MEK1重组抗体[Y77]_MEK1抗体(ab32576)| Abcam中文官网 (2024)

• Western blot - Anti-MEK1 antibody [Y77] (ab32576)

  All lanes : Anti-MEK1 antibody [Y77] (ab32576) at 1/10000 dilution

  Lane 1 : Wild-type A549 cell lysate
  Lane 2 : MAP2K1 knockout A549 cell lysate
  Lane 3 : Wild-type HAP1 cell lysate
  Lane 4 : MAP2K1 knockout HAP1 cell lysate

  Lysates/proteins at 20 µg per lane.

  Performed under reducing conditions.

  Predicted band size: 43 kDa
  Observed band size: 45 kDa why is the actual band size different from the predicted?

  Western blot: Anti-MAP2K1 antibody [Y77] (ab32576) staining at 1/10000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32576 was shown to bind specifically to MAP2K1. A band was observed at 45 kDa in wild-type A549 cell lysates with no signal observed at this size in MAP2K1 knockout cell line. To generate this image, wild-type and MAP2K1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

• Western blot - Anti-MEK1 antibody [Y77] (ab32576)

  Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
  Lane 2:MEK1 knockout HAP1 whole cell lysate (20 µg)
  Lane 3: A431 whole cell lysate (20 µg)
  Lane 4: HepG2 whole cell lysate (20 µg)

  Lanes 1 - 4: Merged signal (red and green). Green - ab32576 (unpurified) observed at 43 kDa. Red - loading control, ab18058, observed at 130 kDa.

  ab32576 was shown to specifically react with MEK1 in wild-type HAP1 cells as signal was lost in MEK1 knockout cells. Wild-type and MEK1 knockout samples were subjected to SDS-PAGE. Ab32576 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

• Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 antibody [Y77] (ab32576)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling MEK1 with purified ab32576 at 1/100 dilution (3.28 µg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Western blot - Anti-MEK1 antibody [Y77] (ab32576)

  All lanes : Anti-MEK1 antibody [Y77] (ab32576) at 1/10000 dilution (Purified)

  Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
  Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates

  Lysates/proteins at 20 µg per lane.

  Secondary
  All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

  Predicted band size: 43 kDa
  Observed band size: 45 kDa why is the actual band size different from the predicted?

• Immunocytochemistry/ Immunofluorescence - Anti-MEK1 antibody [Y77] (ab32576)

  Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling MEK1 with purified ab32576 at 1/50 dilution (6.6 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Immunoprecipitation - Anti-MEK1 antibody [Y77] (ab32576)

  ab32576 (purified) at 1/20 dilution (2ug) immunoprecipitating MEK1 in Jurkat whole cell lysate. Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10ug
  Lane 2 (+): ab32576 & Jurkat whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32576 in Jurkat whole cell lysate
  For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.
  Blocking and diluting buffer: 5% NFDM/TBST.

• Flow Cytometry (Intracellular) - Anti-MEK1 antibody [Y77] (ab32576)

  Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling MEK1 with purified ab32576 at 1/30 dilution (10µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• Immunocytochemistry/ Immunofluorescence - Anti-MEK1 antibody [Y77] (ab32576)

  Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling MEK1 with purified ab32576 at 1/500 dilution (5 µg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEK1 antibody [Y77] (ab32576)

  ab32576 (unpurified)at a 1/250 dilution staining MEK1 in human urinary bladder carcinoma tissue by IHC-P.

• Western blot - Anti-MEK1 antibody [Y77] (ab32576)

  Anti-MEK1 antibody [Y77] (ab32576) at 1/10000 dilution (unpurified) + Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate

  Predicted band size: 43 kDa
  Observed band size: 45 kDa why is the actual band size different from the predicted?

• Anti-MEK1 antibody [Y77] (ab32576)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

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ab32576 被引用在 19 文献中.

• Kim M et al. Exosomes Derived from Colon Cancer Cells Promote Tumor Progression and Affect the Tumor Microenvironment. J Clin Med 12:N/A (2023). PubMed: 37373600
• Owens AE et al. High-Throughput Cellular Thermal Shift Assay Using Acoustic Transfer of Protein Lysates. ACS Chem Biol 17:322-330 (2022). PubMed: 35119255
• Tian J et al. MIR503HG impeded ovarian cancer progression by interacting with SPI1 and preventing TMEFF1 transcription. Aging (Albany NY) 14:5390-5405 (2022). PubMed: 35771155
• Cao H et al. PPP1R14D promotes the proliferation, migration and invasion of lung adenocarcinoma via the PKCα/BRAF/MEK/ERK signaling pathway. Int J Oncol 61:N/A (2022). PubMed: 36263632
• Zhang F et al. Gastric cancer cell-derived extracellular vesicles elevate E2F7 expression and activate the MAPK/ERK signaling to promote peritoneal metastasis through the delivery of SNHG12. Cell Death Discov 8:164 (2022). PubMed: 35383161

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