Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Fibronectin knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab45688 observed at 262 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab45688 was shown to react with Fibronectin in wild-type HAP1 cells as signal was lost in Fibronectin knockout cells. Wild-type and Fibronectin knockout samples were subjected to SDS-PAGE. Ab45688 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/2000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with purified ab45688 at 1:150 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with Purified ab45688 at 1:500 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Anti-Fibronectin antibody [F14] (ab45688) at 1/10000 dilution (purified) + Human serum lysates at 15 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 263 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

All lanes : Anti-Fibronectin antibody [F14] (ab45688) at 1/10000 dilution (purified)

Lane 1 : Mouse serum lysates
Lane 2 : Rat serum lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 263 kDa
Observed band size: 263 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Anti-Fibronectin antibody [F14] (ab45688) at 1/5000 dilution (unpurified) + human serum

Predicted band size: 263 kDa
Observed band size: 263 kDa

ICC/IF image of unpurified ab45688 stained HepG2 (Human liver hepatocellular carcinoma cell line) cells. The cells were fixed in 4% formaldehyde (10 minutes) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab45688 at 1/250 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 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.