Recombinant 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 (Intra), ICC/IF
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Nck antibody [Y531]
See all Nck primary antibodies -
Description
Rabbit monoclonal [Y531] to Nck -
Host species
Rabbit -
Tested applications
Suitable for: WB, IHC-P, Flow Cyt (Intra), ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- Hela cell lysate and human breast carcinoma ICC/IF: HeLa cells
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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
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
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Form
Liquid -
Storage instructions
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y531 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32120 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
1/2000 - 1/10000. Detects a band of approximately 47 kDa.
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
1/1000.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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ICC/IF |
1/250.
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Notes |
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WB
1/2000 - 1/10000. Detects a band of approximately 47 kDa. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Flow Cyt (Intra)
1/1000. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
ICC/IF
1/250. |
Target
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Function
Adapter protein which associates with tyrosine-phosphorylated growth factor receptors or their cellular substrates. Maintains low levels of EIF2S1 phosphorylation by promoting its dephosphorylation by PP1. Plays a role in the DNA damage response, not in the detection of the damage by ATM/ATR, but for efficient activation of downstream effectors, such as that of CHEK2. -
Sequence similarities
Contains 1 SH2 domain.
Contains 3 SH3 domains. -
Post-translational
modificationsPhosphorylated on Ser and Tyr residues. -
Cellular localization
Cytoplasm. Endoplasmic reticulum. Nucleus. Mostly cytoplasmic, but shuttles between the cytoplasm and the nucleus. Import into the nucleus requires the interaction with SOCS7. Predominantly nuclear following genotoxic stresses, such as UV irradiation, hydroxyurea or mitomycin C treatments. - Information by UniProt
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Database links
- Entrez Gene: 4690 Human
- Entrez Gene: 17973 Mouse
- Entrez Gene: 300955 Rat
- Omim: 600508 Human
- SwissProt: P16333 Human
- SwissProt: Q99M51 Mouse
- Unigene: 477693 Human
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Alternative names
- Cytoplasmic protein NCK1 antibody
- Melanoma Nck protein antibody
- MGC12668 antibody
see all
Images
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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.
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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.
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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.
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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.
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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.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (5)
ab32120 has been referenced in 5 publications.
- Guan T et al. Aristolochic acid inhibits Slit2-induced migration and tube formation via inactivation of Robo1/Robo2-NCK1/NCK2 signaling pathway in human umbilical vein endothelial cells. Toxicol Lett 300:51-58 (2019). PubMed: 30381256
- Xia P et al. NCK1 promotes the angiogenesis of cervical squamous carcinoma via Rac1/PAK1/MMP2 signal pathway. Gynecol Oncol N/A:N/A (2018). PubMed: 30442385
- Wu X et al. Activation of diverse signalling pathways by oncogenic PIK3CA mutations. Nat Commun 5:4961 (2014). PubMed: 25247763
- He B et al. A remote cis-acting variant at 3q links glomerular NCK1 to diabetic nephropathy. PLoS One 8:e56414 (2013). WB ; Mouse . PubMed: 23441190
- de Castro RO et al. Once phosphorylated, tyrosines in carboxyl terminus of protein-tyrosine kinase Syk interact with signaling proteins, including TULA-2, a negative regulator of mast cell degranulation. J Biol Chem 287:8194-204 (2012). WB . PubMed: 22267732