Anti-Fibronectin antibody [F14] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [F14] - BSA and Azide free (AB206928)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45688).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [F14] - BSA and Azide free (AB206928)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45688).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Fibronectin antibody [F14] - BSA and Azide free (AB206928)

Intracellular 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45688).

• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] - BSA and Azide free (AB206928)

This data was developed using the same antibody clone in a different buffer formulation (ab45688).

Western blot : Rabbit Monoclonal[F14] to Fibronectin ab45688 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 238-268 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in FN1 knockout MCF7 cell line (ab326019). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Fibronectin antibody [F14] (<a href='/en-us/products/primary-antibodies/fibronectin-antibody-f14-ab45688'>ab45688</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human FN1 knockout MCF7 cell line (ab286324) at 20 µg

Lane 2:

Western blot - Human FN1 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-fn1-knockout-mcf7-cell-line-ab326019'>ab326019</a>) at 20 µg

Lane 3:

HepG2 at 20 µg

Lane 4:

PC-3 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 272 kDa

Observed band size: 238-268 kDa

false

• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] - BSA and Azide free (AB206928)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45688).

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.

All lanes:

Western blot - Anti-Fibronectin antibody [F14] (<a href='/en-us/products/primary-antibodies/fibronectin-antibody-f14-ab45688'>ab45688</a>)

Predicted band size: 262 kDa

false

• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] - BSA and Azide free (AB206928)

This data was developed using ab45688, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[F14] to Fibronectin ab45688 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta.

A band was observed at 272 kDa in Wild-type A549 cell lysates with no signal observed at this size in FN1 knockout A549 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.

Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Fibronectin antibody [F14] (<a href='/en-us/products/primary-antibodies/fibronectin-antibody-f14-ab45688'>ab45688</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

FN1 knockout A549 at 20 µg

Lane 2:

Western blot - Anti-Calnexin antibody [CANX/1543] (<a href='/en-us/products/primary-antibodies/calnexin-antibody-canx-1543-ab238078'>ab238078</a>) at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 272 kDa

Observed band size: 272 kDa

false