The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
1/100. Antigen retieval: boil tissue section in 1mM EDTA, pH 8.0 for 10 minutes followed by cooling at room temperature for 20 minutes. Primary antibody incubation: 30 minutes at room temperature.
功能Component of the post-replicative DNA mismatch repair system (MMR). Heterodimerizes with MSH2 to form MutS alpha, which binds to DNA mismatches thereby initiating DNA repair. When bound, MutS alpha bends the DNA helix and shields approximately 20 base pairs, and recognizes single base mismatches and dinucleotide insertion-deletion loops (IDL) in the DNA. After mismatch binding, forms a ternary complex with the MutL alpha heterodimer, which is thought to be responsible for directing the downstream MMR events, including strand discrimination, excision, and resynthesis. ATP binding and hydrolysis play a pivotal role in mismatch repair functions. The ATPase activity associated with MutS alpha regulates binding similar to a molecular switch: mismatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts MutS alpha into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. This transition is crucial for mismatch repair. MutS alpha may also play a role in DNA homologous recombination repair.
疾病相关Defects in MSH6 are the cause of hereditary non-polyposis colorectal cancer type 5 (HNPCC5) [MIM:600678]. Mutations in more than one gene locus can be involved alone or in combination in the production of the HNPCC phenotype (also called Lynch syndrome). Most families with clinically recognized HNPCC have mutations in either MLH1 or MSH2 genes. HNPCC is an autosomal, dominantly inherited disease associated with marked increase in cancer susceptibility. It is characterized by a familial predisposition to early onset colorectal carcinoma (CRC) and extra-colonic cancers of the gastrointestinal, urological and female reproductive tracts. HNPCC is reported to be the most common form of inherited colorectal cancer in the Western world. Cancers in HNPCC originate within benign neoplastic polyps termed adenomas. Clinically, HNPCC is often divided into two subgroups. Type I: hereditary predisposition to colorectal cancer, a young age of onset, and carcinoma observed in the proximal colon. Type II: patients have an increased risk for cancers in certain tissues such as the uterus, ovary, breast, stomach, small intestine, skin, and larynx in addition to the colon. Diagnosis of classical HNPCC is based on the Amsterdam criteria: 3 or more relatives affected by colorectal cancer, one a first degree relative of the other two; 2 or more generation affected; 1 or more colorectal cancers presenting before 50 years of age; exclusion of hereditary polyposis syndromes. MSH6 mutations appear to be associated with atypical HNPCC and in particular with development of endometrial carcinoma or atypical endometrial hyperplasia, the presumed precursor of endometrial cancer. Defects in MSH6 are also found in familial colorectal cancers (suspected or incomplete HNPCC) that do not fulfill the Amsterdam criteria for HNPCC. Defects in MSH6 are a cause of susceptibility to endometrial cancer (ENDMC) [MIM:608089].
序列相似性Belongs to the DNA mismatch repair mutS family. Contains 1 PWWP domain.
翻译后修饰The N-terminus is blocked. Phosphorylated upon DNA damage, probably by ATM or ATR. Phosphorylated by PRKCZ, which may prevent MutS alpha degradation by the ubiquitin-proteasome pathway.
Western blot - Anti-MSH6 antibody [SP93] (ab99889)
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: MSH6 knockout HAP1 cell lysate (20 µg) Lane 3: HeLa cell lysate (20 µg) Lane 4: A431 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab99889 observed at 160 kDa. Red - loading control, ab18058, observed at 124 kDa.
ab99889 was shown to specifically react with MSH6 when MSH6 knockout samples were used. Wild-type and MSH6 knockout samples were subjected to SDS-PAGE. ab99889 and ab18058 (loading control to Vinculin) were diluted at 1 μg/ml and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.