Overview

  • Product name

    Anti-MSH6 antibody [EPR20316]
    See all MSH6 primary antibodies
  • Description

    Rabbit monoclonal [EPR20316] to MSH6
  • Host species

    Rabbit
  • Specificity

    This antibody recognizes isoforms 1 and 4 of MSH6.
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human MSH6 aa 1250-1350. The exact sequence is proprietary.
    Database link: P52701

  • Positive control

    • WB: Human testis lysate; A431, SW480, HepG2 and HeLa whole cell lysates. IHC-P: Human endometrium and colon cancer tissues. ICC/IF: HeLa cells, HAP1 cells (HAP1-MSH6 knockout cells used as negative cell line). Flow Cyt: HeLa cells. IP: HeLa whole cell lysate.
  • General notes

    To see more of the key markers and tools you need to study the hallmarks of cancer, including genome instability and mutation, please visit the following page.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR20316
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab208940 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000. Detects a band of approximately 160, 120 kDa (predicted molecular weight: 153 kDa).
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 µg/ml.
Flow Cyt 1/500.
IP 1/40.

Target

  • Function

    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.
  • Involvement in disease

    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].
  • Sequence similarities

    Belongs to the DNA mismatch repair mutS family.
    Contains 1 PWWP domain.
  • Post-translational
    modifications

    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.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Database links

  • Alternative names

    • DNA mismatch repair protein Msh6 antibody
    • G/T mismatch binding protein antibody
    • G/T mismatch-binding protein antibody
    • GTBP antibody
    • GTMBP antibody
    • hMSH6 antibody
    • HNPCC 5 antibody
    • HNPCC5 antibody
    • HSAP antibody
    • MSH 6 antibody
    • MSH6 antibody
    • MSH6_HUMAN antibody
    • mutS (E. coli) homolog 6 antibody
    • MutS alpha 160 kDa subunit antibody
    • MutS homolog 6 (E. coli) antibody
    • mutS homolog 6 antibody
    • MutS-alpha 160 kDa subunit antibody
    • p160 antibody
    • Sperm associated protein antibody
    see all

Images

  • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: MSH6 knockout  HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: A431 whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab208940 observed at 165 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab208940 was shown to specifically react with MSH6 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when MSH6 knockout samples were used. Wild-type and MSH6 knockout samples were subjected to SDS-PAGE.  Ab208940 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • ab208940 staining MSH6 in wild-type HAP1 cells (top panel) and MSH6 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab208940 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling MSH6 with ab208940 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 

    Nuclear staining on tumor cells of human endometrium is observed [PMID: 8942985].

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MSH6 with ab208940 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

    Confocal image showing nuclear staining on HeLa cell line.

    The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

  • Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling MSH6 with ab208940 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 

    Nuclear staining on tumor cells of human colon cancer is observed [PMID: 26315971].

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

  • Anti-MSH6 antibody [EPR20316] (ab208940) at 1/1000 dilution + Human testis lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

    Predicted band size: 153 kDa
    Observed band size: 120 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-MSH6 antibody [EPR20316] (ab208940) at 1/1000 dilution

    Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
    Lane 2 : SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate
    Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
    Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 153 kDa
    Observed band size: 120,160 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1,2 and 3: 3 minutes; Lane 4: 15 seconds.

    This antibody recognizes isoforms 1 and 4 of MSH6. The observed molecular weight corresponds to isoform 1 (160kDa) and isoform 4 (120kDa) which is consistent with the Uniprot annotation.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MSH6 with ab208940 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

  • MSH6 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208940 at 1/40 dilution.

    Western blot was performed from the immunoprecipitate using ab208940 at 1/1000 dilution.

    VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

    Lane 1: HeLa whole cell lysate, 10 µg (Input).

    Lane 2: ab208940 IP in HeLa whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208940 in HeLa whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 8 seconds.

    The observed molecular weight corresponds to isoform 1 (160kDa) and isoform 4 (120kDa) which is consistent with the Uniprot annotation.

References

ab208940 has not yet been referenced specifically in any publications.

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