The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/2000 - 1/10000. Detects a band of approximately 105 kDa (predicted molecular weight: 87 kDa).
Use at 2-5 µg/mg of lysate.
Use a concentration of 1 µg/ml.
Hydrolase that can remove conjugated ubiquitin from target proteins such as p53/TP53, SNX3 and CFTR. Acts as an essential regulator of p53/TP53 stability: in unstressed cells, specifically deubiquitinates p53/TP53 in the cytoplasm, leading to counteract MDM2 action and stabilize p53/TP53. Following DNA damage, translocates to the nucleus and deubiquitinates p53/TP53, leading to regulate the p53/TP53-dependent DNA damage response. Does not deubiquitinate MDM2. Deubiquitinates CFTR in early endosomes, enhancing its endocytic recycling.
Belongs to the peptidase C19 family. USP10 subfamily.
Phosphorylated by ATM following DNA damage, leading to stablization and translocation it to the nucleus.
Cytoplasm. Nucleus. Early endosome. Cytoplasmic in normal conditions. After DNA damage, translocates to the nucleus following phosphorylation by ATM.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue labelling USP10 with ab70895 at 1/1000 (0.2µg/ml). Detection: DAB.
Western blot - USP10 antibody (ab70895)
All lanes : Anti-USP10 antibody (ab70895) at 0.04 µg/ml
Lane 1 : Whole cell lysate from HeLa cells at 50 µg Lane 2 : Whole cell lysate from HeLa cells at 15 µg Lane 3 : Whole cell lysate from HeLa cells at 5 µg Lane 4 : Whole cell lysate from 293T cells at 50 µg
Detection of Human USP10 by Immunoprecipitation in Whole cell lysate from HeLa cells (1 mg for IP, 20% of IP loaded), using ab70895 at 3 µg/mg lysate for IP, and at 1 µg/ml for subsequent Western blot detection.
Western blot - Anti-USP10 antibody (ab70895)
All lanes : Anti-USP10 antibody (ab70895) at 0.1 µg/ml
Lane 1 : TCMK-1 whole cell lysate Lane 2 : 4T1 whole cell lysate Lane 3 : CT26.WT whole cell lysate
ICC/IF image of ab70895 stained HeLa cells. The cells were 4% formaldehyde fixed (10 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 (ab70895, 1µg/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.
IHC image of ab70895 staining in human normal tonsil formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab70895, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Katoh H et al. Japanese encephalitis virus core protein inhibits stress granule formation through an interaction with Caprin-1 and facilitates viral propagation. J Virol87:489-502 (2013).
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