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Anti msh6

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Anti-MSH6 is a laboratory reagent used in various research applications. It is an antibody that specifically recognizes the MSH6 protein, which is a component of the DNA mismatch repair system. The core function of Anti-MSH6 is to detect and identify the presence of the MSH6 protein in biological samples.

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Lab products found in correlation

Anti msh2, by BD (3 mentions) Anti mlh1, by BD (3 mentions) Anti pms2, by BD (3 mentions) Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Anti actin, by Merck Group (1 mentions) Phospho histone h2a x, by Cell Signaling Technology (1 mentions) Phospho chk1, by Cell Signaling Technology (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions) Cell lytic m, by Merck Group (1 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Anti alk, by Cell Signaling Technology (1 mentions) Anti total caspase 3, by Cell Signaling Technology (1 mentions) Anti msh2, by Cell Signaling Technology (1 mentions) Anti phospho tyrosine, by Cell Signaling Technology (1 mentions) Anti flag, by Merck Group (1 mentions) Th5487, by Bio-Techne (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)

6 protocols using anti msh6

1

Protein Expression Analysis of DNA Damage Response

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Cells were treated with DMSO, TMZ (100 μM), ABT-888 (100 μM), or the combination of TMZ (100 μM) and ABT-888 (100 μM) and harvested at various time points. Whole cells were lysed in standard NP-40 lysis buffer (Life Technologies) with 1X protease inhibitors tablet, 0.1 mM NaVO3, 1 mM DTT, and 1 μM PMSF (Roche Applied Science). Samples were subjected to SDS-PAGE gel electrophoresis using standard protocols transferring 30 μg of protein onto a PVDF membrane (Millipore), followed by blocking with 10% nonfat milk powder for 30 min and overnight incubation at 4°C with either anti-MSH6 (Cat# 610918, BD Biosciences), anti-actin (Cat# MAB1501R Millipore), phospho-CHK1 (Ser 317, Cat# 12302S, Cell Signaling Technologies), CHK1 (Cat# 2360S, Cell Signaling Technologies) phospho-histone H2A.X (Ser 139, 80312S, Cell Signaling Technologies), or H2A.X (Cat# 2595S, Cell Signaling Technology) occording to manufacturer’s specifications. Membranes were incubated with appropriate secondary antibody for 1 h followed by enhanced chemiluminescence visualization and substrate detection (Thermo Scientific).
Yuan A.L., Ricks C.B., Bohm A.K., Lun X., Maxwell L., Safdar S., Bukhari S., Gerber A., Sayeed W., Bering E.A., Pedersen H., Chan J.A., Shen Y., Marra M., Kaplan D.R., Mason W., Goodman L.D., Ezhilarasan R., Kaufmann A.B., Cabral M., Robbins S.M., Senger D.L., Cahill D.P., Sulman E.P., Cairncross J.G, & Blough M.D. (2018). ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts. PLoS ONE, 13(8), e0202860.
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2

Evaluating Mismatch Repair Deficiency

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We used the presence of MSI to assess the loss of function of mismatch repair gene activity. Immunohistochemistry (IHC) and/or Multiplex polymerase chain reaction method was used to evaluate the status of MSI. We conducted IHC analysis of MSH2, MSH6, PMS2, and MLH1 proteins with formalin-fixed paraffin-embedded tumor samples. After the tumor area adjacent to normal mucosa and/or lymphocytic infiltration was marked, 4 mm of paraffinized tissue was removed, and multiple tissue blocks were prepared. Finally, 4-µm thick sections were obtained for IHC following standard protocols. The mouse monoclonal antibodies used were anti-MSH2, anti-MSH6, anti-MLH1, and anti-PMS2 (BD Pharmingen). Tumors showing a proportion of stained nuclei higher than 10% were classified as staining positive; all others were regarded as negative (Figure 1).
MSI test was performed by multiplex polymerase chain reaction and analysis with a 3130×1 genetic analyzer. MSI testing of DNA samples was based on five dinucleotide markers (NR27, NR21, BAT26, BAT25, and NR24). Tumors that showed instability in ≥2/5 of markers tested were classified as a high MSI and 1/5 of markers were classified as low MSI. Tumors that showed instability in 0/5 of markers were designated as microsatellite stable (MSS) cancers. Only high MSI cases were considered MSI positive.
Kim S.T., Lee S.J., Lee J., Park S.H., Park J.O., Lim H.Y., Kang W.K, & Park Y.S. (2017). The Impact of Microsatellite Instability Status and Sidedness of the Primary Tumor on the Effect of Cetuximab-Containing Chemotherapy in Patients with Metastatic Colorectal Cancer. Journal of Cancer, 8(14), 2809-2815.
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3

Immunohistochemical Analysis of Mismatch Repair Proteins

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Immunohistochemical (IHC) analysis of MSH2, MSH6, PMS2, and MLH1 proteins was performed as previously described [16 (link), 19 (link), 20 (link)]. Briefly, after the tumor area adjacent to normal mucosa and/or lymphocytic infiltration had been marked, the paraffinized tissue was removed and multiple tissue blocks were prepared. Finally, 4 μm-thick sections were obtained for IHC following standard protocols. The mouse monoclonal antibodies used were anti-MSH2, anti-MSH6, anti-MLH1, and anti-PMS2 (BD Pharmingen, CA, USA).
Qiu Y.Y., Zeng Y.X, & Cheng Y. (2023). Are High Levels of Microsatellite Instability and Microsatellite Stability Identical in DNA Mismatch Repair-Deficient Colorectal Cancer Patients?. Canadian Journal of Gastroenterology & Hepatology, 2023, 8370262.
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4

Immunohistochemical Analysis of Mismatch Repair Proteins

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We conducted IHC analysis of MSH2, MSH6, PMS2, and MLH1 proteins with formalin-fixed paraffin-embedded tumor samples. After the tumor area adjacent to normal mucosa and/or lymphocytic infiltration was marked, 4 mm of paraffinized tissue was removed, and multiple tissue blocks were prepared. Finally, 4-µm thick sections were obtained for IHC following standard protocols. The mouse monoclonal antibodies used were anti-MSH2, anti-MSH6, anti-MLH1, and anti-PMS2 (BD Pharmingen). Adjacent normal tissues from each sample served as positive controls (Figure 1).
Yan W.Y., Hu J., Xie L., Cheng L., Yang M., Li L., Shi J., Liu B.R, & Qian X.P. (2016). Prediction of biological behavior and prognosis of colorectal cancer patients by tumor MSI/MMR in the Chinese population. OncoTargets and therapy, 9, 7415-7424.
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5

Coimmunoprecipitation and Biotin-based Purification

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All coimmunoprecipitation and immunoprecipitation experiments were performed as previously described using 1000 μg of lysate in Cell Lytic M (Sigma Aldrich, Oakville, Ontario, Canada).4 (link) No-antibody controls were included, although not always shown. Biotin-based protein purification was performed as previously described using 500 μg of lysate in RIPA buffer.18 (link) These experiments were repeated at least three times. The following antibodies were used: anti-MSH2 (EMD, Billerica, MA, USA, IP/co-IP, catalog number NA27), anti-MSH6 (BD Biosciences, San Jose, CA, USA, catalog number 610919), anti-MSH2, anti-phospho-tyrosine, anti-ALK, anti-total caspase 3 (Cell Signaling Technologies, Danvers, MA, USA, catalog numbers 2017, 9416, 3633 and 14220, respectively) and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA; sc-47778).
Bone K.M., Wang P., Wu F., Wu C., Li L., Bacani J.T., Andrew S.E, & Lai R. (2015). NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair. Blood Cancer Journal, 5(5), e311-.
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6

DNA Damage Response Pathway Analysis

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The following antibodies were used: anti-phospho-ATM (Ser1981) (CST; 5883), anti-ATM (CST; 2873), anti–phospho-CHK2 (Thr68) (CST; 2197), anti-CHK2 (CST; 6334), anti-MSH6 (BD Biosciences; 610919), anti-MSH2 (CST; 2017), anti-H3K36me3 (Abcam; Ab9050), anti-8-oxoG (Trevigen; 4354-MC-050), anti-FLAG (Sigma; F7425), goat anti-rabbit IgG (Thermo; A10034), goat antimouse IgG (Thermo; A10036), anti-FLAG M2 Affinity Gel (Sigma; A2220).
Inhibitors used to block OGG1 and ATM were TH5487 (TOCRIS; 6749) and KU-55933 (Selleck), respectively.
Guo S., Fang J., Xu W., Ortega J., Liu C.Y., Gu L., Chang Z, & Li G.M. (2022). Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells. The Journal of Biological Chemistry, 298(7), 102102.
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