The antibody binds to Calpain-10, but does not cross react with the other calpain family members (Calpain-1, Calpain-2, Calpain-3, etc.).
The antibody binds to the aminoterminal end of domain-III and recognizes latent and amino-processed Calpain-10.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. Predicted molecular weight: 74.9 kDa. 1/1000 when using colorimetric substrates such as BCIP/NBT, and 1:5000 for chemiluminescent substrates. Higher concentrations of antibody may be needed for samples from more distantly related species. Post-translational modifications such as phosphorylation can produce isoforms with larger apparent MW on SDS PAGE gels. When used against the reduced protein, ab28226 identifies bands at 78 kD, 66 kD, and a series of further cleaved forms. Dilution optimised using Chromogenic detection.
Use a concentration of 1 - 5 µg/ml.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
Involvement in disease
Defects in CAPN10 are a cause of susceptibility to diabetes mellitus non-insulin-dependent type 1 (NIDDM1) [MIM:601283]. It is a multifactorial disorder of glucose homeostasis caused by a lack of sensitivity to the body's own insulin. Affected individuals usually have an obese body habitus and manifestations of a metabolic syndrome characterized by diabetes, insulin resistance, hypertension and hypertriglyceridemia. The disease results in long-term complications that affect the eyes, kidneys, nerves, and blood vessels.
Belongs to the peptidase C2 family. Contains 1 calpain catalytic domain.
ICC/IF image of ab28226 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 (ab28226, 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 ab28226 staining in normal human pancreas 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 ab28226, 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.
Panis C et al. The positive is inside the negative: HER2-negative tumors can express the HER2 intracellular domain and present a HER2-positive phenotype. Cancer Lett357:186-95 (2015).
Read more (PubMed: 25434795) »
Smith MA & Schnellmann RG Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury. Arch Biochem Biophys517:144-52 (2012).
Read more (PubMed: 22179018) »