Phospho-HER2 / ErbB2 (Y1139) Recombinant Rabbit Monoclonal Antibody [JE60-90]

☑ Cell treatment (CT)

Western blot analysis of Phospho-HER2 / ErbB2 (Y1139) on different lysates with Rabbit anti-Phospho-HER2 / ErbB2 (Y1139) antibody (HA722201) at 1/1,000 dilution.

Lane 1: A431 cell lysate
Lane 2: A431 treated with 100ng/mL EGF for 10 minutes cell lysate
Lane 3: A431 treated with 100ng/mL EGF for 10 minutes cell lysate, then the membrane treated with λpp for 1 hour
Lane 4: SK-Br-3 cell lysate
Lane 5: SK-Br-3 starved for 4 hours add 200ng/mL EGF for 15 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 138 kDa
Observed band size: 180 kDa

Exposure time: 10 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722201) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunocytochemistry analysis of A431 cells treated with or without 100ng/mL EGF for 10 minutes labeling Phospho-HER2 / ErbB2 (Y1139) with Rabbit anti-Phospho-HER2 / ErbB2 (Y1139) antibody (HA722201) at 1/5,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-HER2 / ErbB2 (Y1139) antibody (HA722201) at 1/5,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Cell treatment (CT)

Flow cytometric analysis of A431 cells (left) and A431 cells treated with 100ng/mL EGF for 10 minutes (right) labeling Phospho-HER2 / ErbB2 (Y1139).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722201, 1/10,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).