Phospho-MEK1/2 (S218 + S222) Recombinant Rabbit Monoclonal Antibody [ST0490]
cat.: ET1609-50
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IF-Tissue, IHC-P, IP
Clonality: Monoclonal
Clone number: ST0490
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: 43 kDa
Isotype: IgG
Immunogen: Synthetic phospho-peptide corresponding to residues surrounding Ser218 and 222 of Human MEK1 aa 200-249 / 393.
Positive control: NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate, C6 treated with 200nM PMA for 30 minutes cell lysate, Hela cell lysate, A431 cell lysate, 293T cell lysate, NIH/3T3 cells treated with 200nM PMA for 30 minutes, Hela, HepG2, human tonsil tissue, human liver carcinoma tissue, human spleen tissue, human breast carcinoma tissue.
Subcellular location: Cytoplasm, Cytoskeleton, Membrane, Nucleus.
Recommended Dilutions:
  WB
  IF-Cell
  IF-Tissue
  IHC-P
  IP
1:500-1:2,000
1:50-1:200
1:50-1:200
1:50-1:200
Use at an assay dependent concentration.
Uniprot #: SwissProt: Q02750 Human | P31938 Mouse | Q01986 Rat
Alternative names: Dual specificity mitogen activated protein kinase kinase 1 Dual specificity mitogen-activated protein kinase kinase 1 ERK activator kinase 1 MAP kinase kinase 1 MAP kinase/Erk kinase 1 MAP2K1 MAPK/ERK kinase 1 MAPKK 1 MAPKK1 MEK 1 Mek1 MEKK1 Mitogen activated protein kinase kinase 1 MKK 1 MKK1 MP2K1_HUMAN PRKMK1 Protein kinase mitogen activated kinase 1 (MAP kinase kinase 1) Protein kinase mitogen activated, kinase 1 hide
Images
ET1609-50_1.jpg Fig1: Western blot analysis of Phospho-MEK1/2 (S218 + S222) on different lysates with Rabbit anti-Phospho-MEK1/2 (S218 + S222) antibody (ET1609-50) 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: C6 cell lysate
Lane 4: C6 treated with 200nM PMA for 30 minutes cell lysate
Lane 5: NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour
Lane 6: C6 treated with 200nM PMA for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 43 kDa
Observed band size: 43 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 (ET1609-50) 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.
ET1609-50_2.jpg Fig2: Western blot analysis of Phospho-MEK1/2 (S218 + S222) 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 (ET1609-50, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: Hela cell lysate
Lane 2: A431 cell lysate
Lane 2: 293T cell lysate
ET1609-50_3.jpg Fig3: Western blot analysis of Phospho-MEK1/2 (S218 + S222) on Hela cell lysates.

Lane 1: Hela cells, whole cell lysate, 10ug/lane
Lane 2/3: Hela cells treated with 200 nM PMA for 20 minutes, whole cell lysates, 10ug/lane
Lane 4: Hela cells treated with 200 nM PMA for 20 minutes, then treated with 2.8ug/ul lambda-PP for 30 minutes, whole cell lysates, 10ug/lane

All lanes :
Anti-Phospho-MEK1/2 (S218 + S222) antibody (ET1609-50) at 1/500 dilution. Anti-MEK1 antibody (ET1603-20) at 1/500 dilution. Anti-GAPDH antibody (ET1601-4) at 1/10,000 dilution. Goat Anti-Rabbit IgG H&L (HRP) (HA1001) at 1/200,000 dilution.

Predicted band size:43 kDa
Observed band size:43 kDa

Blocking and diluting buffer: 5% BSA.

Exposure time: Lane1/2 5 minutes
Lane3/4 1 minutes 32 seconds
ET1609-50_4.jpg Fig4: Immunocytochemistry analysis of NIH/3T3 cells treated with 200nM PMA for 30 minutes labeling Phospho-MEK1/2 (S218 + S222) with Rabbit anti-Phospho-MEK1/2 (S218 + S222) antibody (ET1609-50) 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-MEK1/2 (S218 + S222) antibody (ET1609-50) 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.
ET1609-50_5.jpg Fig5: ICC staining of Phospho-MEK1/2 (S218 + S222) in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1609-50, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1609-50_6.jpg Fig6: ICC staining of Phospho-MEK1/2 (S218 + S222) in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1609-50, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
ET1609-50_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-MEK1/2 (S218 + S222) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-50, 1/50) for 30 minutes 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.
ET1609-50_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human liver carcinoma tissue using anti-Phospho-MEK1/2 (S218 + S222) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-50, 1/50) for 30 minutes 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.
ET1609-50_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-Phospho-MEK1/2 (S218 + S222) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-50, 1/50) for 30 minutes 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.
ET1609-50_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-Phospho-MEK1/2 (S218 + S222) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1609-50, 1/50) for 30 minutes 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.