Recombinant Anti-MSH6 antibody [EPR3945] (ab92471)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR3945] to MSH6
- Suitable for: WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-MSH6 antibody [EPR3945]
See all MSH6 primary antibodies -
Description
Rabbit monoclonal [EPR3945] to MSH6 -
Host species
Rabbit -
Tested applications
Suitable for: WB, IHC-P, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide within Human MSH6 aa 1-100 (N terminal). The exact sequence is proprietary.
Database link: P52701 -
Positive control
- WB: A431, HeLa, HAP1 and SW480 cell lysates; Rat brain lysate. IHC-P: Human colon and colonic adenocarcinoma tissue; Rat liver tissue ICC/IF: HeLa and HAP1 cells.
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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. Stable for 12 months at -20°C. -
Dissociation constant (KD)
KD = 2.30 x 10 -9 M Learn more about KD -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR3945 -
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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KO cell lines
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KO cell lysates
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab92471 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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WB | (3) |
1/1000 - 1/10000. Predicted molecular weight: 153 kDa.
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IHC-P | (1) |
1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
See IHC antigen retrieval protocols. For unpurified, use 1/100 - 1/250. |
ICC/IF | (1) |
Use a concentration of 1 µg/ml.
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Notes |
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WB
1/1000 - 1/10000. Predicted molecular weight: 153 kDa. |
IHC-P
1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. See IHC antigen retrieval protocols. For unpurified, use 1/100 - 1/250. |
ICC/IF
Use a concentration of 1 µg/ml. |
Target
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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
modificationsThe 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
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Database links
- Entrez Gene: 2956 Human
- Entrez Gene: 17688 Mouse
- Entrez Gene: 100360342 Rat
- Omim: 600678 Human
- SwissProt: P52701 Human
- SwissProt: P54276 Mouse
- Unigene: 445052 Human
- Unigene: 18210 Mouse
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Alternative names
- DNA mismatch repair protein Msh6 antibody
- G/T mismatch binding protein antibody
- G/T mismatch-binding protein antibody
see all
Images
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Immunohistochemical staining of paraffin embedded human colon with purified ab92471 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
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All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MSH6 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 153 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab92471 observed at 160 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.
ab92471 was shown to react with MSH6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255410 (knockout cell lysate ab263763) was used. Wild-type HeLa and MSH6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab92471 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.
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ab92471 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 ab92471 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).
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Immunohistochemical staining of paraffin embedded rat liver with purified ab92471 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
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ab92471 staining MSH6 in HeLa cells. 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 ab92471 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).
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Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution (purified) + Rat brain at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 153 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution
Lane 1 : HAP1 WT cell lysate
Lane 2 : MSH6 KO HAP1 cell lysate
Lysates/proteins at 50 µg per lane.
Secondary
All lanes : donkey anti-rabbit IgG-HRP at 1/3000 dilution
Developed using the ECL technique.
Predicted band size: 153 kDa
Exposure time: 5 minutes
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purified at 1/6000 dilution + SW480 cell lysate at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 153 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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Different batches of ab92471 were tested on Rat brain lysate at 0.2 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 160 kDa.
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Anti-MSH6 antibody [EPR3945] (ab92471) at 1/2000 dilution (unpurified) + SW480 cell lysate at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 153 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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Unpurified ab92471, at a 1/100 dilution, detecting MSH6 in paraffin embedded Human colonic adenocarcinoma tissue by immunohistochemistry. Detection used HRP conjugated anti rabbit antibody.
Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.
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Unpurified ab92471 (1/500) staining MSH6 in asynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see abreview.
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All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution (unpurified)
Lane 1 : A431 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : SW480 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : HRP labelled goat anti-rabbit antibody at 1/2000 dilution
Predicted band size: 153 kDaSecondary antibody - goat anti-rabbit HRP (ab6721)
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Tissue Microarrays stained for "Anti-MSH6 antibody [EPR3945]” using "ab92471" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with ab92471 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
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Tissue Microarrays stained for "Anti-MSH6 antibody [EPR3945]” using "ab92471" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with ab92471 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (52)
ab92471 has been referenced in 52 publications.
- Takano S et al. Next-generation sequencing of endoscopically obtained tissues from patients with all stages of pancreatic cancer. Cancer Sci 113:1069-1077 (2022). PubMed: 34962016
- Koster S et al. Modelling Chlamydia and HPV co-infection in patient-derived ectocervix organoids reveals distinct cellular reprogramming. Nat Commun 13:1030 (2022). PubMed: 35210413
- Liao Y et al. Efficacy of PD-1 Inhibitor Combined with Bevacizumab in Treatment of Advanced Endometrial Cancer Patients with Mismatch Repair Deficiency (dMMR)/High-Level Microsatellite Instability (MSI-H). Med Sci Monit 28:e934493 (2022). PubMed: 35322001
- Guan B et al. Identification of Germline Mutations in Upper Tract Urothelial Carcinoma With Suspected Lynch Syndrome. Front Oncol 12:774202 (2022). PubMed: 35372080
- Cardin DB et al. Safety and Efficacy of Avelumab in Small Bowel Adenocarcinoma. Clin Colorectal Cancer 21:236-243 (2022). PubMed: 35450836