Phospho-HER2/ErbB2 Antibody Sampler Kit
cat.: HAK21162
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Uniprot #: SwissProt: P04626 Human | P04626 Human | P04626 Human | P04626 Human
Images
HAK21162_1.jpg Fig1: Western blot analysis of Phospho-HER2 / ErbB2 (Y1248) on different lysates with Rabbit anti-Phospho-HER2 / ErbB2 (Y1248) antibody (HA722084) at 1/1,000 dilution.

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

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 24 seconds;
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 (HA722084) 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.
HAK21162_2.jpg Fig2: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue with Rabbit anti-HER2 / ErbB2 antibody (HA721178) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA721178) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21162_3.jpg Fig3: Western blot analysis of Phospho-HER2 / ErbB2 (Y1221 + Y1222) on different lysates with Rabbit anti-Phospho-HER2 / ErbB2 (Y1221 + Y1222) antibody (HA721433) at 1/1,000 dilution.

Lane 1: SK-Br-3 whole cell lysate
Lane 2: SK-Br-3 treated with λpp for 1 hour whole cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 138 kDa
Observed band size: 250 kDa

Exposure time: 3 minutes;

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 (HA721433) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
HAK21162_4.jpg Fig4: mIHC analysis of human ovarian cancer tissue (Formalin/PFA-fixed paraffin-embedded sections) with Rabbit anti-HER2 / ErbB2 antibody (HA721178) at 1/2,000 dilution. The immunostaining was performed with the IRISKitCmTSA Kit (900808). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.
HAK21162_5.jpg Fig5: 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.
HAK21162_6.jpg Fig6: 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.
HAK21162_7.jpg Fig7: 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).
HAK21162_8.jpg Fig8: Immunocytochemistry analysis of HeLa cells starved for 4 hours add 200ng/mL EGF for 15 minutes labeling Phospho-HER2 / ErbB2 (Y1248) with Rabbit anti-Phospho-HER2 / ErbB2 (Y1248) antibody (HA722084) at 1/100 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 (Y1248) antibody (HA722084) at 1/100 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.