Anti-EGFR antibody [13/EGFR]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [13/EGFR] (AB289889)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-EGFR antibody [13/EGFR] (AB289889)

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.

All lanes:

Immunoprecipitation - Anti-EGFR antibody [13/EGFR] (ab289889)

Predicted band size: 134 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-EGFR antibody [13/EGFR] (AB289889)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-EGFR antibody [13/EGFR] (ab289889) at 1/1000 dilution

All lanes:

A431 (human epidermoid carcinoma epithelial cell), whole cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 134 kDa

Observed band size: 170 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-EGFR antibody [13/EGFR] (AB289889)

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 38kDa.

All lanes:

Western blot - Anti-EGFR antibody [13/EGFR] (ab289889) at 1/1000 dilution

Lane 1:

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

Lane 2:

EGFR knockout HeLa whole cell lysate at 20 µg

Lane 3:

NCI-H1975 (Human adenocarcinoma lung epithelial cell) whole cell lysate at 20 µg

Lane 4:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Mouse IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 134 kDa

Observed band size: 170 kDa

false