All lanes : Anti-PKC epsilon (phospho S729) antibody (ab88241) at 1 µg/mlLane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 2 : NIH 3T3 treated with Vanadate and PDGF; Whole Cell Lysate Lane 3 : NIH 3T3 Whole Cell Lysate with Immunising Peptide at 1 µg/mlLane 4 : NIH 3T3 treated with Vanadate and PDGF; Whole Cell Lysate with Immunising Peptide at 1 µg/mlLane 5 : NIH 3T3 Whole Cell Lysate with Non- Modified Control Peptide at 1 µg/mlLane 6 : NIH 3T3 treated with Vanadate and PDGF; Whole Cell Lysate with Non- Modified Control Peptide at 1 µg/mlLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab88241 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab88241, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.