Anti-N WASP antibody [EPR6959]

9 product images

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  Western blot - Anti-N WASP antibody [EPR6959] (ab126626)

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: N WASP knockout HAP1 cell lysate (20 μg)
  Lane 3: A549 cell lysate (20 μg)
  Lane 4: Human thyroid tissue lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab126626 observed at 67 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  ab126626 was shown to recognize N WASP when N WASP knockout samples were used, along with additional cross-reactive bands. Wild-type and N WASP knockout samples were subjected to SDS-PAGE. ab126626 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 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 1/10000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-N WASP antibody [EPR6959] (ab126626)

  Predicted band size: 55 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-N WASP antibody [EPR6959] (ab126626)

  Immunofluorescence staining of K562 cells with purified ab126626 at a working dilution of 1/300, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), used at a dilution of 1/1000. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab126626 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at a dilution of 1/500. For negative control 2, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at a dilution of 1/400.

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  Flow Cytometry (Intracellular) - Anti-N WASP antibody [EPR6959] (ab126626)

  Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling N WASP with purified ab126626 at 1/150 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluorr^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077)was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-N WASP antibody [EPR6959] (ab126626)

  Immunohistochemical staining of paraffin embedded human kidney with purified ab126626 at a working dilution of 1/100. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

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  Western blot - Anti-N WASP antibody [EPR6959] (ab126626)

  All lanes: Western blot - Anti-N WASP antibody [EPR6959] (ab126626) at 1/10000 dilution

  All lanes: L929 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: 55 kDa

  Observed band size: 65 kDa

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  Western blot - Anti-N WASP antibody [EPR6959] (ab126626)

  Blocking buffer: 5% NFDM/TBST
  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-N WASP antibody [EPR6959] (ab126626) at 1/10000 dilution

  Lane 1: HeLa whole cell lysate at 10 µg

  Lane 2: K562 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: 35 kDa, 38 kDa, 42 kDa, 50 kDa, 55 kDa

  Observed band size: 42 kDa, 65 kDa

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  Western blot - Anti-N WASP antibody [EPR6959] (ab126626)

  All lanes: Western blot - Anti-N WASP antibody [EPR6959] (ab126626) at 1/1000 dilution

  Lane 1: NIH 3T3 cell lysate at 10 µg

  Lane 2: F9 cell lysate at 10 µg

  Lane 3: L929 cell lysate at 10 µg

  Lane 4: HeLa cell lysate at 10 µg

  Lane 5: K562 cell lysate at 10 µg

  Secondary

  All lanes: Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

  Predicted band size: 55 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-N WASP antibody [EPR6959] (ab126626)

  Unpurified ab126626, at 1/50, staining N WASP in formalin fixed, paraffin embedded human papillary carcinoma tissue by Immunohistochemistry
  

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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  OI-RD Scanning - Anti-N WASP antibody [EPR6959] (ab126626)

  We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
  Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.