ET1610-13

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

cat.: ET1610-13

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IP, IHC-P, IF-Cell, IF-Tissue, FC
Clonality:	Monoclonal
Clone number:	SC58-01

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 41/43 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human aa 166-215 / 379.
Positive control:	SH-SY5Y cell lysate, SH-SY5Y treated with 100ng/mL hβ-NGF for 10 minutes cell lysate, PC-12 treated with 100ng/mL hβ-NGF for 10 minutes cell lysate, SK-Br-3 whole cell lysate, NIH/3T3 cell lysate, NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate, HeLa treated with 200nM PMA for 30 minutes cell lysate, human thyroid carcinoma tissue.
Subcellular location:	Cytoplasm, Nucleus.
Recommended Dilutions: WB IP IHC-P IF-Cell IF-Tissue FC	1:5,000 Use at an assay dependent concentration. 1:1,000 1:100 1:200 1:1,000
Uniprot #:	SwissProt: P27361 Human \| P28482 Human \| P63085 Mouse \| Q63844 Mouse \| P21708 Rat \| P63086 Rat
Alternative names:	ERK 1 ERK 2 ERK-1 ERK-2 ERK1 erk1/2 ERK2 ERT1 ERT2 Extracellular signal regulated kinase 1 Extracellular signal-regulated kinase 1 Extracellular signal-regulated kinase 2 HS44KDAP HUMKER1A Insulin-stimulated MAP2 kinase MAP kinase 1 MAP kinase 2 MAP kinase 3 MAP kinase isoform p42 MAP kinase isoform p44 MAPK 1 MAPK 2 MAPK 3 Mapk1 MAPK2 MAPK3 Microtubule-associated protein 2 kinase Mitogen-activated protein kinase 1 Mitogen-activated protein kinase 2 Mitogen-activated protein kinase 3 MK01_HUMAN p38 p40 p41 p41mapk p42-MAPK P42MAPK p44-ERK1 p44-MAPK p44ERK1 p44MAPK PRKM 2 PRKM1 PRKM2 PRKM3 protein tyrosine kinase ERK2

Images

Fig1: 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;

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% 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.

Fig2: Western blot analysis of Phospho-Erk1 (T202 + Y204) + Erk2 (T185 + Y187) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1610-13, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: Untreated SK-Br-3 whole cell lysates
Lane 2: SK-Br-3 treated with λ Protein Phosphatase whole cell lysates

Fig3: 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 1/1,000 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: 41/43kDa

Exposure time: 13 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 (ET1610-13) 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.

Fig4: 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) 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.

Fig5: 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.

Fig6: 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.

Fig7: 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).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.