All lanes : Anti-Cdc4 / Fbw7 / hSel 10 antibody (ab12292) at 1/500 dilution

Lane 1 : HeLa nuclear lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : A6431 whole cell lysate
Lane 4 : Jurkat whole cell lysate
Lane 5 : HEK293 whole cell lysate
Lane 6 : HeLa nuclear lysate with Human Cdc4 / Fbw7 / hSel 10 peptide (ab12311) at 1 µg/ml
Lane 7 : HeLa whole cell lysate with Human Cdc4 / Fbw7 / hSel 10 peptide (ab12311) at 1 µg/ml
Lane 8 : A431 whole cell lysate with Human Cdc4 / Fbw7 / hSel 10 peptide (ab12311) at 1 µg/ml
Lane 9 : Jurkat whole cell lysate with Human Cdc4 / Fbw7 / hSel 10 peptide (ab12311) at 1 µg/ml
Lane 10 : HEK293 whole cell lysate with Human Cdc4 / Fbw7 / hSel 10 peptide (ab12311) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Rabbit Anti-Goat IgG H&L (HRP) (ab6741) at 1/5000 dilution

Predicted band size: 80 kDa


Exposure time: 10 seconds

Additional bands seen at 45, 55 and 150 kDa. We are uncertain as to the identity of these bands.

ab12292 (1/2000) detecting Cdc4 in Hela cells (green). Cells were fixed in methanol, permeabilised with 0.5% Triton X100/ PBS and counterstained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.

See Abreview

ICC/IF image of ab12292 stained HeLa 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 (ab12292, 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% PFA fixed (10 min) HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, HepG2 and MCF7 cells at 5µg/ml.

All lanes : Anti-Cdc4 / Fbw7 / hSel 10 antibody (ab12292) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Nuclear Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 110,37 kDa why is the actual band size different from the predicted?


Exposure time: 5 minutes