Anti-Cdc4 / Fbw7 / hSel 10 antibody [FBOX 3a9/1] (ab74054)

  Immunocytochemistry/ Immunofluorescence - Cdc4 / Fbw7 / hSel 10 antibody [FBOX 3a9/1] (ab74054)

ab74054 at 1/100 dilution staining Cdc4 / Fbw7 / hSel 10 in human Hela cells by Immunocytochemistry/ Immunofluorescence. The cells were fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, blocked with 1% bovine serum albumin in PBS for 1 h. A FITC-conjugated anti-mouse secondary antibody was used at 1/100 dilution. The nuclei were stained with 0.5 μg/ml 4′,6-diamidino-2-phenylindole. A mouse IgG was used as isotype control from other source. 

Image from Liu N, et. al., J Biol Chem. 2010 Jun 11;285(24):18858-67. (Fig 3G)

  Western blot - Cdc4 / Fbw7 / hSel 10 antibody [FBOX 3a9/1] (ab74054)

All lanes : Anti-Cdc4 / Fbw7 / hSel 10 antibody [FBOX 3a9/1] (ab74054) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 4 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 80 kDa
Observed band size : 45 kDa (why is the actual band size different from the predicted?)
Additional bands at : 100 kDa,36 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 16 minutes

The 45 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to Cdc4 / Fbw7 / hSel 10.

  Flow Cytometry-Anti-Cdc4 / Fbw7 / hSel 10 antibody [FBOX 3a9/1](ab74054)

Overlay histogram showing HeLa cells stained with ab74054 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab74054, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.