Lanes 1 & 3 & 5 & 7 : Untreated L-929 (mouse connective tissue fibroblast cell line) whole cell lysateLanes 2 & 6 : L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 8 hours whole cell lysateLanes 4 & 8 : L-929 (mouse connective tissue fibroblast cell line) treated with 20 ng/ml TNF-a, 100 nM Smac mimetic and 20 µM z-VAD for 6 hours whole cell lysate
Lysates/proteins at 20 µg per lane.
SecondaryLanes 1-4 : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/20000 dilutionLanes 5-8 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 57 kDaObserved band size: 53 kDa why is the actual band size different from the predicted?
Exposure time: 180 seconds
Blocking and diluting buffer concentration: 5% NFDM/TBTS.
ab181602 GAPDH was used as a loading control at 1/1000000 dilution.
We recommend involving downstream protein p-MLKL as a control to validate the stimulation of p-RIP3.