Contents |
water
50 mM Tris, 2 mM EDTA, 1% Nonidet P-40 (NP40), 1% Triton X-100
1. Lyse cells grown in flasks by incubating in L1 or L2 for 30 minutes on ice. Lyse and pool cells grown on 3.5-cm dishes by adding 0.3 mL of L1 or L2, scraping cells using a cell scraper, and transferring the lysate to a second dish andsubsequently to a third dish. Leave for 30 minutes on ice. 2. Transfer lysates into 15-mL plastic tubes (when flasks were used) or 2-mL centrifuge tubes (when 3.5-cm dishes were used). 3. Add 3.2 volumes of MeOH and vortex mix. 4. Add 0.8 volumes of ClCH3 and vortex mix. 5. Add 2.4 volumes water, vortex mix vigorously for 1 minute, and spin for 2 minutes. 6. Remove and discard upper phase. 7. Add 2.4 volumes MeOH, vortex mix, and spin for 5 minutes. 8. Remove supernatant and air-dry pellet. 9. Resuspend in 1× SDS running buffer and boil for 2 minutes. SDS gel electrophoresis and western blotting can then be performed.
The following primary antibodies were used: mouse anti-HA (12CA5 from Roche Moleculer Biochemicals, Mannheim, Germany), polyclonal antibody 514 anti- MAP2 (a gift from C. Sanchez, Centro de Biologia Molecular, Madrid), and polyclonal anti-human APP (kindly provided by C. Haass, München).
1. Fix cells grown on glass coverslips with 4% paraformaldehyde in phosphatebuffered saline (PBS) for 15 minutes. 2. Quench paraformaldehyde with 50 mM ammonium chloride in PBS for 10 minutes. 3. Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes.
4. Then incubate with blocking solution (2% bovine serum albumin [BSA], 2% fetal calf serum [FCS], 0.2% fish skin gelatin) in PBS for 30 minutes. 5. For double labeling, incubate the cells with the primary antibodies diluted in 10% blocking solution in PBS for 1 hour at room temperature or overnight at 4°C. 6. Wash with PBS 3 times. 7. Incubate with appropriate fluorochrome- conjugated secondary antibodies diluted in 10% blocking solution in PBS for 20 to 30 minutes at room temperature. 8. Mount coverslips with mowiol containing 100 mg/mL DABCO (Sigma) as an antifading agent.
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