Anti-EGFR 抗体 [13/EGFR] - BSA and Azide free (ab289896)

This data was developed using ab289889, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. 

False colour image of Western blot: Anti-EGFR antibody [13/EGFR] (ab289889 staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody [16891] (ab181602) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab289889 was shown to bind specifically to EGFR. A band was observed at 170 kDa in wild-type HeLa cell lysates with no signal observed at this size in the EGFR knockout cell line. To generate this image, wild-type and EGFR knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS 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-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution.

Performed under reducing conditions.

Low expression: SW480(PMID: 19789347)
We observe an unknown band at around 38k. 

This data was developed using ab289889, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer: 5% NFDM/TBST. 

This data was developed using ab289889, the same antibody clone in a different buffer formulation. 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A-431 (Human epidermoid carcinoma cell line) labeling EGFR with ab289889 at 1/50 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150117) preadsorbed secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and membranous staining in A431 cell line. 

Low expression：SW480 (PMID: 27729020). The nuclear counter stain is DAPI (blue).

Tubulin is counterstained with Anti-alpha Tubulin mouse MAb (ab179513) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150088) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab289889 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150088) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab179513) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150117) secondary antibody at 1/1000 dilution.

This data was developed using ab289889, the same antibody clone in a different buffer formulation. 

EGFR was immunoprecipitated from A431 (human epidermoid carcinoma epithelial cell) whole cell lysate 10 μg with ab289889 at 1/30 diultion. Western Blot was performed from immunoprecipitate using ab289889 at 1/1000 diultion. Mouse IgG for IP (HRP) (ab131368) was used as a secondary antibody at 1/5000 dilution. 

Lane 1 (Input): A431 (human epidermoid carcinoma epithelial cell) whole cell lysate) 

Lane 2 (+): A431 whole cell lysate

Lane 3 (-): Mouse IgG1 monoclonal (ab18443) instead of ab289889 in A431 whole cell lysate 

Blocking and dilution buffer: 5% NFDM/TBST.