HOMOGENIZATION OF THE NERVOUS TISSUE IN SDS BUFFER
Homogenize with a Potter at a ratio of 1 gram of tissue plus 10 ml of SDS buffer.
Boil the sample during 10 minutes at 100°C.
Solution of SDS according to Laemmli for 1 litre:
SDS 50g
Tris 7,57 g
EDTA 1,12g
Glycerol 100 ml pH 6,8
H20 1000 ml final
Monoclonal and Polyclonal antibodies
Detection of pathological tau proteins:
AD2 is a protein A-purified monoclonal antibody raised against a crude PHF preparation obtained from an AD brain. It specifically recognizes phosphorylated serines 396 and 404 (numbering according to the longest human brain Tau isoform) in the carboxy-terminal part of Tau (Buée-Scherrer et al., 1996a). AD2 was used at 0.2 µg/ml in Tris-buffered saline containing 0.05 % Tween-20 (TBS-T), as described in (Sergeant et al., 1997).
Detection of normal and pathological tau proteins
Use a phospho-independent tau antibody.
Monodimensional (1-D) and two-dimensional (2-D) gel electrophoresis
1D and 2D-gels
For 1-D and 2-D gel electrophoresis, frontal brain tissue samples were freshly homogenized in Laemmli sample buffer (1/10) containing 5% of SDS. Brain homogenates were aliquoted and frozen at -80°C until used. Each aliquot was used not more than twice. 1-D and 2-D were performed as already described (Sergeant et al., 1997 ; Delacourte et al., 1998a). Tau isoforms distribution was investigated using 10-20 % polyacrylamide gradient gels.
For 1-D and 2-D gel electrophoresis, brain tissue samples were freshly homogenized in Laemmli sample buffer (1/10) containing 5% (wt/wt) SDS and boiled during 5 minutes. The protein concentration was determined using the BCA protein assay (Pierce) and brain tissue homogenates were aliquoted and frozen at -80°C until used. Each aliquot was used not more than twice. A same amount of total brain protein (90 µg) was loaded and, 1-D and 2-D gels were performed as already described (Sergeant et al., 1997 ; Delacourte et al., 1998a). Tau isoforms distribution was investigated using 10-20% polyacrylamide gradient gels.
1D-gels
For mono-dimensional slab gels, 10 µl of each AD brain homogenate was loaded onto 15 well 10-20% sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS-PAGE), according to Laemmli (Laemmli, 1970).
2D-gels
On 2-D gels, isoelectric points (pI) were determined using the carbamylated CPK (MW = 45 kDa ; pI range [4.9-7.1]) of the Carbamylyte™ calibration kit (Pharmacia).
Two-dimensional gel electrophoresis was performed according to a modified O'Farrell's method (O'Farrell, 1975). Isofocusing gels contained 4% (w/v) acrylamide and 2.5% (w/v) of bis-acrylamide as cross-linker, 9.5M urea, 2% (v/v) Triton X-100, 4% (v/v) pH 3-10 PharmalytesTM and 1% (v/v) pH 4-6.5 PharmalytesTM (Pharmacia).
Total brain homogenates (15 µl) were heat treated (100° Celsius / 5 min) and then centrifuged (10000 r.p.m. / 10 min). The supernatant was added with 1 volume of a solution containing 8M urea and 4% (v/v) Triton X-100. Isoelectric focusing was run with a total of 10500 Vh on 12 cm long and 3 mm i.d. capillary tubes. Isoelectric focusing capillary gels were then extruded and equilibrated for 5 mn in an equilibration buffer containing 3% (w/v) SDS, 0.25 M Tris-HCl pH 6,8 and 0.5% (w/v) DTT. The second dimension was performed as for mono-dimensional slab gels, onto 10-20% gradient SDS-PAGE. The two-dimensional gel electrophoresis pH gradient was calibrated using the CarbamylyteTM calibration kit (Pharmacia). Carbamylated creatine phosphokinase (CPK) was always load with each brain sample homogenate, in accordance with the manufacturer's instructions.
TRANSFER:
Proteins resolved on mono and two-dimensional gel electrophoresis were transferred onto nitrocellulose membranes (0.45 µm pore size, Amersham-life Science) for 90 min (current: 0.8 mA per square centimeter) using an LKB Multiphor II Nova Blot. Proteins were stained with Ponceau Red (2 mg/ml) in order to control the quality of resolution of the mono or two-dimensional gels and to visualise the carbamylated CPK.
2D: Membranes were scanned or photographed and isoelectric points (pI) were calculated based upon the theorical pI given by manufacturer for the carbamylated CPK. Blocking was carried out with TBS containing 5% (w/v) dry milk and 0.05% (w/v) Tween-20.
Monoclonal and polyclonal immunological probes were revealed with horseradish peroxidase-labelled sheep anti-mouse and anti-rabbit immunoglobulins (Sigma Immuno Chemicals), respectively. Those antibodies are adsorbed with human serum proteins and do not cross-react with human brain proteins (data not shown). All were detected by chemiluminescence with ECL Western blotting system (Amersham).
Quantitative and statistical analyses
For each brain samples, AD2 immunoblots from monodimensional gels were assessed for densitometric quantitative analyses. Blots were digitized on Prolinea COMPAQ PC engin with a film scan unit SHARP JX-32F6. The scanner was calibrated for optical densities (O.D) using the photographic calibration step tablet no. 3 (KODAK) with the IMAGE MASTER V1.2 1-D Software (Pharmacia). The images of AD2 immunoblots were processed with the IMAGE MASTER V1.2 1-D Software. Each pathological Tau components (Tau 55, 64 and 69) was quantitated without substracting background. This quantitation was performed using a pre-established calibration curve and data were collected in the linear range of O.D. This pathological Tau proteins calibration curve was realized with increasing Laemmli sample volumes (e.g 1µl to 20µl) from the temporal brain region of a well characterized AD patient.
Statistical assessment were performed by calculating the Spearman's rank correlation coeficient or a Mann and Whitney U test.
Western blots for cells
2. IMMUNOEMPREINTE
NT2 cells
Les échantillons cellulaires de NT2, repris dans la solution de Laemmli sont chauffés pendant 10 minutes à 100°C. Les protéines sont ensuite séparées par eléctrophorèse monodimensionnelle sur un gel de polyacrylamide à 15% (37,5 :1) durant 1h30 à 100 volts. Les protéines sont ensuite transférées sur membrane de nitrocellulose 0,45mm (Amersham) par électrotransfert semi-sec à 200 mA pendant 45 minutes. Après coloration au rouge Ponceau (Sigma), les membranes sont saturées dans du tampon TNT (10mM Tris, 150mM NaCl, 0,05% Tween 20) contenant 5% de lait délipidé puis sont incubées avec lĠanticorps primaire pendant 1h30mn. Après trois rinçages, lĠanticorps secondaire anti-souris ou anti-lapin (Sigma) couplé à la peroxydase est dilué au 1/4000 et incubé pendant 45 minutes. Le marquage est révélé par chimioluminescence (ECL, Amersham).
Sigma products if possible.