Recombinant Anti-MLH1 antibody [EPR20522] (ab223844)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR20522] to MLH1
- Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF, IP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-MLH1 antibody [EPR20522]
See all MLH1 primary antibodies -
Description
Rabbit monoclonal [EPR20522] to MLH1 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF, IPmore details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: SW480 and HeLa whole cell lysates; human colon tissue lysate. IHC-P: Human colon, colon cancer and ovarian cancer tissues, human breast tissue ICC/IF: Hap1 (MLH1 knockout Hap1 cells used as a negative control) cells. Flow cyto (intra): SW480 cells, Hap1 cells. IP: HeLa whole cell lysate.
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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.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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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
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR20522 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
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 |
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Flow Cyt (Intra) |
1/600.
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WB |
1/1000. Detects a band of approximately 84 kDa (predicted molecular weight: 84 kDa).
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IHC-P |
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
Use a concentration of 0.2 µg/ml.
This product gave a positive signal in Hap1 (MLH1 knockout Hap1 cells used as a negative control) fixed with 4% formaldehyde (10 min). |
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IP |
1/30.
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Notes |
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Flow Cyt (Intra)
1/600. |
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
Use a concentration of 0.2 µg/ml. This product gave a positive signal in Hap1 (MLH1 knockout Hap1 cells used as a negative control) fixed with 4% formaldehyde (10 min). |
IP
1/30. |
Target
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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
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Database links
- Entrez Gene: 4292 Human
- Omim: 120436 Human
- SwissProt: P40692 Human
- Unigene: 195364 Human
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Alternative names
- COCA 2 antibody
- COCA2 antibody
- DNA mismatch repair protein Mlh1 antibody
see all
Images
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Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labelling MLH1 with ab223844 at 1/100 (7.79 μg/ml) dilution for 10 minutes at room temperature, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on human breast carcinoma is observed. Counter stained with hematoxylin.The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Secondary antIbody only control - Secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use.
Perform heat mediated antigen retrieval with Tris/EDTA buffer (pH 9.0,Epitope Retrieval Solution2) for 10 minutes before commencing with IHC staining protocol.
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Flow cytometry overlay histogram showing wild-type Hap1 (green line) and MLH1 knockout Hap1 stained with ab223844 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab223844) (1x 106 in 100μl at 1.0 μg/ml (1/2070)) for 30min at 22°C.
The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, MLH1 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
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All lanes : Anti-MLH1 antibody [EPR20522] (ab223844) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : MLH1 CRISPR-Cas9 edited A549 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HCT 116 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 84 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-MLH1 antibody [EPR20522] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab223844 was shown to bind specifically to MLH1. A band was observed at 85 kDa in wild-type A549 cell lysates with no signal observed at this size in MLH1 CRISPR-Cas9 edited cell line ab276105 (CRISPR-Cas9 edited cell lysate ab283566). The band observed in the CRISPR-Cas9 edited lysate lane below 85 kDa is likely to represent a truncated form of MLH1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MLH1 CRISPR-Cas9 edited A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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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 kDaLanes 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. -
ab223844 staining MLH1 in wild-type Hap1 cells, with negative expression in MLH1 knockout Hap1 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised 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 overnight at +4°C with ab223844 at 0.2 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.
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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
Predicted band size: 84 kDa
Observed band size: 84 kDaExposure 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).
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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.
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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.
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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.
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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. -
Intracellular 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).
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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.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (1)
ab223844 has been referenced in 1 publication.
- Cannavo E et al. Regulation of the MLH1-MLH3 endonuclease in meiosis. Nature 586:618-622 (2020). PubMed: 32814904