All lanes : Anti-IGF1 Receptor antibody [EPR23027-80] (ab263907) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IGF1R knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 154 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

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.

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 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.

 

All lanes : Anti-IGF1 Receptor antibody [EPR23027-80] (ab263907) at 1/1000 dilution

Lane 1 : A431 (human epidermoid carcinoma epithelial cell) whole cell lysate
Lane 2 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 154 kDa
Observed band size: 130,200 kDa why is the actual band size different from the predicted?

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).

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.