Anti-Fibronectin 抗体 [F14]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [F14] (AB45688)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Fibronectin antibody [F14] (AB45688)

Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Fibronectin with purified ab45688 at 1/150 dilution (10μg/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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody [F14] (AB45688)

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.

• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] (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 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] (ab45688) 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='/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

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• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] (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. 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] (ab45688) 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 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 kDa,268 kDa

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• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] (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] (ab45688)

Predicted band size: 262 kDa

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• WB

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Western blot - Anti-Fibronectin antibody [F14] (AB45688)

All lanes:

Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/5000 dilution

All lanes:

human serum

Predicted band size: 262 kDa

Observed band size: 263 kDa

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• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] (AB45688)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/10000 dilution

All lanes:

Human serum lysates at 15 µg

Secondary

All lanes:

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

Predicted band size: 262 kDa

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• WB

Lab

Western blot - Anti-Fibronectin antibody [F14] (AB45688)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Fibronectin antibody [F14] (ab45688) at 1/10000 dilution

Lane 1:

Mouse serum lysates at 15 µg

Lane 2:

Rat serum lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 262 kDa

Observed band size: 263 kDa

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• WB

CiteAb

Western blot - Anti-Fibronectin antibody [F14] (AB45688)

Fibronectin western blot using anti-Fibronectin antibody [F14] ab45688. Publication image and figure legend from Liu, B., Ding, Y., et al., 2020, BMC Urol, PubMed 33287818.

ab45688 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab45688 please see the product overview.

FN1 expression in EAE-induced fibrotic bladder tissue. a The expression of FN1 in bladder fibrosis with different scores was analyzed by immunohistochemistry. b The expression of FN1 in bladder fibrosis with different scores was analyzed by Western blot. c The expression of FN1 in bladder fibrosis with different scores was analyzed by RT-qPCR. The findings were from three separate experiments. The data are expressed as the mean ± SD. NC, normal control group; CS, clinical symptoms; FN1, fibronectin 1. nsP > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. Scale bar = 200 μm

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