Overview

  • Product name
    Anti-MLH1 antibody [G168-15]
    See all MLH1 primary antibodies
  • Description
    Mouse monoclonal [G168-15] to MLH1
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, IHC-P, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Human
  • Immunogen

    Recombinant full length protein.

  • Positive control
    • Tonsil, colon carcinoma. This antibody gave a positive result in IF/ICC when used in the following formaldehyde fixed cell lines: HepG2

Properties

Applications

Our Abpromise guarantee covers the use of ab14206 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
Flow Cyt 1/50.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

ICC/IF 1/30. PubMed: 17543860
IHC-P 1/25 - 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use at an assay dependent concentration. Predicted molecular weight: 85 kDa. PubMed: 29488881

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

  • Formalin fixed paraffin embedded human tonsil stained with MLH1 using ABC and DAB chromogen.
  • Overlay histogram showing HeLa cells stained with ab14206 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab14206, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human colon carcinoma tissue, staining MLH1 with ab14206.
  • ICC/IF image of ab14206 stained HepG2 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 ab14206 at 1/50 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) 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.

References

This product has been referenced in:
  • Hodel KP  et al. Explosive mutation accumulation triggered by heterozygous human Pol e proofreading-deficiency is driven by suppression of mismatch repair. Elife 7:N/A (2018). WB . Read more (PubMed: 29488881) »
  • Hajkova N  et al. Germline mutation in the TP53 gene in uveal melanoma. Sci Rep 8:7618 (2018). Read more (PubMed: 29769598) »
See all 11 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

Merci de nous avoir contactés.

Je suis désolé d'apprendre que le produit ab14206 que vous avez reçu ne fonctionne pas comme attendu. Les informations que vous m'avez fournies vont nous permettre de chercher l'origine du problème rencontré avec cet anticorps et de prendre les dispositions nécessaires pour assurer une bonne qualité de nos produits.

Comme convenu, j'ai mis en place l'envoi d'une unité d'un lot différent, le numéro de commande de remplacement gratuit de ce produit est *****.

Concernant la date de livraison, le système n'avait pas été mis à jour lorsque j'ai regardé la disponibilité du produit. La livraison ne pourra malheureusement pas se faire demain comme je vous l'ai dit par téléphone mais en début de semaine prochaine. Désolé pour cette erreur.

Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

N'hésitez pas à me communiquer les résultats obtenus avec ce nouveau lot de ab14206.

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Question
Answer

Thank you for your call today and for letting us know about the trouble with this antibody.

As we discussed, I'm sending a free of charge vial of ab92312 on the order *** which should arrive shortly.

Please keep me updated about the results after using this new antibody, and let me know if you have any further questions or if there is anything else that we can do for you. Have a wonderful day!

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Answer

Thank you for contacting us.
The immunogen for ab14206 is the full length protein. However, the epitope of this antibody is not mapped. We do not know the sequence homology of the epitope with the chicken protein. The homology of the full length protein between human and chicken is 79.6%. We have not tested this antibody in chicken so we do not have any empirical data to support its use in the species.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your response. I have not had a chance to use MLH1 in my experiments. I recommend changes in fixation based on Abcams recommended protocols for ICC-IF. We recommend that users fix samples either in ice-cold methanol, acetone (1-10 min) or in 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature. Often when a customer is experiencing issues with a product, changing fixation procedures, permeabilization steps or antigen retrieval will yield positive results. Please let us know if you have any other questions or if there is anything else that Abcam can do to help you reach your research goals.

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Question

DESCRIPTION OF THE PROBLEM non-specific staining and high backgroud SAMPLE rat esticula rtissue:seminiferous tubules PRIMARY ANTIBODY MLH1, mouse monoclonal antibody DETECTION METHOD The procedure we follow the prepublised paper Gamma-irradiation increased meiotic cross overs in mouse spermatocytes, and with little remedied. In briefly, MLH1 was diluted at 1:30, applied to each slide,covered with a glass cover slip and sealed with finger oil,and the slides were incubated for 24h at 37 in a humid chamber. On the following day,after removing covers lips,slides were washed in 0.5% tween in PBS solution for 10 min three times at room temperature soaked in blocking solution in a humid chamber for 6h and repeated 0.5% tween in PBS washing for 10min three times at room temperature and for 3min three times at 4 washing for 3 times.Subsequent to the wash, secondary antibody (goat anti-mouse Alexa Fluor 488, 1:100) was applied to the slides. Slides were incubated at 37 in a humid chamber for 90min and were washed three times in 0.5% tween in PBS solution for 10,20 and 30min.Subsequently,slides were stained with DAPI at room temperature for 15 min.And then slides were washed in 0.5% tween in PBS solution 3 times for 5min. At last, a glass cover slip was then applied and sealed . POSITIVE AND NEGATIVE CONTROLS USED no. i dont know how to make positive control. we make negative control,which indicated that goat anti-mouse Alexa Fluor 488 was ok. ANTIBODY STORAGE CONDITIONS 4 centigrade FIXATION OF SAMPLE 1%paraformaldehyde(PFA),pH9.2, solution containing 0.15%TritonX-100 ANTIGEN RETRIEVAL no PERMEABILIZATION STEP Testiculartissue wasshreddedwithtwopairsofforceps,andthereleasedpachytenecellswere spreadevenlyovermicroscopeslideslayeredwithparaformaldehyde(

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Answer

Thank you for contacting Abcam regarding this issue. I am sorry to here of the problems that you have been experiencing using ab14206. Often, changing the fixation step of your tissue can lead to better results. A methanol fixation may work well for your tissue. I have attached a protocol detailing different fixation options for you. Other have had success using antigen retrieval and I would recommend an antigen retrieval step such as Tris/EDTA pH 9.0 heat induced antigen retrieval. I've attached pdfs of Abcams IHC protocols for you. I would highly recommend using a positive control tissue, tonsil and colon carcinoma are the recommended tissue for this product. I hope that you find these tips useful and wish you the success with your experiments. If you have any questions, please feel free to contact us.

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Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of a different lot of ab14206. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

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Answer

Thanks for the additional information. I agree that the vial of ab14206 you received is not working as expected. If you have purchased this antibody within the last 6 months or so I would be happy to offer a replacement, credit or refund. In your reply, please note the purchase order number or Abcam order reference number associated with the order so I can process your request.

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Answer

Thank you for the images. Does your sentence "Besides the same second antibody can be work in another IHC detection with Cat#ab3366" mean that the secondary antibody works well? The colon tissue must be colon carcinoma, not normal colon tissue as the latter does not have high levels of MLH1. Please provide a detailed protocol of the customer's staining (using the Abcam questionnaire pasted in the body of an e-mail or via our website). In the future please provide this information as soon as you contact us so we minimise the delay and inconvenience for the customer, Thank you for your support,

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Question
Answer

Thank you for your enquiry. Yes, this antibody has been applied against rat cells using immunohistochemistry (ABC method). I am not aware of its application against spermatocytes, nor the expression profile of MLH1. However, I am assuming its expression given that it is part of the repair machinery. We guarantee the antibody to behave as detailed on the datasheet. Should the antibody not behave we operate a 90 day refund or replacement vial policy from the date of delivery. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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