Anti-RSK1 p90 antibody [E4]

9 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RSK1 p90 antibody [E4] (ab32114)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast cancer tissue sections labeling RSK1 p90 with purified ab32114 at 1/100 dilution (15.4 μg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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.

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  Western blot - Anti-RSK1 p90 antibody [E4] (ab32114)

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: RSK1 p90 knockout HAP1 cell lysate (20 μg)
  Lane 3: MCF7 cell lysate (20 μg)
  Lane 4: K562 cell lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab32114 (unpurified) observed at 88 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  ab32114 was shown to recognize RSK1 p90 when RSK1 p90 knockout samples were used, along with additional cross-reactive bands. Wild-type and RSK1 p90 knockout samples were subjected to SDS-PAGE. ab32114 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted at 1/500 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-RSK1 p90 antibody [E4] (ab32114)

  Predicted band size: 82 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-RSK1 p90 antibody [E4] (ab32114)

  Immunocytochemistry/ Immunofluorescence analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling RSK1 p90 with purified ab32114 at 1/150 dilution (10 μg/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) at 1:200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (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.

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  Immunoprecipitation - Anti-RSK1 p90 antibody [E4] (ab32114)

  ab32114 (purified) at 1/70 dilution (2ug) immunoprecipitating RSK1 p90 in K-562 whole cell lysate. K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10ug
  Lane 2 (+): ab32114 & K-562 whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32114 in K-562 whole cell lysate
  For western blotting, VeriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/1000 dilution.
  Blocking and diluting buffer: 5% NFDM/TBST.

  All lanes: Immunoprecipitation - Anti-RSK1 p90 antibody [E4] (ab32114)

  Predicted band size: 82 kDa

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  Western blot - Anti-RSK1 p90 antibody [E4] (ab32114)

  All lanes: Western blot - Anti-RSK1 p90 antibody [E4] (ab32114) at 1/1000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

  Lane 3: Mouse lung lysates at 20 µg

  Lane 4: PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 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: 82 kDa

  Observed band size: 83 kDa

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  Flow Cytometry (Intracellular) - Anti-RSK1 p90 antibody [E4] (ab32114)

  Overlay histogram showing HeLa cells stained with (unpurified)ab32114 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32114, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RSK1 p90 antibody [E4] (ab32114)

  ab32114 (unpurified), at a 1/100 dilution, staining RSK1 p90 in paraffin embedded human colon tissue by immunohistochemistry.

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  Western blot - Anti-RSK1 p90 antibody [E4] (ab32114)

  All lanes: Western blot - Anti-RSK1 p90 antibody [E4] (ab32114) at 1/1000 dilution

  All lanes: MCF7 cell lysate at 10 µg

  Predicted band size: 82 kDa

  Observed band size: 90 kDa

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  Flow Cytometry (Intracellular) - Anti-RSK1 p90 antibody [E4] (ab32114)

  Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RSK1 p90 with purified ab32114 at 1/150 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 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).