mTOR (7C10) Rabbit mAb #2983

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

Cells were rinsed twice with ice-cold PBS and lysed in ice-cold lysis buffer (40 mM HEPES pH 7.4, 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, and 0.3% CHAPS, and one tablet of EDTA-free protease inhibitors (#11873580001 from Roche) per 25 mL. The soluble fractions from cell lysates were isolated by centrifugation at 13,000 rpm for 10 min in a microfuge. For immunoprecipitations, primary antibodies were added to the lysates and incubated with rotation for 2 h at 4°C. Twenty microliters of a 50% slurry of protein G-sepharose (GE Healthcare #17-0618-01) or A-sepharose (Repligen #IPA-300) were added and the incubation continued for an additional 2 h. In some experiments, just HA-beads (#PI88836, from Fisher) or Myc-beads (#PI20168, from fisher) were used for the immunoprecipitation. Immunoprecipitates were washed three times with lysis buffer. Immunoprecipitated proteins were denatured by addition of 50 μl of sample buffer and boiling for 5 min, resolved by 10–15% SDS-PAGE, and analyzed via western blot analysis.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for mTOR (7C10) Rabbit mAb #2983 below.

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