Atg5 (D5F5U) Rabbit mAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß mercaptoethanol	- Dilute primary Ab at 1:1000 - Incubate primary antibody for 16 hours

Upstream tips
- Lyse cells in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß mercaptoethanol
Protocol tips
- Dilute primary Ab at 1:1000 - Incubate primary antibody for 16 hours

Publication protocol

Western blot analysis was conducted as previously described [45]. Briefly, A549Pt and A549cisR cells were plated to approximately 70% confluency. Next day, cells were exposed to the designated treatment as described in the “Results” section. At the end of the incubation time, cells were lysed in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß-mercaptoethanol and boiled for approximately 5 minutes. Whole protein lysates were separated on a 10% polyacrylamide gel by SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA), then blocked for 1 hour in 10% milk in TBST (20 mM Tris, 137 mM NaCl, 0.1% Tween 20, pH 7.5) and probed with appropriate primary antibodies for 16 hours. Membranes were then incubated with HRP-conjugated secondary antibodies (1:5000) for one hour prior to signal development using ECL2. Kodak BioMax XAR film was used for chemiluminescence detection.

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Manufacturer protocol

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

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