Atg5 (D5F5U) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF) containing complete protease inhibitor cocktail	Dilute primary antibody at 1:1,000 dilutionand incubate at 25°C for 2 h

Upstream tips
Lyse cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF) containing complete protease inhibitor cocktail
Protocol tips
Dilute primary antibody at 1:1,000 dilutionand incubate at 25°C for 2 h

Publication protocol

Cells were washed with PBS and then incubated with lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF) containing complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) for 1 h at 4°C. The resulting supernatants were collected for protein ßquantitation using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Crude proteins (20 µg) were subjected to 12.5% SDS-PAGE, transferred to a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA) and then incubated with 5% w/v skimmed milk/PBS at room temperature for 1 h to block nonspecific binding. The blocked membrane was then incubated with primary antibodies (1:1,000 dilution) at 25°C for 2 h, followed by incubation with peroxidase-conjugated anti-IgG secondary antibodies (1:2,000 dilution) at 25°C for 1 h. Detailed information for antibodies were described in the reagents. The blocked membrane without incubation with primary antibodies was also incubated with secondary antibodies for specificity test, and a nonsignificant signal was detected as the control. Signal development was performed using an enhanced chemiluminescence reagent (EMD Millipore). Luminescent signals were acquired and quantified using an image analysis system (LAS-4000 with Image Reader LAS-4000 version 2.1, Fuji, Tokyo, Japan). GAPDH was used as an internal control.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Atg5 (D5F5U) Rabbit mAb below.

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