Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) Recombinant Rabbit Monoclonal Antibody [SC58-01] - BSA and Azide free

☑ Cell treatment (CT)

This data was developed using ET1610-13, the same antibody clone in a different buffer formulation.
Western blot analysis of Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) on different lysates with Rabbit anti-Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution.

Lane 1: SH-SY5Y cell lysate
Lane 2: SH-SY5Y treated with 100ng/mL hβ-NGF for 10 minutes cell lysate
Lane 3: PC-12 cell lysate
Lane 4: PC-12 treated with 100ng/mL hβ-NGF for 10 minutes cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 41/43 kDa
Observed band size: 41/43 kDa

Exposure time: 1 minute 50 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 (ET1610-13) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution were used in 5% BSA at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

This data was developed using ET1610-13, the same antibody clone in a different buffer formulation.
Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue with Rabbit anti-Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (ET1610-13) at 1/1,000 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.

☑ Cell treatment (CT)

This data was developed using ET1610-13, the same antibody clone in a different buffer formulation.
Western blot analysis of Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) on different lysates with Rabbit anti-Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) at 1/5,000 dilution.

Lane 1: NIH/3T3 cell lysate
Lane 2: NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate
Lane 3: HeLa cell lysate
Lane 4: HeLa treated with 200nM PMA for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 41/43 kDa
Observed band size: 41/43 kDa

Exposure time: 13 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 (ET1610-13) at 1/5,000 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)

This data was developed using ET1610-13, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of HeLa cells treated with 200nM PMA for 30 minutes labeling Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) with Rabbit anti-Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) 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-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) 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.

☑ Cell treatment (CT)

This data was developed using ET1610-13, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of NIH/3T3 cells treated with 200nM PMA for 30 minutes labeling Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) with Rabbit anti-Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) 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-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) antibody (ET1610-13) 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.

☑ Cell treatment (CT)

This data was developed using ET1610-13, the same antibody clone in a different buffer formulation.
Flow cytometric analysis of HeLa cells untreated (left) or treated (right) with PMA for 30 minutes labeling Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187).

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1610-13, 1μg/mL) (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).