All lanes : Anti-Fibrinogen gamma chain antibody [EP24539-127] (ab281924) at 1/5000 dilution

Lane 1 : Human plasma lysate
Lane 2 : Human plasma lysate treated with PNGase F whole cell lysate
Lane 3 : Mouse plasma lysate
Lane 4 : Mouse plasma lysate treated with PNGase F whole cell lysate
Lane 5 : Rat plasma lysate
Lane 6 : Rat liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/20000 dilution

Predicted band size: 52 kDa
Observed band size: 50,100 kDa why is the actual band size different from the predicted?

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 21480334).

Exposure time: Lanes 1-5: 10 seconds

Lane 6: 37 seconds.

 

All lanes : Anti-Fibrinogen gamma chain antibody [EP24539-127] (ab281924) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
Lane 2 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 3 : DU145 (human prostate carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 52 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

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

Low expression: A549/DU145 (PMID: 17949478).

 Exposure time: 26 seconds.

 

All lanes : Anti-Fibrinogen gamma chain antibody [EP24539-127] (ab281924) at 1/1000 dilution

Lane 1 : Human liver lysate
Lane 2 : Mouse liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

Predicted band size: 52 kDa
Observed band size: 50,100 kDa why is the actual band size different from the predicted?

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

 Exposure time: Lanes 1: 1 seconds.

Lane 2: 26 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelling Fibrinogen gamma chain with ab281924 at 1/4000 (0.135 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human liver (PMID:24648543). The section was incubated with ab281924 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labelling Fibrinogen gamma chain with ab281924 at 1/4000 (0.135 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in the blood plasma and stroma of human pancreas (PMID:16510577). The section was incubated with ab281924 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labelling Fibrinogen gamma chain with ab281924 at 1/4000 (0.135 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human hepatocellular carcinoma (PMID:30863175). The section was incubated with ab281924 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling Fibrinogen gamma chain with ab281924 at 1/100 (5.38 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green) Confocal image showing cytoplasmic staining in HepG2 cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Fibrinogen gamma chain was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug with ab281924 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab281924 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug.

Lane 2: ab281924 IP in HepG2 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab281924 in HepG2 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds.

 

Fibrinogen gamma chain was immunoprecipitated from 0.35 mg Mouse plasma lysate 10 ug with ab281924 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab281924 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse plasma lysate 10 ug.

Lane 2: ab281924 IP in Mouse plasma lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab281924 in mouse plasma lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds.