概述

• 产品名称Anti-NEDD8抗体[Y297]
     

• 描述

  兔单克隆抗体[Y297] to NEDD8

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

• 经测试应用WB,IHC-P,ICC/IF,IP

• 种属反应性

  与反应: Mouse, Rat, Human    
   

• 免疫原

  Synthetic peptide corresponding to residues in the N terminal of human NEDD8.

• 阳性对照    

• Hela and K562 cell lysates and human lung carcinoma.

• 常规说明

  This product is a recombinant rabbit monoclonal antibody.

   

  Produced using Abcam’s RabMAb^® technology. RabMAb^® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

  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.

  Alternative versions available:
  Anti-NEDD8 antibody (Alexa Fluor^® 647) [Y297] (ab200849)

  Anti-NEDD8 antibody (Alexa Fluor^® 488) [Y297] (ab201249)

性能

• 形式Liquid

• 存放说明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.

• 存储溶液PBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%

• 
  

• 纯度Protein A purified

• 克隆单克隆

• 克隆编号Y297

• 同种型IgG

• 研究领域

• Cell Biology

• Proteolysis / Ubiquitin

• Proteasome / Ubiquitin

• Ub-like Proteins

Anti-NEDD8 antibody [Y297] 图像

• 

  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, HRP Veriblot for IP (ab131366) was used as the secondary antibody (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST  

• 

  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.

   

• 

  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.  

• 

  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
  Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
  
  Predicted band size : 9 kDa
   

• 

  Western blot - 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
  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 - 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) - 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.