Anti-Mark3 antibody [EPR633Y]

8 product images

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  Immunoprecipitation - Anti-Mark3 antibody [EPR633Y] (ab52626)

  ab52626 (purified) at a dilution of 1/20 immunoprecipitating Mark3 in K562 whole cell lysate.
  

  Lane 1 (input): K562 whole cell lysate (10μg)

  Lane 2 (+): ab52626 + K562 whole cell lysate.

  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab52626 in K562 whole cell lysate.

  For western blotting, VeriBlot for IP Detection Reagent (HRP) ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Predicted band size: 87 kDa

  Observed band size: 86 kDa

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  Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Lane 1: Wild-type HAP1 cell lysate (40 μg)
  Lane 2: MARK3 knockout HAP1 cell lysate (40 μg)
  Lane 3: HeLa cell lysate (20 μg)
  Lane 4: K562 cell lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab52626 observed at 85 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  ab52626 was shown to recognize Mark3 when Mark3 knockout samples were used, along with additional cross-reactive bands. Wild-type and Mark3 knockout samples were subjected to SDS-PAGE. ab52626 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1:10,000 dilution respectively and incubated overnight at 4C. 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 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Predicted band size: 87 kDa

• 

  Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Blocking and dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626) at 1/2000 dilution

  Lane 1: K562 whole cell lysate at 20 µg

  Lane 2: HeLa whole cell lysate 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: 87 kDa

  Observed band size: 86 kDa

• 

  Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Blocking and dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626) at 10 µg

  All lanes: NIH/3T3 whole cell lysate at 10 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: 87 kDa

  Observed band size: 86 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling Mark3 with purified ab52626 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.
  

  Control 1: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

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  Flow Cytometry (Intracellular) - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Intracellular Flow Cytometry analysis ofHeLa cells labelling Mark3 with purified ab52626 at a dilution of 1/50 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluorr^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
  

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  Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626)

  All lanes: Western blot - Anti-Mark3 antibody [EPR633Y] (ab52626) at 1/2000 dilution

  All lanes: HeLa cell lysate at 10 µg

  Secondary

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

  Predicted band size: 87 kDa

  Observed band size: 86 kDa

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  Flow Cytometry (Intracellular) - Anti-Mark3 antibody [EPR633Y] (ab52626)

  Overlay histogram showing HeLa cells stained with unpurified ab52626 (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 (unpurified ab52626, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 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) (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.