10 cm dishes were lysed with 50–150 μl (depending on cell density) of boiling 1% SDS Lysis Buffer [50 ml-10% SDS (5 ml) 5 M NaCl (1 ml), 1 M Tris pH 7.5 (500 μl) dH20 (43.5 ml)] and put at 95°C for 5 minutes. Protein concentration was assessed in a 96 well format by BCA Protein assay Kit (PIERCE Catalog number: 23225). 30 μg of protein extract was loaded onto 10% Tris-Glycine SDS-PAGE gels and run at 100 Volts for 90 minutes. The gels were transferred onto PVDF membrane and incubated overnight at 4°C with primary antibody,1:1000 rabbit Anti-MRE11 Antibody (NOVUS Biologicals Catalog number: NB100-276 diluted in 5% non fat dry milk/TBS-T. The secondary antibody used was a 1:5000 dilution of ECL Anti-rabbit IgG Horseradish Peroxidase-Linked Species specific F(ab’)2 Fragment (donkey) (Amersham Catalog number: NA9340). Membranes were incubated for 1 hour in secondary antibody and developed using Pierce Supersignal West Dura Extended Duration Substrate (Catalog number: 34705).
Anti mre11 antibody
Manufactured by Novus Biologicals
Anti-MRE11 antibody is a laboratory reagent used for the detection and quantification of MRE11 protein in biological samples. MRE11 is a key component of the DNA double-strand break repair complex. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the expression and localization of MRE11 in cells and tissues.
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Lab products found in correlation
Quick starttm bradford 1x dye reagent, by Bio-Rad (1 mentions)
Ecltm anti mouse igg, by GE Healthcare (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Nb100 142, by Novus Biologicals (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Anti γh2ax antibody, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Gapdh, by GeneTex (1 mentions)
4 protocols using anti mre11 antibody
1
Western Blot Analysis of MRE11 Protein
2
Western Blot Analysis of DNA Damage Response
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Cells were harvested by trypsinization, and resuspended in urea lysis buffer (9 M urea, 150 mM β-mercaptoethanol, 75 mM Tris, pH 7.4). The lysate were sheared by sonication, centrifuged at 15000 rpm for 30 min, and the soluble fractions were collected. Protein concentration was determined using Quick StartTM Bradford 1x Dye Reagent (BioRad) and equal amounts of proteins were separated on 7.5% Tris-HCl SDS-polyacrylamid gels and transferred to Immobilon-P membrane (IPVH00010; EMD Millipore). The membrane was blocked in 5% milk in TBS with 0.02% Tween-20 and incubated with anti-ATM antibody (NB100–309; Novus), anti-ATM (phosphor S1981) antibody (ab81292; abcam), anti-MRE11 antibody (NB100–142; Novus), and anti-GAPDH antibody (#5174; Cell Signaling). ECLTM Anti-rabbit IgG, horseradish peroxidase linked whole antibody (NA934V; GE Healthcare) and ECLTM Anti-mouse IgG, horseradish peroxidase linked whole antibody (NA931V; GE Healthcare) were used as secondary antibodies. ECL western blotting substrate (Pierce) was used for detection by FluorChemTM E (Protein Simple).
3
Colon Carcinoma Cell Line Characterization
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The human colon carcinoma cell lines HT29, SW480, HCT8, and HCT116 cells were purchased from American Type Culture Collection (ATCC) and routinely verified by morphology and growth characteristics. Cells were cultured in 10% FBS-supplemented RPMI medium with L-glutamine and maintained at 37°C with 5% CO2. For Western blot analysis, antibodies against GLI1, NBS1, MRE11, RAD50, γ-H2AX, cleaved caspase-3, and β-Actin were purchased from Cell Signaling. For confocal microscopy, anti-γ-H2AX antibody was obtained from Millipore; anti-NBS1 mouse monoclonal antibody and anti-MRE11 antibody were purchased from Novus; and AlexaFluor 488 goat anti-rabbit and AlexaFluor 633 goat anti-mouse secondary antibodies were obtained from Invitrogen. GANT61 and 5-FU were purchased from Sigma. SRI-38832 was synthesized in-house at Southern Research (Birmingham, AL).
4
Gamma Irradiation Response in Cell Lines
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We exposed control, A-T and NBS lymphoblastoid cell lines to varying doses of gamma irradiation and collected them at the time points indicated. Total cell extracts were prepared by using cell lysis buffer (50 mM Tris pH 7.4, 0.15 M NaCl, 10% Glycerol, 0.5% Tween 20, 50 mM β-glycerophosphate, 1 mM DTT, 1 mM PMSF, 5–10 μg/ml Aprotinin, 5 μg/ml Leupeptin, 5 μg/ml Pepstatin, 1mM Na3VO4, 1 mM NaF). MRE11 immunoprecipitations were performed using anti-MRE11 antibody (Novus). Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).
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