Anti-Fibronectin antibody [10/Fibronectin]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling Fibronectin with ab281575 at 1/2000 (0.551 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Positive staining in stromal cells of human colon cancer.The section was incubated with ab281575 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^™ RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) .

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Fibronectin with ab281575 at 1/2000 (0.551 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) . Positive staining in stromal cells of human testis.The section was incubated with ab281575 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) .

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling Fibronectin with ab281575 at 1/2000 (0.551 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) . Positive staining in stromal cells of human ovarian cancer.The section was incubated with ab281575 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) .

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• WB

Lab

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Western blot : Mouse Monoclonal[10/Fibronectin] to Fibronectin ab281575 staining at 1/500 dilution, shown in green; Rabbit anti-ACTN4 (ab108198) 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-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (ab281575) at 1/500 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-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

Predicted band size: 272 kDa

Observed band size: 238-268 kDa

false

• WB

Lab

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Western blot : Mouse Monoclonal[10/Fibronectin] to Fibronectin ab281575 staining at 1/500 dilution, shown in green; Rabbit anti-ACTN4 (ab108198) 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 [10/Fibronectin] (ab281575) at 1/500 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

FN1 knockout A549

Lane 2:

Western blot - Human FN1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-fn1-knockout-a549-cell-line-ab286514'>ab286514</a>) at 20 µg

Lane 3:

HepG2 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

• WB

Lab

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Samples are non-boiled as boiling may cause protein aggregates.

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 28367057).

Exposure time : lane 1 : 3 secondslane 2 : 37 seconds

All lanes:

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (ab281575) at 1/1000 dilution

Lane 1:

Human plasma lysate at 20 µg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate at 40 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 262 kDa

Observed band size: 285 kDa

false

• WB

Lab

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (AB281575)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 28367057).

Lanes 1-3 : Merged signal (red and green). Green - ab281575 observed at 285kDa. Red - loading control ab52901(Rabbit monoclonal [EP1331Y] to beta Tubulin) observed at 50 kDa.

Lanes 1-2 : ab281575 Anti-Fibronectin antibody was shown to react with Fibronectin in HAP1 cells in Western blot. Loss of signal was observed when Fibronectin knockout sample was used. Wild-type and Fibronectin knockout samples were subjected to SDS-PAGE.

ab281575 and Anti-beta Tubulin antibody [EP1331Y] - Loading Control (ab52901) were incubated at 4℃ overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) preadsorbed (ab216772) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Fibronectin antibody [10/Fibronectin] (ab281575) at 1/500 dilution

Lane 1:

Wild type HAP1(human chronic myelogenous leukemia near-haploid cell line), whole cell lysate at 20 µg

Lane 2:

Fibronectin knockout HAP1(human chronic myelogenous leukemia near-haploid cell line), whole cell lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Mouse IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 262 kDa

Observed band size: 285 kDa

false