Antibodies, Proteins, Kits and Reagents for Life Science

351 792 ₸ Product size

100 µl 351 792 ₸ 10 µl 140 716 ₸

Order now and get it on Tuesday March 02, 2021

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, WB, IHC-P, ICC/IF, IP
Reacts with: Mouse, Rat, Human

Product name

Anti-NEDD8 antibody [Y297]
See all NEDD8 primary antibodies
Description

Rabbit monoclonal [Y297] to NEDD8
Host species

Rabbit
Specificity

ab81264 detects NEDD8 bound to cullins proteins (neddylated cullins) as well as free NEDD8.

Tested Applications & Species

Application	Species
Flow Cyt	Human
ICC/IF	Human
IHC-P	Human
IP	Human
WB	Rat Human

See all applications and species data

Immunogen

Synthetic peptide within Human NEDD8 (N terminal). The exact sequence is proprietary.
Positive control
- Hela and K562 cell lysates and human lung carcinoma. Flow Cyt: A-673
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.

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...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

Y297
Isotype

IgG
Research areas

Western blot - Anti-NEDD8 antibody [Y297] (ab81264)

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
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEDD8 antibody [Y297] (ab81264)

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.
Immunocytochemistry/ Immunofluorescence - Anti-NEDD8 antibody [Y297] (ab81264)

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.
Flow Cytometry - Anti-NEDD8 antibody [Y297] (ab81264)

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 Fluor^®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).
Western blot - Anti-NEDD8 antibody [Y297] (ab81264)

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/TBST

The band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.
Western blot - Anti-NEDD8 antibody [Y297] (ab81264)

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/TBST

The band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.
Western blot - Anti-NEDD8 antibody [Y297] (ab81264)

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/TBST

The band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.
Immunoprecipitation - Anti-NEDD8 antibody [Y297] (ab81264)

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
Western blot - Anti-NEDD8 antibody [Y297] (ab81264)

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
Immunoprecipitation - Anti-NEDD8 antibody [Y297] (ab81264)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEDD8 antibody [Y297] (ab81264)

Unpurified ab81264, at 1/100 dilution, staining NEDD8 in human lung carcinoma tissue by Immunohistochemistry using formalin-fixed, paraffin-embedded section.
Anti-NEDD8 antibody [Y297] (ab81264)

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