Western blot - anti-VEGF antibody (ab46154)

All lanes : Anti-VEGF antibody (ab46154) at 0.5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)
Lane 4 : HEK293 Human embryonic kidney cell line Whole Cell Lysate (ab7902)
Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate (ab7900)
Lane 6 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 7 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
Lane 8 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with VEGF peptide (ab46160) at 1 µg/ml
Lane 9 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899) with VEGF peptide (ab46160) at 1 µg/ml
Lane 10 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909) with VEGF peptide (ab46160) at 1 µg/ml
Lane 11 : HEK293 Human embryonic kidney cell line Whole Cell Lysate (ab7902) with VEGF peptide (ab46160) at 1 µg/ml
Lane 12 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate (ab7900) with VEGF peptide (ab46160) at 1 µg/ml
Lane 13 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate with VEGF peptide (ab46160) at 1 µg/ml
Lane 14 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate with VEGF peptide (ab46160) at 1 µg/ml

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 24, 45 kDa
Observed band size : 43 kDa (why is the actual band size different from the predicted?)
Additional bands at : 52 kDa. We are unsure as to the identity of these extra bands.

Immunocytochemistry/ Immunofluorescence - VEGF antibody (ab46154)

ICC/IF image of this anti-VEGF antibody (ab46154) stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab46154, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VEGF antibody (ab46154)

ab46154 staining kidney tissue sections (human, mouse and rat) by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking with sequential peroxidase and protein block (prediluted) for 20 minutes at 20°C.  The anti-VEGF antibody was diluted 1/100 and incubated with the samples for 45 minutes at 20°C.  A HRP-conjugated goat anti-rabbit antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Antibody Solutions Ltd

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VEGF antibody (ab46154)

IHC image of VEGF staining in human heart formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab46154, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Immunocytochemistry/ Immunofluorescence-Anti-VEGF antibody(ab46154)

ICC/IF image of ab46154 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab46154, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.