Anti-NEDD8 抗体 [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.

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). 

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

The band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.

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

The band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.

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

The band at 12kDa is free NEDD8, and at 90kDa is Neddylated cullins.

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 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.

Unpurified ab81264, at 1/100 dilution, staining NEDD8 in human lung carcinoma tissue by Immunohistochemistry using formalin-fixed, paraffin-embedded section.