Immunohistochemical staining of paraffin embedded human cervical carcinoma tissue using ab51599 (1/100). Goat anti-rabbit HRP (1/2000) used for secondary staining.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ICC/IF image of ab51599 stained A431 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 ab51599 at 1/100 dilution overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Anti-Cytokeratin 4 antibody [EP1599Y] (ab51599) at 1/1000 dilution + A431 cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 57 kDa
Observed band size: 59 kDa why is the actual band size different from the predicted?

Purified ab51599 at 1/50 dilution (2µg) immunoprecipitating Cytokeratin 4 in HaCaT whole cell lysate.
Lane 1 (input): HaCaT (Human skin keratinocyte) whole cell lysate 10µg
Lane 2 (+): ab51599 + HaCaT whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab51599 in HaCaT whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 59 kDa

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cytokeratin 4 with purified ab51599 at 1/100 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cells without incubation with primary antibody and secondary antibody (Blue).