All lanes : Anti-VEGF Receptor 1 antibody [Y103] (ab32152) at 1/1000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Human brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 151 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?


Exposure time: 40 seconds

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

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling VEGF with ab32152, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human gastric carcinoma. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

VEGF Receptor 1 was immunoprecipitated from 0.35 mg mouse brain lysate 10μg with ab32152 at 1:30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab32152 1:1000 dilution (2 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:1000 dilution.

Lane 1: Mouse brain lysate 10μg.

Lane 2: ab32152 IP in mouse brain lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32152 in mouse brain lysate.

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

Exposure time: 1 second.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human abdominal aortic aneurysm (AAA) wall tissue sections labeling VEGF Receptor 1 with ab32152 at 1/100 dilution.

Resected aortic tissues were immersed in 10% neutral buffered formalin for at least 24 h for immunohistochemical staining. Tissue sample was embedded in paraffin; 4 µm sections were cut and mounted onto MAS-coated slides. The sections were deparaffinized, dehydrated, and boiled in a pressure cooker in 0.01 M citric acid buffer (pH 6.0) for 20 min. The sections were washed with phosphate-buffered saline and incubated with 3% H₂O₂ in absolute methanol for 5 min to inhibit any endogenous peroxidase activity. Sections were preincubated with 3% normal goat serum for 20 min to minimize nonspecific binding to VEGF Receptor 1, and incubated with ab32152 at 4°C overnight in a moist chamber. The section was washed with phosphate-buffered saline and then incubated with the appropriate secondary antibody for 30 min at room temperature. Staining was visualized with Vector DAB, and tissue section was then counterstained with hematoxylin.

Anti-VEGF Receptor 1 antibody [Y103] (ab32152) at 1/10000 dilution + mouse brain tissue

Predicted band size: 151 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?