Notch 1 Antibody (C-20)

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	Dilutions:immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml of cell lysate)], immunofluorescence (starting dilution 1:50, dilution range 1:50-1:500), For immunoprecipitation, incubate cells for overnight after Ab treatment. For Immunofluorescence staining, incubate cells for 1 hr

Protocol tips
Dilutions:immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml of cell lysate)], immunofluorescence (starting dilution 1:50, dilution range 1:50-1:500), For immunoprecipitation, incubate cells for overnight after Ab treatment. For Immunofluorescence staining, incubate cells for 1 hr

Publication protocol

Immunoblot analysis
Immunoblotting analyses were carried out as described previously [19]. The cultured cells were lysed in 1 ml of radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM DTT, and 2 μg/ml each of leupeptin and aprotinin] for 30 min at 4°C. The cell lysates were then subjected to 15 min of centrifugation at 12,000 g at 4°C. The resultant soluble fraction was boiled in Laemmli buffer [63 mM Tris-HCl (pH 6.8), 10% Glycerol, 2% SDS, 5% β-mercaptoethanol, 0.0025% Bromophenol blue] and subjected to SDS-PAGE. After gel electrophoresis, the separated proteins were transferred via electroblotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with phosphate-buffered saline solution [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4] containing 0.1% Tween 20 and 5% nonfat milk. The blotted proteins were then probed with anti-Notch1, anti-β-actin, anti-p62, anti-Fbw7 (Cdc4), anti-GFP, anti-LC3, anti-Myc, anti-HA, or anti-Flag, followed by incubation with anti-mouse horseradish peroxidase-conjugated secondary antibodies and anti-rabbit horseradish peroxidase-conjugated secondary antibodies. The blots were then developed using enhanced chemiluminescence (ECL).

Co-immunoprecipitation assay
The cells were lysed in 1 ml of lysis buffer [20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 25 mM NaF, 1% Triton X-100, 2 μg/ml each of leupeptin and aprotinin] for 30 min at 4°C. After centrifugation at 12,000 g for 15 min, the supernatants were subjected to immunoprecipitation with specific antibodies. After overnight incubation, Protein A agarose beads (GE healthcare) were added, and the samples were incubated for 1 h at 4°C on the rotator. The resulting immunoprecipitates were washed three times with PBS, and the immune complexes were eluted with Laemmli buffer for 5 min at 95°C and visualized by immunoblotting.

Immunofluorescence staining
HEK293 cells were seeded onto coverslips in 6-well culture plates. Cells were washed with PBS and fixed by 4% paraformaldehyde for 15 min at room temperature. Cells were then washed three times with PBS and permeabilized with 1% BSA in PBS solution with 0.1% Triton X-100 at 4°C for 5 min. Cells were blocked with 1% BSA in PBS for 30 min. Thereafter, cells were incubated with specified primary and secondary antibodies for 1 h respectively with three washes in between. The primary antibodies against p62 and Notch1-IC V1744 were diluted in 1% BSA/PBS. The fluorescent-conjugated secondary antibodies were anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 488, and anti-rabbit Alexa Fluor 532 and were diluted in 1% BSA/PBS. The nuclei were stained with ToPro3. Cells were mounted with anti-fade solution and visualized using confocal microscopy with LAS AF software (Leica). Scale bars represent 25 μm as indicated.

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