All lanes : Anti-Fibronectin antibody [EPR23110-25] (ab268021) at 1/1000 dilution

Lane 1 : Human colon lysate at 20 µg
Lane 2 : Human stomach lysate at 20 µg
Lane 3 : Wild-type HAP1 whole cell lysate at 40 µg
Lane 4 : Fibronectin knockout HAP1 whole cell lysate at 40 µg

Secondary
Lanes 1-2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 3-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 262 kDa
Observed band size: 285 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 44 seconds.

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

ab268021 was shown to specifically 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. ab268021 and ab179475 (Rabbit anti-Integrin alpha V loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/5000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Fibronectin with ab268021 at 1/2000 dilution (0.23 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the perivascular tissue of sinusoids of rat liver (PMID:650101). Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of frozen section of  4% PFA-fixed, 0.2% Triton X-100 permeabilized rat colon tissue labeling Fibronectin with ab268021 at 1/100 dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Positive staining mainly on smooth muscle of rat colon (PMID: 17881565). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Flow cytometric analysis of  NIH/3T3 (mouse embryonic fibroblast) cells labeling Fibronectin with ab268021 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

 

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling Fibronectin with ab268021 at 1/2000 dilution (0.23 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the smooth muscle of mouse stomach (PMID:650101). Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human esophageal squamous cell carcinoma tissue labeling Fibronectin with ab268021 at 1/2000 dilution (0.23 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the stroma of human esophageal squamous cell carcinoma (PMID:30314454). Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling Fibronectin with ab268021 at 1/2000 dilution (0.23 µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in the stroma of human mammary gland (PMID:650101). Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-Fibronectin antibody [EPR23110-25] (ab268021) at 1/1000 dilution

Lane 1 : Human serum
Lane 2 : Human plasma
Lane 3 : Mouse plasma
Lane 4 : Rat plasma

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 3-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 262 kDa
Observed band size: 285 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3.25 seconds.

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

Immunohistochemical analysis of frozen section of  4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse colon tissue labeling Fibronectin with ab268021 at 1/100 dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Positive staining mainly on smooth muscle of mouse colon (PMID: 17881565). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).