Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-Nck antibody [Y531] (ab32120)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y531] to Nck
  • Suitable for: WB, IHC-P, Flow Cyt, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-Nck antibody [Y531]
    See all Nck primary antibodies

  • Description

    Rabbit monoclonal [Y531] to Nck

  • Host species

    Rabbit

  • Specificity

    ab32120 recognises Nck.

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    A synthetic peptide corresponding to residues close to the SH2 domain of human Nck

  • Positive control

    • Hela cell lysate and human breast carcinoma ICC/IF: HeLa cells

  • General notes

     

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Images

  • Western blot - Anti-Nck antibody [Y531] (ab32120)
    Western blot - Anti-Nck antibody [Y531] (ab32120)
    All lanes : Anti-Nck antibody [Y531] (ab32120) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : NCK1 knockout HeLa cell lysate
    Lane 3 : NIH/3T3 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

    Observed band size: 50 kDa
    why is the actual band size different from the predicted?



    Lanes 1-3: Merged signal (red and green). Green - ab32120 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa. 

     ab32120 Anti-Nck antibody [Y531] was shown to specifically react with Nck in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265323 (knockout cell lysate ab258059) was used.  Wild-type and Nck knockout samples were subjected to SDS-PAGE.  ab32120 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and 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.

  • Immunocytochemistry/ Immunofluorescence - Anti-Nck antibody [Y531] (ab32120)
    Immunocytochemistry/ Immunofluorescence - Anti-Nck antibody [Y531] (ab32120)

    Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Nck with purified ab32120 at 1/250 dilution (8 µg/mL). Cells were fixed in 100% Methanol. 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.

  • Flow Cytometry - Anti-Nck antibody [Y531] (ab32120)
    Flow Cytometry - Anti-Nck antibody [Y531] (ab32120)
    Overlay histogram showing Jurkat cells stained with ab32120 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab32120, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (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 Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nck antibody [Y531] (ab32120)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nck antibody [Y531] (ab32120)

    ab32120 (1/100), staining human breast carcinoma by immunohistochemistry, Paraffin embedded tissue.

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

  • Western blot - Anti-Nck antibody [Y531] (ab32120)
    Western blot - Anti-Nck antibody [Y531] (ab32120)
    Anti-Nck antibody [Y531] (ab32120) at 1/10000 dilution + Hela cell lysate

    Observed band size: 47 kDa why is the actual band size different from the predicted?
    Additional bands at: 35 kDa. We are unsure as to the identity of these extra bands.

  • Anti-Nck antibody [Y531] (ab32120)
    Anti-Nck antibody [Y531] (ab32120)

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