Anti-NEK9 antibody [EP7361]

9 product images

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  Western blot - Anti-NEK9 antibody [EP7361] (ab138488)

  Lanes 1 - 4: Merged signal (red and green). Green - ab138488 observed at 120 kDa. Red - loading control, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
  

  ab138488 was shown to react with NEK9 in wild-type A431 cells in western blot. Loss of signal was observed when NEK9 knockout sample was used. Wild-type and NEK9 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab138488 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-NEK9 antibody [EP7361] (ab138488) at 1/1000 dilution

  Lane 1: Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: NEK9 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: Western blot - Human NEK9 knockout A-431 cell line (Human NEK9 knockout A-431 cell line ab270474)

  Lane 3: Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

  Lane 4: MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 107 kDa

  Observed band size: 120 kDa

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  Western blot - Anti-NEK9 antibody [EP7361] (ab138488)

  Blocking and diluting buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-NEK9 antibody [EP7361] (ab138488) at 1000 Cells

  All lanes: Rat spleen lysates at 15 µg

  Secondary

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

  Predicted band size: 107 kDa

  Observed band size: 120 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)

  Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling NEK9 with Purified ab138488 at 1:100 dilution (3.8μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

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  Flow Cytometry (Intracellular) - Anti-NEK9 antibody [EP7361] (ab138488)

  Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified Anti-Sonic Hedgehog antibody [EP1190Y] ab53281 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - cells without incubation with primary antibody and secondary antibody (Blue).
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling NEK9 with Purified ab138488 at 1:100 dilution (3.84 μg/ml). Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

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  Western blot - Anti-NEK9 antibody [EP7361] (ab138488)

  Blocking and diluting buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-NEK9 antibody [EP7361] (ab138488) at 1/5000 dilution

  Lane 1: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

  Lane 2: THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 15 µg

  Secondary

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

  Predicted band size: 107 kDa

  Observed band size: 120 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] (ab138488)

  Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling NEK9 with unpurified ab138488 at 1/50 dilution.

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  Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] (ab138488)

  Immunofluorescent analysis of HeLa cells labelling NEK9 with unpurified ab138488 at 1/250 dilution.

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  Western blot - Anti-NEK9 antibody [EP7361] (ab138488)

  All lanes: Western blot - Anti-NEK9 antibody [EP7361] (ab138488) at 1/1000 dilution

  Lane 1: NIH3T3 cell lysate at 10 µg

  Lane 2: HepG2 cell lysate at 10 µg

  Lane 3: HeLa cell lysate at 10 µg

  Lane 4: THP1 cell lysate at 10 µg

  Secondary

  All lanes: HRP labelled goat anti-rabbit at 1/2000 dilution

  Predicted band size: 107 kDa