Cell Signaling Technology
p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695
4695
N/A
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MDA-MB-415 cells were treated with 0.3µM Neratinib and RSL3 for 3 days. The immunoblotting procedure is described below. Lyse cells by adding RIPA (with Proteinase and Phosphatase inhibitors) buffer. Heat 20µg of protein to 95–100°C for 5 min. Load 20µg of protein onto SDS-PAGE gel. Electrotransfer to PVDF membrane (Semi-dry, 20V, 1 hour). Incubate membrane in blocking buffer (1X TBST with 5% nonfat dry milk) for 1 hour at room temperature. Incubate membrane with diluted (1:1,000) primary antibody in 5% BSA, 1X TBS, 0.1% Tween20 at 4°C with gentle shaking, overnight. Incubate membrane with the Rabbit-specific HRP-conjugated secondary antibody in blocking buffer with gentle agitation for 1 hour at room temperature.
Western Blot
Human, MDA-MB-415
Incubate membrane with diluted (1:1,000) primary antibody in 5% BSA, 1X TBS, 0.1% Tween20 at 4°C with gentle shaking, overnight.
1X TBST with 5% nonfat dry milk.
Rabbit-specific HRP-conjugated secondary antibody in blocking buffer with gentle agitation for 1 hour at room temperature.
None
SuperSignal™ West Pico PLUS Chemiluminescent Substrate.
MDA-MB-415 cells were treated with 0.3µM Neratinib and RSL3 for 3 days.Immunoblotting procedure as described below.Lyse cells by adding RIPA (with Proteinase and Phosphatase inhibitors) buffer. Heat 20µg of protein to 95–100°C for 5 min. Load 20µg of protein onto SDS-PAGE gel. Electrotransfer to PVDF membrane (Semi-dry, 20V, 1 hour).Incubate membrane in blocking buffer (1X TBST with 5% nonfat dry milk) for 1 hour at room temperature.Incubate membrane with diluted (1:1,000) primary antibody in 5% BSA, 1X TBS, 0.1% Tween20 at 4°C with gentle shaking, overnight.Incubate membrane with the Rabbit-specific HRP-conjugated secondary antibody in blocking buffer with gentle agitation for 1 hour at room temperature.
Sensitivity.
None.
Found best Total Erk 1/2.