Overview

  • Product name

    Anti-MLH1 antibody [EPR20522]
    See all MLH1 primary antibodies
  • Description

    Rabbit monoclonal [EPR20522] to MLH1
  • Host species

    Rabbit
  • Tested applications

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

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human MLH1 aa 300-400. The exact sequence is proprietary.
    Database link: P40692

  • Positive control

    • WB: SW480 and HeLa whole cell lysates; human colon tissue lysate. IHC-P: Human colon, colon cancer and ovarian cancer tissues. ICC/IF: SW480 cells. Flow: SW480 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

Applications

Our Abpromise guarantee covers the use of ab223844 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 84 kDa (predicted molecular weight: 84 kDa).
IHC-P 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/100.
Flow Cyt 1/600.
IP 1/30.

Target

  • Function

    Heterodimerizes with PMS2 to form MutL alpha, a component of the post-replicative DNA mismatch repair system (MMR). DNA repair is initiated by MutS alpha (MSH2-MSH6) or MutS beta (MSH2-MSH6) binding to a dsDNA mismatch, then MutL alpha is recruited to the heteroduplex. Assembly of the MutL-MutS-heteroduplex ternary complex in presence of RFC and PCNA is sufficient to activate endonuclease activity of PMS2. It introduces single-strand breaks near the mismatch and thus generates new entry points for the exonuclease EXO1 to degrade the strand containing the mismatch. DNA methylation would prevent cleavage and therefore assure that only the newly mutated DNA strand is going to be corrected. MutL alpha (MLH1-PMS2) interacts physically with the clamp loader subunits of DNA polymerase III, suggesting that it may play a role to recruit the DNA polymerase III to the site of the MMR. Also implicated in DNA damage signaling, a process which induces cell cycle arrest and can lead to apoptosis in case of major DNA damages. Heterodimerizes with MLH3 to form MutL gamma which plays a role in meiosis.
  • Tissue specificity

    Colon, lymphocytes, breast, lung, spleen, testis, prostate, thyroid, gall bladder and heart.
  • Involvement in disease

    Defects in MLH1 are the cause of hereditary non-polyposis colorectal cancer type 2 (HNPCC2) [MIM:609310]. 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, and accounts for 15% of all colon cancers. 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. The term 'suspected HNPCC' or 'incomplete HNPCC' can be used to describe families who do not or only partially fulfill the Amsterdam criteria, but in whom a genetic basis for colon cancer is strongly suspected.
    Defects in MLH1 are a cause of mismatch repair cancer syndrome (MMRCS) [MIM:276300]; also known as Turcot syndrome or brain tumor-polyposis syndrome 1 (BTPS1). MMRCS is an autosomal dominant disorder characterized by malignant tumors of the brain associated with multiple colorectal adenomas. Skin features include sebaceous cysts, hyperpigmented and cafe au lait spots.
    Defects in MLH1 are a cause of Muir-Torre syndrome (MuToS) [MIM:158320]; also abbreviated MTS. MuToS is a rare autosomal dominant disorder characterized by sebaceous neoplasms and visceral malignancy.
    Note=Defects in MLH1 may contribute to lobular carcinoma in situ (LCIS), a non-invasive neoplastic disease of the breast.
    Defects in MLH1 are a cause of susceptibility to endometrial cancer (ENDMC) [MIM:608089].
    Note=Some epigenetic changes can be transmitted unchanged through the germline (termed 'epigenetic inheritance'). Evidence that this mechanism occurs in humans is provided by the identification of individuals in whom 1 allele of the MLH1 gene is epigenetically silenced throughout the soma (implying a germline event). These individuals are affected by HNPCC but does not have identifiable mutations in MLH1, even though it is silenced, which demonstrates that an epimutation can phenocopy a genetic disease.
  • Sequence similarities

    Belongs to the DNA mismatch repair mutL/hexB family.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Database links

  • Alternative names

    • COCA 2 antibody
    • COCA2 antibody
    • DNA mismatch repair protein Mlh1 antibody
    • FCC 2 antibody
    • FCC2 antibody
    • hMLH 1 antibody
    • hMLH1 antibody
    • HNPCC 2 antibody
    • HNPCC antibody
    • HNPCC2 antibody
    • MGC5172 antibody
    • MLH 1 antibody
    • MLH1 antibody
    • MLH1_HUMAN antibody
    • MutL homolog 1 (E. coli) antibody
    • MutL homolog 1 antibody
    • MutL homolog 1 colon cancer nonpolyposis type 2 antibody
    • MutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) antibody
    • MutL protein homolog 1 antibody
    • MutL, E. coli, homolog of, 1 antibody
    see all

Images

  • All lanes : Anti-MLH1 antibody [EPR20522] (ab223844) at 1 µg/ml

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : MLH1 knockout HAP1 whole cell lysate
    Lane 3 : HCT116 whole cell lysate
    Lane 4 : Hek293 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 84 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab223844 observed at 85 kDa. Red - loading control, ab9484, observed at 37 kDa

    ab223844 was shown to specifically react with MLH1 in wild-type HAP1 cells as signal was lost in MLH1 knockout cells. Wild-type and MLH1 knockout samples were subjected to SDS-PAGE. Ab223844 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 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.

  • All lanes : Anti-MLH1 antibody [EPR20522] (ab223844) at 1/1000 dilution

    Lane 1 : SW480 (human colorectal adenocarcinoma cell line) whole cell lysate
    Lane 2 : HCT 116 (human colorectal carcinoma epithelial cell line) whole cell lysate
    Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 4 : Human colon tissue lysate

    Lysates/proteins at 20 µg per lane.

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

    Predicted band size: 84 kDa
    Observed band size: 84 kDa



    Exposure times. 

    Lanes 1-3: 8 seconds 
    Lane 4: 3 minutes.

    The expression profile observed is consistent with the literature (PMID:15249596). The HCT116 cell line is a negative control for MLH1 (PMID: 23724141).

  • Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labelling MLH1 with ab223844 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in tumor cells of human colon cancer is observed (PMID: 22608206). Counter stained with hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human colon tissue labelling MLH1 with ab223844 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in human colon is observed (PMID: 10535979). Counter stained with hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human ovary cancer tissue labelling MLH1 with ab223844 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in tumor cells of human ovarian cancer (PMID: 10778972) is observed. Counter stained with hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunofluorecent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilised SW480 (human colorectal adenocarcinoma cell line) cells labelling MLH1 with ab223844 at 1/100 dilution, followed by AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution (green). Confocal image showing nuclear staining in SW480 cell line. 

    DAPI was used as the Nuclear counterstain (blue). Alpha Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

    The HCT 116 (human colorectal carcinoma epithelial) cell line is a negative control for MLH1 (PMID: 23724141). 

    Secondary antibody only control: PBS instead of ab223844, followed by AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HCT 116 (human colorectal carcinoma epithelial cell line, left) / SW480 (human colorectal adenocarcinoma cell line, right) cells labelling MLH1 with ab223844 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

    The HCT 116 (human colorectal carcinoma epithelial) cell line is a negative control for MLH1 (PMID: 23724141)(left panel).

  • MLH1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab223844 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223844 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/10000 dilution.

    Lane 1: HeLa whole cell lysate 10 μg (input).
    Lane 2: ab223844 IP in HeLa whole cell lysate.
    Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab223844 in HeLa whole cell lysate.

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

    Exposure time: Less than 1 second.

     

References

ab223844 has not yet been referenced specifically in any publications.

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