Recombinant Anti-NEDD8 antibody [Y297] (ab81264)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y297] to NEDD8
- Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF, IP
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-NEDD8 antibody [Y297]
See all NEDD8 primary antibodies -
Description
Rabbit monoclonal [Y297] to NEDD8 -
Host species
Rabbit -
Specificity
This antibody detects NEDD8 bound to cullins proteins (neddylated cullins) as well as free NEDD8.
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Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF, IPmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide within Human NEDD8 (N terminal). The exact sequence is proprietary.
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Positive control
- Hela and K562 cell lysates and human lung carcinoma. Flow Cyt (intra): A-673
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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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C. -
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
Y297 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Alexa Fluor® 647 Anti-NEDD8 antibody [Y297] (ab200849)
- Alexa Fluor® 488 Anti-NEDD8 antibody [Y297] (ab201249)
- Alexa Fluor® 594 Anti-NEDD8 antibody [Y297] (ab207247)
- Alexa Fluor® 405 Anti-NEDD8 antibody [Y297] (ab207248)
- Alexa Fluor® 555 Anti-NEDD8 antibody [Y297] (ab207249)
- Anti-NEDD8 antibody [Y297] - BSA and Azide free (ab220816)
- Alexa Fluor® 568 Anti-NEDD8 antibody [Y297] (ab312568)
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Compatible Secondaries
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Isotype control
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab81264 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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Flow Cyt (Intra) |
1/300.
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WB | (3) |
1/1000 - 1/10000. Detects a band of approximately 9 kDa (predicted molecular weight: 9 kDa).
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IHC-P |
1/100 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
1/100 - 1/250.
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IP |
1/20 - 1/50.
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Notes |
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Flow Cyt (Intra)
1/300. |
WB
1/1000 - 1/10000. Detects a band of approximately 9 kDa (predicted molecular weight: 9 kDa). |
IHC-P
1/100 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
1/100 - 1/250. |
IP
1/20 - 1/50. |
Target
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Function
Ubiquitin-like protein which plays an important role in cell cycle control and embryogenesis. Covalent attachment to its substrates requires prior activation by the E1 complex UBE1C-APPBP1 and linkage to the E2 enzyme UBE2M. Attachment of NEDD8 to cullins activates their associated E3 ubiquitin ligase activity, and thus promotes polyubiquitination and proteasomal degradation of cyclins and other regulatory proteins. -
Tissue specificity
Highly expressed in heart, skeletal muscle, spleen, thymus, prostate, testis, ovary, colon and leukocytes. -
Sequence similarities
Belongs to the ubiquitin family. -
Post-translational
modificationsCleavage of precursor form by UCHL3 or SENP8 is necessary for function. -
Cellular localization
Nucleus. Mainly nuclear. - Information by UniProt
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Database links
- Entrez Gene: 4738 Human
- Entrez Gene: 18002 Mouse
- Entrez Gene: 25490 Rat
- Omim: 603171 Human
- SwissProt: Q15843 Human
- SwissProt: P29595 Mouse
- SwissProt: Q71UE8 Rat
- Unigene: 531064 Human
see all -
Alternative names
- FLJ43224 antibody
- MGC104393 antibody
- MGC125896 antibody
see all
Images
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All lanes : Anti-NEDD8 antibody [Y297] (ab81264) at 1/1000 dilution (Unpurified)
Lane 1 : C6 Cell Lysate
Lane 2 : Raw264.7 Cell Lysate
Lane 3 : PC-12 Cell Lysate
Lane 4 : 3T3 Cell Lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 9 kDa -
Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab81264 at a working dilution of 1/400. The secondary antibody used is 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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Immunofluorescence staining of HeLa cells with purified ab81264 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with 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 ab81264 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.
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Intracellular Flow Cytometry analysis of A-673 (Human muscle Ewing's Sarcoma) cells labeling NEDD8 (red) with purified ab81264 at a 1/300 dilution (1ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluorr®488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).
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Anti-NEDD8 antibody [Y297] (ab81264) at 1/1000 dilution (purified) + PC-12 whole cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 9 kDa
Observed band size: 12,90 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBSTThe band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.
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Anti-NEDD8 antibody [Y297] (ab81264) at 1/1000 dilution (purified) + K562 whole cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 9 kDa
Observed band size: 12,90 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBSTThe band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.
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Anti-NEDD8 antibody [Y297] (ab81264) at 1/1000 dilution (purified) + HEK293 cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 9 kDa
Observed band size: 12,90 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBSTThe band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.
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ab81264 (purified) at 1/20 immunoprecipitating NEDD8 in 10 μg K562 cell lysate (Lanes 1 and 2, observed at 10 and 90 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730). For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST
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All lanes : Anti-NEDD8 antibody [Y297] (ab81264) at 1/500 dilution (Unpurified)
Lane 1 : HeLa cell lysate
Lane 2 : K562 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution
Predicted band size: 9 kDa
Observed band size: 9 kDa
Other Bands: 90kDa Neddylated cullins -
Western blot analysis of immunoprecipitation pellet from HeLa cell lysate using unpurified ab81264 at 1/500 dilution. Band at 90 kDa shows Neddylated cullins, 50 kDa the IgG fraaction and 9 kDa Free NEDD8.
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Unpurified ab81264, at 1/100 dilution, staining NEDD8 in human lung carcinoma tissue by Immunohistochemistry using formalin-fixed, paraffin-embedded section.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (39)
ab81264 has been referenced in 39 publications.
- Jones TM et al. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism. Cell Death Dis 13:350 (2022). PubMed: 35428778
- Zhou Q et al. Neddylation inhibition induces glutamine uptake and metabolism by targeting CRL3SPOP E3 ligase in cancer cells. Nat Commun 13:3034 (2022). PubMed: 35641493
- Serrano-Maciá M et al. Neddylation tunes peripheral blood mononuclear cells immune response in COVID-19 patients. Cell Death Discov 8:316 (2022). PubMed: 35831294
- Yang G et al. Neddylation Inhibition Causes Impaired Mouse Embryo Quality and Blastocyst Hatching Failure Through Elevated Oxidative Stress and Reduced IL-1β. Front Immunol 13:925702 (2022). PubMed: 35860255
- Xu T et al. A small molecule inhibitor of the UBE2F-CRL5 axis induces apoptosis and radiosensitization in lung cancer. Signal Transduct Target Ther 7:354 (2022). PubMed: 36253371