All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/ml

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : FOXO3 knockout HEK-293 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 71 kDa
Observed band size: 82 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab23683 observed at 71 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab23683 was shown to recognize FOXO3A in wild-type HEK-293 cells as signal was lost at the expected MW in FOXO3 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and FOXO3 knockout samples were subjected to SDS-PAGE. Ab23683 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/ml

Lane 1 : Jurkat whole cell lysate (ab7899)
Lane 2 : Jurkat whole cell lysate (ab7899) with Human FOXO3A peptide (ab24031) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Predicted band size: 71 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?
Additional bands at: 50 kDa (possible cleavage fragment), 50 kDa (possible cross reactivity)



ab23683 detects a band at approximately 90 kDa that corresponds in size to that seen for FOXO3A. This protein has been shown to migrate at a size larger than the predicted molecular weight (see Yin et al., J. Biol. Chem., Vol. 279, Issue 44, 45721-45727). It also detects a band at 50 kDa which could be a cleavage fragment. Both bands are partially blocked by the addition of the immunizing peptide.

All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/ml

Lane 1 : Heart (Mouse) Tissue Lysate - normal tissue
Lane 2 : Mouse skeletal muscle tissue lysate - total protein (ab29711)
Lane 3 : Heart (Rat) Tissue Lysate - normal tissue

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 71 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?
Additional bands at: 16 kDa, 27 kDa. We are unsure as to the identity of these extra bands.

ab23683 detects a band at approximately 90 kDa that corresponds in size to that seen for FOXO3A. This protein has been shown to migrate at a size larger than the predicted molecular weight (see Yin et al., J. Biol. Chem., Vol. 279, Issue 44, 45721-45727).

ICC/IF image of ab23683 stained MCF7 cells. The cells were 4% PFA fixed (10 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 (ab23683, 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. This antibody also gave a positive result in 4% HepG2 fixed (10 min) HepG2 cells at 5µg/ml.

IHC image of FOXO3A staining in Human normal lung formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab23683, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.