 15: Primer combinations and amplicon sizes for the long-range PCRs used during the study.
Primer A Primer B Amplicon size
Spastin 3’ 23 61 5043 bp
Katanin 5’ 87 123 6226 bp
Katanin 3’ 86 122 3762 bp
The cycling conditions for the individual long-range PCRs are summarized below:
Table 16: cycling conditions used for long-range PCR in this study. The individual annealing temperatures and extension times are highlighted in subcolumns.
Cycling conditions
Temperature in °C Time in min repeats
Spastin 3’ Katanin 5’ Katanin 3’ Spastin 3’ Katanin 5’ Katanin 3’
Initial denaturation
95 0.5 -
Denaturation 95 0.5 40
Annealing 60 57 57 0.5
Extension 70 4 5 3
Final extension 70 10 -
Cooling 4 ∞ -
MATERIAL AND METHODS
harvested cells were incubated for 30 min on ice under occasional vortexing and subsequently centrifuged at 1,000 g. The supernatant containing the soluble protein fraction was either used for immediate protein analysis or was directly frozen using liquid nitrogen and kept frozen at -80 °C.
After cultivation and transfection in culture dishes, the lysis of cells (primary neurons / HeLa cells) was performed on ice. The cells were washed with ice-cold 1x PBS. Then, an appropriate amount of cell lysis buffer was added to the cells, which were subsequently scraped off the dishes using a cell scraper. After optional sonication (duty cycle = 20 %, output control = 0.2) cells were rotated for 20 min at 4 °C to complete cell lysis. Cell debris was removed by centrifugation for 5 min at 4
°C at 10,000 g.
3.4.2 Preparation of cellular fractions from brain lysates by differential centrifugation In order to obtain different cellular fractions, the differential centrifugation method was used (Table 17).
Table 17: Overview of the cell fractionation by differential centrifugation.
Step Centrifugation speed
Time Rotor used Pellet Pellet components
1 1,000 x g 10 min JA 20 or JS
13.1 Beckman
P1 cell-membrane debris, mitochondria and nuclei 2 10,000 x g 10 min JA 20 or JS
13.1 Beckman
P2 small cell-membrane debris, mitochondria, large vesicular cell organelles (like ER) and the plasma membrane 3 100,000 x g 60 min SW40 Ti Ultra P3 Golgi, transport vesicles,
microsomes etc.
4 400,000 x g 60 min TLA 100, Ultra P4 small vesicles and protein complexes
Initially, eight postnatal day 11 (P11) mice were sacrificed by decapitation. The brains were isolated, and put into 7.5 mL tubes supplemented with IMAC buffer (20 mM HEPES, 100 mM potassium acetate, 40 mM KCl, 5 mM EGTA, 5 mM MgCl2, pH 7.2 freshly supplemented with protease inhibitor (Roche cOmplete), 2 mM Mg-ATP, 5 mM DTT and 1 mM PMSF). The brains were homogenized with 8 strokes at 900 rpm. The lysates were centrifuged for 10 min at 1,000 x g (JA 20 or JS 13.1, Beckman), and the supernatant S1 was transferred into a new tube. The pellet P1 containing cell-membrane debris, mitochondria and nuclei was put aside.
The supernatant S1 was then centrifuged for 10 min at 10,000 x g (JA 20 or JS 13.1 Beckman), and the resulting supernatant S2 was transferred into a new tube. The pellet P2 consisting of small cell-membrane debris, mitochondria, large vesicular cell organelles (like ER) and the plasma membrane was put aside.
The supernatant S2 was then centrifuged for 1 h at 100,000 x g (SW40 Ti Ultra), and the supernatant (S3) was transferred to a new tube. The pellet P3 consisting of Golgi, transport vesicles, microsomes was put aside for further usage.
MATERIAL AND METHODS
56 After the centrifugation of the supernatant S3 for 1 h at 400,000 x g (TLA 100, Ultra), the supernatant S4 was transferred into a new tube. The pellet P4 contained small vesicles and protein complexes.
After the differential centrifugation, each pellet was resuspended in 2 mL IMAC buffer.
3.4.3 Preparation of synaptosomal fractions
Synaptosomes are artificial fusions of neuronal pre- and postsynaptic membranes. They form in vitro after homogenization of neuronal tissue following several centrifugation steps. This method allows to specifically enrich synaptic proteins for further analysis (Whittaker, Michaelson et al.
1964).
After sacrificing, mice were decapitated and their whole brains were transferred into 4 mL ice-cold sucrose buffer A each. For homogenization, 12 strokes at a rotation speed of 900 rpm were applied in a 15 mL potter S (B Braun). After 6 strokes, 4 mL of sucrose buffer A were added.
Subsequently, the homogenate was centrifuged at 4 °C for 10 min and 1,400 x g ((12 mL centrifuge tubes (Greiner Bio-one); JA20 rotor). The pellet was resuspended in 4 mL of sucrose buffer A by applying 8 strokes at 900 rpm and centrifuged again for 10 min at 4 °C and 700 x g.
The supernatants from both centrifugation steps were then pooled and centrifuged at 13,800 x g for 10 min.
The pellet was resuspended in 2 mL sucrose buffer B and applied on a sucrose gradient (3 mL of 1.2 M, 1.0 M and 0.85 M sucrose and with an increasing sucrose density from top to bottom) and centrifuged for 2 h at 82,500 x g (SW40Ti Rotor). The synaptosome-enriched interface between 1.2 M and 1 M as well as the other interfaces were then collected and used for further analyses (see Figure 16).
Figure 16: Exemplary picture of synaptosomal fractionation. A sucrose density gradient of 1.2 M, 1.0 M and 0.85 M sucrose was used. The separated fractions were used for further analysis.
3.4.4 Co-immunoprecipitation
For co-immunoprecipitation, 30 µL slurry protein G coupled dynabeads (Dynal) were washed three times with 1 mL IP buffer using the Dynal magnetic particle concentrator (Dynal). Then 5 µg of antibody were coupled to the washed beads in 800 µL IP buffer overnight using an overhead shaker at 4 °C. Three washing steps with IP buffer were used to ensure the removal of unbound antibodies. Subsequently, the dynal beads with immobilized antibodies were incubated with cellular fractions obtained from differential centrifugation at 4 °C overnight using an overhead shaker. Then the dynal beads were washed again three times using IP buffer supplemented with 0.5 % Triton-X-100 and cOmplete protease inhibitor cocktail (Roche) for 5-10 min each time. The
MATERIAL AND METHODS
dynabeads were then dissolved in 30-45 µL H2O and supplemented with 10-15 µL 4 x SDS-sample buffer and boiled for 10 min at 95° to elute bound proteins. Finally, the SDS-samples were separated by SDS-PAGE followed by a Western Blotting analysis.
3.4.5 Determination of protein concentration (BCA Assay)
To normalize samples to total protein levels the BCA method (BCA Protein Assay kit from Thermo Scientific) was used. A standard curve was determined by using a BSA solution with a known concentration. The appropriate amount of BSA and 1 – 2 µL from each cell lysate was filled up to 25 µL with distilled water. Ice-cold BCA Protein Assay Reagent A was mixed with BCA Protein Assay Reagent B (ratio 50:1) and 200 µL of this solution was added on ice to each protein sample. The reactions run for 30 min at 37 °C. Afterward, the samples were allowed to cool down to room temperature for 5 min before the OD was measured at 562 nm with a SLT Rainbow Scanner (SLT Labinstruments). The concentration of each protein sample was determined with help of the calculated standard curve and adjusted to 1 µg/µL with protein sample buffer and lysis buffer. Samples were stored at -20 °C until further usage.
3.4.6 SDS-PAGE
Proteins were separated by their molecular weight by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with the Mini-Protean III system (Biorad). In order to prepare the gels, a 40 % (w/v) acrylamide-/bis-acrylamide solution (Carl Roth) was used (Sambrook, Fritsch et al. 1989). Depending on the target molecular weight, different final percentages of the acrylamide-/bis-acrylamide in the gel were selected. In the following table an exemplary composition of a typical SDS-PA gel is presented.
Table 18: Typical composition of SDS polyacrylamide gels.
Components Stacking gel 7.5 % Separating gel 15 %
Acrylamide/Bisacrylamide (37.5:1) 1.25 mL 6 mL
0.5 M Tris-HCl, pH 6.8 1.25 mL -
1 M Tris-HCl pH 8.8 - 3 mL
10 % SDS (w/v) 50 µL 120 µL
H2O (Millipore) 2.45 mL 2.75 mL
10 % APS (w/v) 50 µL 120 µL
TEMED 5 µL 6 µL
After mixing the protein solutions with 4 x SDS-loading buffer and boiling at 95 °C for 5 min, the samples were loaded onto the gel to separate the proteins. The electrophoresis was buffered in 1 x SDS-Running buffer. For the initial separation in the stacking gel, a voltage of 90 V was applied followed by a voltage of 120 V in the separating gel. The Precision Plus Protein
MATERIAL AND METHODS
58 Standards Dual Color (Biorad) molecular weight marker was used to identify the region of the target molecular weight. After electrophoresis, the gel was either used for Coomassie staining or for Western Blotting.
3.4.7 Western Blotting
In order to detect proteins following separation by SDS-PAGE with specific antibodies, they had to be transferred from the SDS-PA-gel onto a PVDF-membrane (Hybond-P, pore size 0.45 µm, Amersham). The Wet-Blot Mini Trans-Blot Cell System (Biorad) was used for the transfer at 100 V for 75 min.
The PVDF-membrane was activated in Methanol for 15 to 30 s and then washed with transfer buffer. Then, the protein-containing gel was placed onto it. This so-called sandwich was then wrapped from both sides with two layers of filter paper soaked with transfer buffer each and one sponge (Biorad). The protein containing gel was facing the cathode and the PVDF- membrane was facing the anode so that the negatively charged proteins were transferred in the direction of the anode and fixed on the PVDF-membrane.
To identify the immobilized proteins on a PVDF membrane using specific antibodies, the membrane was incubated for 60 min in a 1 % BSA solution in TBST. This step was necessary to prevent nonspecific binding of the antibodies to the protein-free sites on the PVDF-membrane.
Next, the PVDF-membrane was incubated with a specific primary antibody diluted (Table 4) in the same 1 % BSA solution in TBST for 2 h at room temperature or at 4 °C overnight. Then, the membrane was washed three times with TBST for 10 min each time and then incubated with an HRP-conjugated secondary antibody diluted in 1 % BSA in TBST for 30 min at room temperature.
After this incubation, the membrane was washed again three times using TBST and then used for the chemodetection of the specific protein bands using the Immobilon Western HRP substrate (Millipore) following the manufacturer’s instructions.
The documentation of the results was done using the chemiluminescence reader Intas ChemoCam (Intas).
For the specific detection of additional protein bands from the same PVDF-membrane, previously used primary antibodies had to be removed. For that purpose, the membrane was incubated in stripping buffer for 30 min at room temperature and shaking. After two following washing steps using TBST, the membrane was again incubated with the HRP-conjugated secondary antibody to exclude remaining bound primary antibody. In case no remaining signal could be detected, the immunodetection protocol was used again starting with the blocking of the membrane with 1 % BSA in TBST.
MATERIAL AND METHODS