Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: IDH2knockout HAP1 cell lysate (20 µg) Lane 3: K562 cell lysate (20 µg) Lane 4: HepG2 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab94359 observed at 47 kDa. Red - loading control, ab8245, observed at 37 kDa. ab94359 was shown to recognize IDH2 when IDH2 knockout samples were used, along with additional cross-reactive bands. Wild-type and IDH2 knockout samples were subjected to SDS-PAGE. ab94359 at a concentration of 1ug/mL and ab8245 (loading control to GAPDH) diluted to 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.
Western blot - Anti-IDH2 antibody (ab94359)
All lanes : Anti-IDH2 antibody (ab94359) at 1 µg/ml
Lane 1 : Human heart tissue lysate - total protein (ab29431) Lane 2 : Human skeletal muscle tissue lysate - total protein (ab29330) Lane 3 : Human liver tissue lysate - total protein (ab29889) Lane 4 : Human brain tissue lysate - total protein (ab29466) Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 6 : DU 145 (Human prostate carcinoma cell line) Whole Cell Lysate Lane 7 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
The predicted molecular weight of the IDH2 protein is 51-kDa. However, the protein sequence contains a 39 residue transit peptide at the N-terminus. This might explain the migration of the protein at a lower molecular weight than expected.
ICC/IF image of ab94359 stained HepG2 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 (ab94359, 1µg/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. This antibody also gave a positive result in 100% methanol fixed (5 min) MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HepG2 cells at 1µg/ml.