Anti-EGFR (phospho Y1045) antibody [EPR28381-88]

• IP

Supplier Data

Immunoprecipitation - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (AB316155)

EGFR (phospho Y1045) was immunoprecipitated from 0.35 mg A431 (human epidermoid carcinoma epithelial cell) treated with 100ng/ml EGF for 30 minutes whole cell lysate with ab316155 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316155 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : A431 (human epidermoid carcinoma epithelial cell) treated with 100ng/ml EGF for 30 minutes whole cell lysate
Lane 2 : ab316155 IP in A431 (human epidermoid carcinoma epithelial cell) treated with 100ng/ml EGF for 30 minutes whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316155 in A431 treated with 100ng/ml EGF for 30 minutes whole cell lysate

All lanes:

Immunoprecipitation - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (ab316155) at 1/30 dilution

All lanes:

A431 (human epidermoid carcinoma epithelial cell) treated with 100ng/ml EGF for 30 minutes whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>)

false

Exposure time: 84s

• WB

Supplier Data

Western blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (AB316155)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-EGFR antibody - Total protein control (ab32077) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (ab316155) at 1/1000 dilution

Lane 1:

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

Lane 2:

A431 treated with 100 ng/ml EGF for 30 minutes whole cell lysate (untreated membrane) at 20 µg

Lane 3:

A431 treated with 100 ng/ml EGF for 30 minutes whole cell lysate (alkaline phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 175 kDa,36 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (AB316155)

In Western blot, ab316155 was shown to bind specifically to EGFR (phospho Y1045). Target of interest was observed at 175 kDa in wild-type HeLa cell lysates (lane 2) with no signal observed at this size in EGFR knockout cell line (lanes 3-4 KO cell line ab255385/KO cell lysate ab263845).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-EGFR antibody (ab32077) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (ab316155) at 1/1000 dilution

Lane 1:

Untreated Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HeLa treated with 100 ng/ml EGF for 30 minutes whole cell lysate at 20 µg

Lane 3:

Untreated EGFR knockout HeLa whole cell lysate at 20 µg

Lane 4:

EGFR knockout HeLa treated with 100 ng/ml EGF for 30 minutes 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 1/100000 dilution

Observed band size: 175 kDa,36 kDa

false

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (AB316155)

Dot blot analysis of EGFR (phospho Y1045) using ab316155 at 1 : 1000 (0.498 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

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

The antibody recognizes EGFR when phosphorylated at Tyr1045, and shows weak reactivity with ErbB2 when phosphorylated at Tyr1088. The antibody does not cross-react with EGFR phosphorylated at S1046 or S1047.

All lanes:

Dot Blot - Anti-EGFR (phospho Y1045) antibody [EPR28381-88] (ab316155) at 1/1000 dilution

Lane 1:

EGFR (phospho Y1045) peptide a

Lane 2:

EGFR (phospho Y1045) peptide b

Lane 3:

EGFR non-phospho peptide c

Lane 4:

EGFR (phospho S1046) peptide d

Lane 5:

EGFR (phospho S1047) peptide e

Lane 6:

ErbB2 (phospho Y1088) peptide f

Secondary

All lanes:

Dot 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 1/100000 dilution

false

Exposure time: 180s