Anti-VEGFA antibody

• WB

Project

Western blot - Anti-VEGFA antibody (AB46154)

All lanes:

Western blot - Anti-VEGFA antibody (ab46154) at 0.5 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Western blot - Jurkat whole cell lysate (<a href='/en-us/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 10 µg

Lane 3:

Western blot - A-431 whole cell lysate (<a href='/en-us/products/cell-lysates/a-431-whole-cell-lysate-ab7909'>ab7909</a>) at 10 µg

Lane 4:

Western blot - HEK-293 whole cell lysate (<a href='/en-us/products/cell-lysates/hek-293-whole-cell-lysate-ab7902'>ab7902</a>) at 10 µg

Lane 5:

Hep G2 whole cell lysate (<a href='/en-us/products/unavailable/hep-g2-whole-cell-lysate-ab7900'>ab7900</a>) at 10 µg

Lane 6:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

Lane 8:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Lane 9:

Western blot - Jurkat whole cell lysate (<a href='/en-us/products/cell-lysates/jurkat-whole-cell-lysate-ab7899'>ab7899</a>) at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Lane 10:

Western blot - A-431 whole cell lysate (<a href='/en-us/products/cell-lysates/a-431-whole-cell-lysate-ab7909'>ab7909</a>) at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Lane 11:

Western blot - HEK-293 whole cell lysate (<a href='/en-us/products/cell-lysates/hek-293-whole-cell-lysate-ab7902'>ab7902</a>) at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Lane 12:

Hep G2 whole cell lysate (<a href='/en-us/products/unavailable/hep-g2-whole-cell-lysate-ab7900'>ab7900</a>) at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Lane 13:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Lane 14:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg with VEGFA peptide (<a href='/en-us/products/proteins-peptides/vegfa-peptide-ab46160'>ab46160</a>)

Secondary

All lanes:

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

false

• WB

Lab

Western blot - Anti-VEGFA antibody (AB46154)

This blot was produced using a 10% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab46154 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-VEGFA antibody (ab46154) at 1 µg/mL

All lanes:

HCT116 whole cell lysate at 20 µg

Secondary

All lanes:

Rabbit IgG secondary antibody at 1/10000 dilution

Predicted band size: 27 kDa

Observed band size: 23 kDa

true

Exposure time: 2min

• ELISA

Lab

ELISA - Anti-VEGFA antibody (AB46154)

ab46154 was tested using indirect ELISA. The wells were coated with peptide (1 μg x mL^-1 at 100 μL per well) overnight at 4°C, followed by a 1% fat-free milk blocking step for 1 hour at room temperature. The primary antibody (ab46154) was added at a range of dilutions (50 μL per well) for 1 hour at room temperature. ab97080 (Goat Anti-Rabbit IgG H&L-HRP) was used as a secondary antibody at 1 : 50.000 dilution for 1 hour at room temperature and signal was developed with TMB substrate.

• WB

Lab

Western blot - Anti-VEGFA antibody (AB46154)

This blot was produced using a 10% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab46154 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-VEGFA antibody (ab46154) at 1 µg/mL

Lane 1:

Recombinant Human VEGFA protein (His tag) (<a href='/en-us/products/unavailable/recombinant-human-vegfa-protein-his-tag-ab204773'>ab204773</a>) at 0.1 µg

Lane 2:

Western blot - Recombinant mouse VEGFA protein (Active) (<a href='/en-us/products/proteins-peptides/recombinant-mouse-vegfa-protein-active-ab185265'>ab185265</a>) at 0.1 µg

Secondary

All lanes:

Rabbit IgG secondary antibody at 1/10000 dilution

Predicted band size: 27 kDa

Observed band size: 23 kDa

true

Exposure time: 10s

• WB

CiteAb

Western blot - Anti-VEGFA antibody (AB46154)

Western Blotting using Anti-VEGFA antibody, ab46154. Publication image from Bjørås, M. et al., 2017, Nat Commun, 28534495. Legend direct from paper.

HCAR1 regulates VEGFA and capillary density in response to exercise.(a) Collagen IV-labelled capillaries in the sensorimotor cortex grey matter of wild-type or Hcar1 knockout mice exposed to vehicle injections (control), treadmill exercise or lactate injections, 5 days a week for 7 consecutive weeks. Scale bar, 100 µm. (b) Capillary density (per cent of the total area, normalized to wild-type control) in the sensorimotor cortex. Mean±s.e.m. of n=7 wild-type controls, seven wild-type exercise, six wild-type lactate, five knockout controls, four knockout exercise and six knockout lactate mice. Analysis of variance (ANOVA), P=0.001; Fisher's least significant difference (LSD) post hoc test, **P<0.01; ***P<0.001. (c) Collagen IV-labelled capillaries in the dentate gyrus (DG) of the hippocampus of wild-type or Hcar1 knockout mice treated as in a. Stippled line, the inner border of the granule cell layer (G), circumscribing the sampled area, hilus (H). Scale bar, 50 µm. (d) Capillary density (see b) in the hilus. Mean±s.e.m. of n mice (n=4–7, as specified below for diameters). ANOVA, P=0.022; Fisher's LSD post hoc test, **P<0.01. Capillary external diameters (µm) in the same areas were unchanged (mean±s.e.m. (n)) : wild-type control 5.8±0.2 (6), wild-type exercise 5.7±0.1 (7), wild-type lactate 5.7±0.2 (6), knockout control 5.8±0.3 (5), knockout exercise 6.0±0.1 (4), knockout lactate 5.8±0.2 (6). ANOVA, P=0.93. (e) Quantification of VEGFA in hippocampus of wild-type animals. Data are relative toα-tubulin (α-tub), normalized to wild-type control, mean±s.e.m. of n=4 wild-type control mice, five wild-type exercised mice and six mice treated with lactate, ANOVA, P=0.001; Dunnetts's T3 post hoc test, *P<0.05, ***P<0.001. (f) Quantification of VEGFA in hippocampus of knockout animals presented as in e. n=6 mice per group. ANOVA, P=0.88. (g) Western blots of VEGFA underlying e,f (uncropped scans shown in Supplementary Fig. 6a).

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