BECN1 Antibody (H-300)

Protocol tips

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Lyse cells inradioimmunoprecipitation 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]	Dilute primary Ab at 1:200

Upstream tips
Lyse cells inradioimmunoprecipitation 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]
Protocol tips
Dilute primary Ab at 1:200

Publication protocol

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)

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