Anti-IGF1 Receptor antibody [EPR23027-80]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IGF1 Receptor antibody [EPR23027-80] (AB263907)

Immunofluorescent analysis of 100% Methanol-fixed MCF7 (human breast adenocarcinoma epithelial cell) cells labelling IGF1 Receptor with ab263907 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in MCF7 cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

• Flow Cyt

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Flow Cytometry - Anti-IGF1 Receptor antibody [EPR23027-80] (AB263907)

Flow cytometric analysis MCF7 (human breast adenocarcinoma epithelial cell) cells labelling IGF1 Receptor with ab263907 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled 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.

• IP

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Immunoprecipitation - Anti-IGF1 Receptor antibody [EPR23027-80] (AB263907)

IGF1 Receptor was immunoprecipitated from 0.35 mg MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate with ab263907 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263907 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab263907 IP in MCF7 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab263907 in MCF7 whole cell lysate

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

Exposure time : 3 min.

All lanes:

Immunoprecipitation - Anti-IGF1 Receptor antibody [EPR23027-80] (ab263907)

Predicted band size: 154 kDa

Observed band size: 130 kDa

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• WB

Lab

Western blot - Anti-IGF1 Receptor antibody [EPR23027-80] (AB263907)

Lanes 1- 2 : Merged signal (red and green). Green - ab263907 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab263907 was shown to react with IGF1 Receptor in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264801 (knockout cell lysate ab256951) was used. Wild-type HeLa and IGF1R knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab263907 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IGF1 Receptor antibody [EPR23027-80] (ab263907) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

IGF1R knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human IGF1R (IGF1 Receptor) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-igf1r-igf1-receptor-knockout-hela-cell-line-ab264801'>ab264801</a>)

Predicted band size: 154 kDa

Observed band size: 100 kDa

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• WB

Lab

Western blot - Anti-IGF1 Receptor antibody [EPR23027-80] (AB263907)

False colour image of Western blot : Anti-IGF1 Receptor antibody [EPR23027-80] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab263907 was shown to bind specifically to IGF1 Receptor. A band was observed at 105-125 kDa (alpha chain) in wild-type MCF7 cell lysates with no signal observed at this size in IGF1R knockout cell line. To generate this image, wild-type and IGF1R knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IGF1 Receptor antibody [EPR23027-80] (ab263907) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

IGF1R knockout MCF7 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HDLM-2 cell lysate at 20 µg

Observed band size: 105-125 kDa

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• WB

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Western blot - Anti-IGF1 Receptor antibody [EPR23027-80] (AB263907)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 10 seconds.

The expression profile & molecular weight observed is consistent with what has been described in the literature (PMID : 21807868, 28591735).

Note : the bands larger than 200kDa are Pro-IGF1R.

Low expression cell line : MDA-MB-231 (PMID : 28591735).

All lanes:

Western blot - Anti-IGF1 Receptor antibody [EPR23027-80] (ab263907) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 154 kDa

Observed band size: 130 kDa,200 kDa

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