The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/2000 - 1/10000.
1/1000 - 1/5000. Detects a band of approximately 118 kDa (predicted molecular weight: 118 kDa).
Use a concentration of 2 - 20 µg/ml.
Use a concentration of 5 µg/ml.
Activates ubiquitin by first adenylating its C-terminal glycine residue with ATP, and thereafter linking this residue to the side chain of a cysteine residue in E1, yielding an ubiquitin-E1 thioester and free AMP.
Protein modification; protein ubiquitination.
Involvement in disease
Defects in UBA1 are the cause of spinal muscular atrophy X-linked type 2 (SMAX2) [MIM:301830]; also known as X-linked lethal infantile spinal muscular atrophy, distal X-linked arthrogryposis multiplex congenita or X-linked arthrogryposis type 1 (AMCX1). Spinal muscular atrophy refers to a group of neuromuscular disorders characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. SMAX2 is a lethal infantile form presenting with hypotonia, areflexia, and multiple congenital contractures.
ICC/IF image of ab34711 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab34711, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
ab34711 (4µg/ml) staining E1 Ubiquitin activating enzyme in human testis using an automated system (DAKO Autostainer Plus). Using this protocol there is nuclear and weak cytoplasmic staining. Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
Western blot - Anti-E1 Ubiquitin Activating Enzyme antibody (ab34711)
All lanes : Anti-E1 Ubiquitin Activating Enzyme antibody (ab34711) at 1 µg/ml
Lane 1 : Human brain tissue lysate - total protein (ab29466) Lane 2 : Human liver tissue lysate - total protein (ab29889) Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 4 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg/ml per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 118 kDa Observed band size: 118 kDa Additional bands at: 35 kDa, 53 kDa. We are unsure as to the identity of these extra bands.