Atg5 (D5G3) Rabbit mAb

Protocol tips

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	Dilute primary Ab at 1:1000 and incubate 4°C overnight

Protocol tips
Dilute primary Ab at 1:1000 and incubate 4°C overnight

Publication protocol

After the indicated treatment, cells were lysed and the BCA Protein Assay Kit (Sigma, B9643-1L) was used to determine the total protein concentration. Same amounts of protein were subjected to SDS-PAGE with certain concentrations, transferred to a polyvinylidenedifluoride (PVDF) membrane (Millipore, USA) and blocked for 1 hour with 5% nonfat dry milk in Tris buffered saline (TBS-T, 10 mM Tris-HCl [pH 7.5], 100 mM NaCl, and 0.1% Tween 20). Primary antibody incubations were performed at the following dilutions: TBP-2, 1 : 200 (#K0204-3, MBL, USA); LC3B, 1 : 1000 (#L7543, Sigma-Aldrich, USA); P62, 1 : 1000 (#sc-28359, Santa Cruz Biotech, USA); Atg5, 1 : 1000 (#9980, CST, USA); mTOR, 1 : 1000 (#2972, CST, USA); Phospho-mTOR (Ser2448), 1 : 1000 (#2971, CST, USA); GAPDH, 1 : 5000 (#M20006, Abmart, China); Akt (pan), 1 : 1000 (#4691, CST, USA); Phospho-Akt (Ser473), 1 : 2000 (#4060, CST, USA); and β-actin, 1 : 10000 (#A5441, Sigma-Aldrich, USA). All incubations were done at 4°C overnight. Then, the membrane was washed thrice with TBS-T for 30 minutes and followed by respective secondary antibody incubations with goat anti-rabbit/mouse IgG-horseradish peroxidase (Santa Cruz, USA). Immunodetection was performed with the ECL Western Blot Detection System (Millipore, USA). The immunoblot was analyzed with an imaging system (ChemiDoc™ MP System, Bio-Rad, USA). The intensities of the bands were quantified by densitometry using ImageJ (NIH, USA). Values were reported as percentage of the control values ± SEM.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Atg5 (D5G3) Rabbit mAb below.

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