ChIPs were done as previously described with the modifications described below41 (link). The following histone antibodies were used: 1 μl of anti-H3K4me2 (Upstate 07-030); 0.5 μl of anti-H3K36me3 (Abcam 9050); 0.5 μl of anti-acetyl H4 (Upstate 06-598); and 2 μl of anti-H3 (Abcam 1791). All antibodies were bound to Protein A-agarose beads and used for precipitation of formaldehyde-crosslinked chromatin. For anti-H3 antibody, binding was done in FA lysis buffer containing 275 mM NaCl. For other antibodies, binding was done in FA lysis buffer containing 1 M NaCl. Precipitated DNAs were analysed in real-time using SYBR Green Supermix and CFX96 cycler (Bio-Rad). Oligonucleotides for PCR analysis are listed in Supplementary Table 2 .
Anti h3k36me3
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-H3K36me3 is a laboratory tool that detects the presence of trimethylation at lysine 36 of histone H3. This modification is associated with active transcription and chromatin remodeling. The antibody can be used in various techniques, such as Western blotting and immunoprecipitation, to study epigenetic mechanisms and gene expression regulation.
Automatically generated - may contain errors
Lab products found in correlation
Anti h3k4me3, by Abcam (13 mentions)
Anti h3k27me3, by Merck Group (11 mentions)
Anti flag, by Merck Group (8 mentions)
Anti h3k4me3, by Merck Group (8 mentions)
Anti h3, by Abcam (7 mentions)
Anti h3k9me3, by Abcam (5 mentions)
Anti h3k9ac, by Abcam (4 mentions)
Anti h3k27ac, by Abcam (4 mentions)
Anti h3k27me3, by Abcam (4 mentions)
Ab9050, by Abcam (4 mentions)
Anti h3k9me2, by Abcam (3 mentions)
Anti h3k4me1, by Abcam (3 mentions)
Anti histone h3, by Abcam (3 mentions)
Anti h3k36me2, by Abcam (3 mentions)
Cfx96 cycler, by Bio-Rad (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti polii, by Abcam (2 mentions)
H2ak119ub, by Cell Signaling Technology (2 mentions)
Ab4729, by Abcam (2 mentions)
46 protocols using anti h3k36me3
1
ChIP-seq Histone Modification Analysis Protocol
2
Chromatin Extraction and Immunoblotting for Histone Marks
3
Cell Culture and Protein Analysis
4
ChIP-seq Analysis of CTCF Binding
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To investigate the binding of CCCTC-binding factor (CTCF) to chromatin, ChIP analysis was carried out after sonicating the chromatin to an average fragment size of 250-300 bp following the previously described procedure [45 (link), 46 (link)]. The sequences of primers used for qPCR were given in Supplementary Table 5 and their location is depicted in Figure 4B . Although the amplicons are not tiled, the average size of chromatin fragments ensured that CTCF is absent from the entire region under consideration. Epigenetic modifications of histones were studied at nucleosomal level by Nuc-ChIP [31 (link), 32 (link)]. The following antibodies were used: anti-H3K9ac (Abcam, ab-4441); anti-H3K9me3 (Abcam, ab-8898); anti-H3K27ac (Abcam, ab-4729); anti-H3K27me3 (Millipore, 07-449); anti-H3K4me3 (Abcam, ab-8580); anti-H3K36me3 (Abcam, ab-9050); anti-H3K20m (Abcam, ab-9051); anti-β-actin (Abcam, ab-8227).
5
Western Blotting of Protein Expression
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Fresh tissues and cells were lysed with cell lysis buffer (Beyotime Biotechnology) and western blot was performed as described previously [18 (link)]. Briefly, 40 μg total proteins were applied to separation with SDS–PAGE gel. After the electrophoresis, the proteins were transferred to PVDF membranes (Millipore), followed by blocking in the TBST buffer containing 5% fat-free milk. The membranes were then incubated with indicated antibodies overnight at 4 °C, and then washed and incubated with HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 h at room temperature and finally visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following antibodies were used in this work: Anti-GAPDH (Cell Signaling Technology), anti-JMJD2A (Cell Signaling Technology), anti-Histone H3 (Santa Cruz Biotechnology), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-mTOR (Cell Signaling Technology), anti-p-mTOR (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt Thr308 (Cell Signaling Technology), anti-S6K1 (Cell Signaling Technology), anti-p-S6K1 (Cell Signaling Technology).
6
Chromatin Immunoprecipitation (ChIP) Protocol
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Chromatin immunoprecipitation (ChIP) was performed as previously reported[29 (link)]. Briefly, cortex tissue was dissected from 6-8-week-old mice, minced and crosslinked in 1% formaldehyde (wt/vol) and sonicated using a Misonix 3000. Antibody was first bound to Dynabeads and then incubated with sheared chromatin overnight at 4°C. After 4 washes with RIPA buffer and 1 wash with TE buffer, bound chromatin was eluted and reverse crosslinked at 65°C overnight. Eluted DNA was treated with RNase A (Thermo Scientific) and proteinase K (Promega), purified by phenol-chloroform extraction and dissolved in water. Antibodies used were: anti-Flag (Sigma M2), anti-LEDGF (Bethyl A300-847A), anti-H3K36me3 (Abcam ab9050), and anti-PolII (Abcam ab5408). Primer sequence for ChIP-qPCR is provided in S8 Table .
7
Epigenetic Profiling of Gene Regulation
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Histone modification and PolII ChIP was performed as previously described17 (link),62 (link), respectively. Immunoprecipitation was done with antibodies: anti-H3 (Abcam, #ab1791), anti-H3K27me3 (Millipore, #07-449), H2AK119ub (Cell Signaling Technology, #8240), anti-H3K36me3 (Abcam, #ab9050). PolII antibodies were as in64 (link). All ChIP experiments were followed by qPCR with indicated primer pairs (Supplementary Table 2 ).
8
Chromatin Immunoprecipitation for Epigenetic Analysis
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Chromatin immunoprecipitation was performed as previously described using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions [36 (link),55 (link),56 (link),57 (link)]. Sheared chromatin (90 μL) from the prefrontal cortex or hippocampus was used for immunoprecipitation and incubated at 4 °C overnight with primary antibodies (anti-H4R3me2a, anti-H3K4me3, antiH3K36me3, and anti-MeCP2; Abcam, Cambridge, UK) or normal rabbit IgG (negative control, supplied in the kit). Meanwhile, 5 μL of sheared chromatin was saved as input for normalization. Follow-up purified gDNA from each antibody was used at the BDNF exon IV, MBP, and SOX10 promoter region via real-time PCR (Table 2 ). The percentage input was calculated using the following formula: ΔCt [normalized ChIP] = (Ct [ChIP] − (Ct [Input] − Log2 (Input dilution factor), where dilution factor = 5/90 = 18.5. Finally, input% = 100/2ΔCt [normalized ChIP], with this value delegating the enrichment of epigenetic modifications in specific regions.
9
ChIP-seq protocol for Drosophila and mouse
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For ChIP-seq, Drosophila S2 cells and mouse bone marrow-derived macrophages (BMDM) were cross-linked for 10 min with 1% formaldehyde. ChIP material was prepared independently for each cell type as described previously (Henriques et al. 2013 (link)). To ensure proper normalization between RNAPII ChIP-seq of control and Spt5-depleted cells, S2 cells and BMDM ChIP material were pooled in a 10:1 ratio (Drosophila to mice), and immunoprecipitations were carried out with anti-Rpb3 (Drosophila) and total anti-RNAPII antibody (mice; Santa Cruz Biotechnology, H-224). For the remaining ChIP-seq libraries, separate immunoprecipitations were performed with anti-cohesin (gift from D. Dorsett), anti-H3K4me1 (Millipore, 07-436), anti-H3K4me3 (Millipore, 07-473), anti-H3K27ac (Abcam, ab4729), and anti-H3K36me3 (Abcam, ab9050) antibodies. Immunoprecipitated material was purified using the Qiaquick PCR purification kit, and ChIP-seq libraries were prepared using the NEXTflex ChIP-seq kit (Bioo Scientific) according to the manufacturer's instructions.
10
Chromatin Extraction and Immunoblotting for Histone Marks
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For histone marks analyses, whole-cell extracts were prepared as described (Gardner et al., 2005 (link)) with minor changes. Cells from log-phase yeast cultures were harvested by centrifugation and lysed in 400 mL of SUME buffer (8 M urea, 1% SDS, 10 mM MOPS at pH 6.8, 10 mM EDTA, 0.01% bromophenol blue) by mechanical shearing. For RNA Pol II phosphoSer2 and phosphoSer5 analyses, extracts were prepared by treating cell pellets with 0.2M NaOH for 5min and boiling the samples in 2x Laemmli buffer for 5min. Extracts were cleared from debris through centrifugation. Antibodies used in this study were as follows: anti-H3K9ac (Abcam, ab4441), anti-Flag (Sigma, M2), anti-H3K4me3 (Abcam, ab8580), anti-H3K36me3 (Abcam, ab9050), anti-RNA Pol II phosphoSer2 (3E10, Active Motif), anti-RNA Pol II phosphoSer5 (3E8, Active Motif) and anti-Taf4 (gift from P. Anthony Weil).
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