HeLa and SiHa cells in logarithmic phrase were added with precold cell lysis at 4°C for 15 min before centrifugation at 14000 g for 15 min. The supernatant was transferred into a new centrifuge tube for incubation with negative IgG for anti-KDM1A antibody (2139S, 1 : 50, Cell Signaling Technology, Boston, USA) or anti-H3 antibody (9649S, 1 : 25, Cell Signaling Technology, Boston, USA) for overnight at 4°C. The pretreated 10 μL protein A Agarose beads were added into cell lysis for incubation at 4°C for 24 h, with gentle shaking to enable the coupling of antibody with protein A Agarose beads. Then, the cell lysis was centrifuged at 4°C and 3000 rpm for 3 min to allow the Agarose beads down to the tube bottom. The supernatants were removed, and the Agarose beads were washed in lysis buffer for 3 min before boiling water bath with 15 μL 2 × SDS loading buffer. The expressions of DACT1 or Anti-Histone H3 (acetyl K27) in the protein complex were measured by western blot.
Anti h3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-H3 antibody is a laboratory tool used to detect and measure the presence of histone H3 protein in biological samples. Histone H3 is a core component of chromatin, the complex of DNA and proteins that make up the structure of chromosomes in eukaryotic cells. The Anti-H3 antibody can be used in various research applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to investigate chromatin structure and function.
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Lab products found in correlation
Criterion tgx precast gel, by Bio-Rad (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Anti h3k4me3 antibody, by Abcam (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Anti acetylated tubulin antibody, by Merck Group (1 mentions)
Anti acetyl histone h3 lys9, by Merck Group (1 mentions)
Clarity ecl chemiluminescent substrates, by Bio-Rad (1 mentions)
Anti mouse igg hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Thymidine t1895, by Merck Group (1 mentions)
Anti flag m2 f1804, by Merck Group (1 mentions)
M1404, by Merck Group (1 mentions)
Bi2536, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
A00865, by GenScript (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
5 protocols using anti h3 antibody
1
Immunoprecipitation of KDM1A and H3
2
Quantitative Western Blot Analysis
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Cells were lysed with a hypotonic shock. Lysates (10 μg) underwent electrophoresis on Criterion TGX precast gels (10 or 12%; Bio-Rad), gels were blotted against PVDF membranes and blocked in 5% nonfat dry milk. rhPPCA protein (2 and 6 ug) was loaded on Criterion TGX precast gels (10% Bio-Rad), gels were blotted against Polyvinylidene Difluoride (PVDF) membranes. The latter was stained with Coomassie brilliant Blue (Bio-Rad). Membranes were immunoblotted for anti-NEU1 antibody (in house prepared 1:300) and anti-PPCA antibody (in house prepared 1:250), anti-H3 antibody (Cell Signaling #4499 1:1000) and anti-H3K4me3 antibody (Abcam ab8580 1:500) and incubated overnight at 4 °C in 3% BSA–TBS-T solution. The next day, membranes were incubated with HRP-conjugated secondary antibodies and developed using SuperSignal West Femto Maximum Sensitivity Substrate or SuperSignal West Pico (Thermo Fisher Scientific, Carlsbad, CA, USA). Quantitative analyses of the immunoblots were performed with Image Lab software.
3
Quantitative Western Blot Analysis of Protein Acetylation
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After treatments, 4T07 cells were solubilized in Lysis Buffer (20 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 3,5 mM SDS, 13 mM deoxycholic acid) and proteins extracted were separated on a 4–15% Tris/Glycine/SDS gel (BIO RAD). A standard protocol was used for transfer and blotting. Anti-acetylated tubulin antibody was purchased from Sigma-Aldrich (#T7451). Anti-H3 antibody was clone96C10 (Cell Signaling Technology (CST); cat # 3638). Anti-acetyl-histone H3(Lys9) was 07–352 (Merck Millipore), and anti-acetyl-histone H3 (Lys27) was #8173 (CST). Anti-GAPDH antibody was purchased from Abcam (#ab8245) and the anti-mouse IgG HRP-linked secondary antibody was purchased from Cell Signaling Technology and were used at recommended concentrations. Signal was detected using Clarity ECL chemiluminescent substrates (Bio-Rad) and ChemiDoc Imaging System from the same company was used to quantify the intensity of bands. Changes in protein levels were quantified relative to control using Image Lab software (Version 6.0.1) after normalization to GAPDH. Western-blot images presented are three biological replicates.
4
HeLa and HEK293T Cell Culture Protocols
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HeLa and HEK293T cells were grown in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum at a 37 °C incubator with 5% CO2.
Nocodazole (M1404, a final concentration of 340 nM was used throughout this research) and Thymidine (T1895, a final concentration of 2 mM was used) were purchased from Sigma. BI2536, a PLK1 inhibitor (S1109, a final concentration of 1 μM was used), was purchased from Selleck.
Rabbit polyclonal antibodies used for immunoblotting and immunoprecipitation in this study including anti-HA (A190–208A), anti-LSD1 (A300–215A), anti-CoREST (A300–130A), anti-GAPDH (A300–643A) and anti-H3 Ser10 (A301–844A) were from Bethyl Laboratories. Rabbit polyclonal anti-H3 antibody (#9715) was from Cell Signaling Technology. Mouse monoclonal antibody against GST (A00865) was from GenScript Corporation. Mouse monoclonal anti-FLAG M2 (F1804) was from Sigma. Mouse monoclonal anti-HIS (D291-3) was from MBL Biotech.
Nocodazole (M1404, a final concentration of 340 nM was used throughout this research) and Thymidine (T1895, a final concentration of 2 mM was used) were purchased from Sigma. BI2536, a PLK1 inhibitor (S1109, a final concentration of 1 μM was used), was purchased from Selleck.
Rabbit polyclonal antibodies used for immunoblotting and immunoprecipitation in this study including anti-HA (A190–208A), anti-LSD1 (A300–215A), anti-CoREST (A300–130A), anti-GAPDH (A300–643A) and anti-H3 Ser10 (A301–844A) were from Bethyl Laboratories. Rabbit polyclonal anti-H3 antibody (#9715) was from Cell Signaling Technology. Mouse monoclonal antibody against GST (A00865) was from GenScript Corporation. Mouse monoclonal anti-FLAG M2 (F1804) was from Sigma. Mouse monoclonal anti-HIS (D291-3) was from MBL Biotech.
5
Investigating DNA Damage Response Signaling
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CPT was used at a final concentration of 1 µM throughout this study. The ATR inhibitor NU6027 was used at a final concentration of 10 µM. Both reagents were purchased from Sigma. BrdU was used at a final concentration of 20 µM and was purchased from BD Biosciences.
Rabbit polyclonal antibodies used for immunoblotting and immunoprecipitation, including anti-MYC (A190-205A), anti-HA (A190-208A), anti-USP11 (A301-613A), anti-CLASPIN (A300-267A), anti-BRCA1 (A300-000A), and anti-ATR (A300-138A) were purchased from Bethyl Laboratories. Antibodies for Chk1 (sc-8408), and Ub (sc-8408) were obtained from Santa Cruz Biotechnology. The mouse monoclonal antibody against FLAG M2 (F1804) was from Sigma. Phosphorylation-specific antibodies against CHK1 (Ser345) (#2348), the (Ser/Thr) ATM/ATR substrate motif (#2851) and the anti-H3 antibody (#9715) were from Cell Signaling Technology. Anti-BrdU FITC (347583) was used for IF and was purchased from BD Biosciences.
All cultured cells were grown in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, in a humidified 37 °C incubator with 5% CO 2 .
Rabbit polyclonal antibodies used for immunoblotting and immunoprecipitation, including anti-MYC (A190-205A), anti-HA (A190-208A), anti-USP11 (A301-613A), anti-CLASPIN (A300-267A), anti-BRCA1 (A300-000A), and anti-ATR (A300-138A) were purchased from Bethyl Laboratories. Antibodies for Chk1 (sc-8408), and Ub (sc-8408) were obtained from Santa Cruz Biotechnology. The mouse monoclonal antibody against FLAG M2 (F1804) was from Sigma. Phosphorylation-specific antibodies against CHK1 (Ser345) (#2348), the (Ser/Thr) ATM/ATR substrate motif (#2851) and the anti-H3 antibody (#9715) were from Cell Signaling Technology. Anti-BrdU FITC (347583) was used for IF and was purchased from BD Biosciences.
All cultured cells were grown in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, in a humidified 37 °C incubator with 5% CO 2 .
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