Protein biochemical methods

3.2.3.1 Preparation of protein lysates from cell cultures

Primary cortical neurons (plated ~1 · 10⁶ cells) kept in culture for 2 DIV on 10 cm dishes were lysed in RIPA buffer to obtain proteins for subsequent immunoblot analysis. The dish was washed once with cold PBS, 200 μl of RIPA buffer was applied and cells were scraped from the dish with a cell-scraper. The obtained cells were placed into a microfuge tube and left on ice for 20 min to lyse. Cell lysates were centrifuged at 4ºC for 20 min at 13000rpm maximum speed, to pellet the undigested material. The supernatant containing the soluble proteins was transferred into a new tube and kept at -20ºC. The pellet containing the cell debris and unsolubilized material was as well stored at -20ºC.

3.2.3.2. Preparation of a CNS-myelin enriched fraction

Mice were sacrificed by spinal cord dislocation and decapitated. The brains were removed, collected and homogenated in 0.32 M sucrose. This preparation of the CNS-myelin enriched fraction was according to Norton & Poduslo (1973). All centrifugation steps were performed in a SW28 rotor in a Beckman ultracentrifuge. The brains were homogenated with an Ultra-Turrax in 0.32 M sucrose and than layered over a 0.85 M sucrose solution in centrifuging tubes (Beckmann). It was then centrifuged at 23800 rpm for 30 min and the interphase was collected with a Pasteur pipette and put into a clean tube. Then the interphase was washed with dH2O, by filling the tubes with dH2O and centrifuging at 23800 rpm for 15 min. The supernatant was discarded, the pellet was resuspended in dH₂O while vortexing. Then the first osmotic shock took place, by filling the tubes with dH2O, incubating for 10 min and centrifuging at 9500 rpm for 15 min. For the second osmotic shock, the resulting pellet was resuspended in dH₂O and centrifuged again at 9500 rpm for 15 min. The pellet was than resuspended in 0.32 M sucrose and layered over 0.85 M sucrose in a clean centrifuge tube.

This was centrifuged at 23800 rpm for 30 min. The second interphase is collected than, and washed with water, by filling the tubes with dH₂O and centrifuging at 23800 rpm for 15 min.

The resulting pellet was the purified myelin fraction, which was resuspended in TBS with protease inhibitors, aliquoted and kept at -80°C until further use.

3.2.3.3. Protein concentration measurement by Lowry assay

The protein concentrations were assessed in 96-well plates (flat bottom) with the “DC Protein Assay kit” (BioRad) according to the manufacturer’s 'microplate assay' protocol, which is based on the Lowry assay (Lowry et al., 1951). The assay is based on two subsequent reactions between the proteins and a copper in alkaline medium, followed by the reduction of the Folin reagent that produces several reduced species of a characteristic blue colour with a maximum absorbance at 750 nm. The colour development is primarily due to the oxidation of the amino acids tyrosine and tryptophan, and to a lesser extent, cystine, cysteine, and histidine. The absorbance was measured at 650 nm in a microtitre plate reader.

3.2.3.4. SDS-polyacrylamide gel electrophoresis

The separation of proteins was performed using the discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the Mini-Protean-3 system (BioRad), based on the description of Laemmli (1970). The chamber and gels were assembled as described by the manufactures protocol. A separating gel of desired thickness and percentage of acrylamide was layered with isobutanol. Before casting a stacking gel, the residual alcohol was removed and the future interphase between the two gels was rinsed twice with dH₂O. 5 μl of prestained Page Ruler Plus (Fermentas) was loaded on each gel as a molecular weight standard and to monitor the electrophoresis. A maximum of 40 μl sample was loaded in a single pocket and the gels were run with a constant current of 30 mA per gel, and a maximum voltage of 150 V. Gels were subjected to immunoblotting, once the bromophenol blue of the loading dye reached the lower end of the gel.

3.2.3.5. Immunoblot

The proteins were transferred from the SDS-gel onto a PVDF membrane (pore size 0.45 μm, Amersham/Millipore) by electrophoresis with the Invitrogen blotting apparatus (based on the original description of Towbin et al., 1979). PVDF membranes were activated for 30 sec in

methanol and incubated 5 to 15 min in transfer buffer. Blotting paper and blotting pads presoaked in transfer buffer were assembled according to the manufacturer’s protocol, although the blotting buffer differs from the manufacturers recommended. Proteins were transferred at a constant voltage of 38 V and a maximum current of 275 mA, for 1 hour at RT. Afterwards, the immunodetection of the proteins on PVDF membranes was performed.

The membranes were rinsed briefly in TBS and blocked for at least 1 hour at RT in blocking buffer (5 % non-fat dry milk in TBS). Primary antibody diluted in blocking buffer was applied ON at 4°C. After four washes in TBS-T (0.05 % Tween-20 in TBS), HRP-conjugated secondary antibodies were applied for at least 1 hour, followed by four washes with TBS-T.

Membranes were exposed using the Enhanced Chemiluminescence Detection kit (PerkinElmer), according to the manufacturer’s instructions. ECL photographic films (Hyperfilm™, Amersham Biosciences) were used to expose the membranes. The time of exposure varied depending on the signal intensity. To reprobe the same membrane with a second antibody, the membrane was incubated with stripping buffer for 1 hour at 55°C with rigorous shaking. After one wash with TBS-T, the membrane was incubated in blocking buffer for 30 min before probing with the second primary antibody.

3.2.3.6. Silver staining

The silver staining was performed according to the modified protocols of Blum et al. (1987) and Mortz et al. (2001). The proteins were separated by SDS-polyacrylamide gel electrophoresis (section 3.2.3.4.) and than the gel was incubated in different solutions. All steps were performed on a shaking table. The silver staining was performed according to the following incubation steps: fixed in 40 % ethanol, 10 % acetic acid for 1 hour, washed twice with 30 % ethanol for 20 min each, and once with dH₂O for 20 min, sensitized in sodium thiosulfate for 1 min, rinsed trice with dH2O for 20 sec each, impreganated with 0.2 % silver nitrate, 0.05 % formaldehyde solution (37 %) for 20 min and rinsed again trice in dH₂O for 20 sec. Then the development took place, and this step needed to be performed under visual inspection for evaluating when the staining is sufficient. The gel was incubated with 3 % sodium carbonate, 0.05 % formaldehyde solution (37 %) for 2 to 10 min, and once the staining was prominent enough, the gel was rinsed in dH₂O for 20 sec and the staining reaction was stopped by incubation with 5 % acetic acid for 10 min. Finally, the gel was washed trice with dH₂O for 10 min each and could be stored without drying out for long time at RT.