The anti-HP1BP3 antibody was created by injecting guinea pigs with the peptide STRETPPKSKLAEGEEEKPEPD-C corresponding to amino acids 47–68 of the murine protein. Peptide synthesis, keyhole limpet hemocyanin (KLH) conjugation and immunization of guinea pigs were carried out by Peptide Specialty Laboratories GmbH, Heidelberg, Germany. Anti-Histone H3 (ab1791) and anti-HP1α (ab77256) antibodies were from Abcam. Anti H3K9me3 antibody was from Cell signaling (#9754) and anti-Tubulin was purchased from Sigma-Aldrich (T5168).
Anti h3k9me3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-H3K9me3 antibody is a reagent used in molecular biology and biochemistry applications. It specifically recognizes and binds to the trimethylation of lysine 9 on histone H3, a post-translational modification associated with epigenetic regulation of gene expression.
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Lab products found in correlation
Anti tubulin, by Merck Group (1 mentions)
Anti histone h3 ab1791, by Abcam (1 mentions)
Epiquik chromatin immunoprecipitation kit, by Epigentek (1 mentions)
Epiquik total histone extraction kit, by Epigentek (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Anti h3k4me3 antibody, by Cell Signaling Technology (1 mentions)
Bullet blocking one for western blotting, by Nacalai Tesque (1 mentions)
Uvp chemstudio, by Analytik Jena (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
3 protocols using anti h3k9me3 antibody
1
Generation and Validation of Anti-HP1BP3 Antibody
2
ChIP Assays for KDM4B and H3K9me3
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ChIP assays were performed in accordance with the manufacturer's protocols (EpiQuik Chromatin Immunoprecipitation Kit, Epigentek Group Inc.), and repeated at least three times. To examine changes in KDM4B-binding activity at the HAX1 promoter, ChIP assays were performed with an anti-KDM4B antibody (8639; Cell Signaling Technology), using the Millipore ChIP assay protocol. In brief, cells were cross-linked with 1% formaldehyde for 10 min at 37°C, collected in SDS lysis buffer, and DNA was fragmented (200–1000 bp) by sonication. Antibodies against KDM4B or control were added to each aliquot of pre-cleared chromatin and incubated overnight. Protein G-agarose beads were added and incubated for 2 h at 4°C. After a series of washes, cross-linking was reversed and DNA was extracted and purified for PCR. The primer sequences for ChIP are listed in Supplementary Table S2 . PCR values were normalized to input and calculated as percentage of input. To examine the status of H3K9 methylation, ChIP assays were performed using anti-H3K9me3 antibody (9754S; Cell Signaling Technology) as described above.
3
Sarcoma PDX Histone Extraction and Immunoblotting
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The sarcoma PDX mouse models were sacrificed and tumor and femoral muscle tissues were resected before tumors reached a volume of 4000 mm 3 . Then tissues were frozen immediately after resections in liquid nitrogen. To prepare for immunoblotting, the tumors were homogenized and the histones were extracted using a Epiquik Total Histone Extraction Kit (Epigentek, Farmingdale, NY, USA).
Immunoblotting for these histones was performed under the following conditions: 12% SDS-PAGE gels and 0.2 μm polyvinylidene difluoride (PVDF) membranes were used.
Blocking was performed using the Bullet Blocking One for Western Blotting (Nakalai Tesque, Inc. Kyoto, Japan). Anti-H3K4me3 antibody (1:1,000, #9751, Cell Signaling Technology, Danvers, MA, USA); anti-H3K9me3 antibody (1:1,000, #13969, Cell Signaling Technology); and anti-H3 antibody (1:1,500, 17168-1-AP, Proteintech, Rosemont, IL, USA) were used. Total histone H3 was used as a loading control. Horseradish-peroxidase-conjugated anti-rabbit IgG (1:20,000, SA00001-2, Proteintech, Rosemont, IL, USA) was used as a second antibody. Immunoreactivity was visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). The signals were detected with UVP ChemStudio (Analytik Jena, Upland, CA, USA) [18, 19] (link).
Immunoblotting for these histones was performed under the following conditions: 12% SDS-PAGE gels and 0.2 μm polyvinylidene difluoride (PVDF) membranes were used.
Blocking was performed using the Bullet Blocking One for Western Blotting (Nakalai Tesque, Inc. Kyoto, Japan). Anti-H3K4me3 antibody (1:1,000, #9751, Cell Signaling Technology, Danvers, MA, USA); anti-H3K9me3 antibody (1:1,000, #13969, Cell Signaling Technology); and anti-H3 antibody (1:1,500, 17168-1-AP, Proteintech, Rosemont, IL, USA) were used. Total histone H3 was used as a loading control. Horseradish-peroxidase-conjugated anti-rabbit IgG (1:20,000, SA00001-2, Proteintech, Rosemont, IL, USA) was used as a second antibody. Immunoreactivity was visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). The signals were detected with UVP ChemStudio (Analytik Jena, Upland, CA, USA) [18, 19] (link).
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