The HD-11 cell line was transfected with plasmids encoding TY-tagged c-Myb variants (empty vector, 3xTY1-c-Myb, 3xTY-c-Myb D152V) described in [13 (link)]. The ChIP analysis was performed essentially as previously described [53 (link), 54 ], using anti-Ty1 antibodies for IP of c-Myb and anti-H3K27ac antibody (# 39,133, Active Motif) for IP of H3K27ac. The anti-Ty1 monoclonal mouse antibody was produced in our lab from a hybridoma cell line [55 (link)]. Occupancy was determined with qRT-PCR using primers for specific regions of the mim-1 locus (LECT2). Primer sequences are available upon request.
Anti h3k27ac antibody
Manufactured by Active Motif
The Anti-H3K27ac antibody is a tool used to detect and study the acetylation of lysine 27 on histone H3 protein. This modification is associated with active gene transcription and enhancer regions. The antibody can be used in various techniques, such as chromatin immunoprecipitation (ChIP), western blotting, and immunofluorescence, to investigate the distribution and dynamics of this epigenetic mark.
Automatically generated - may contain errors
Lab products found in correlation
Smarter thruplex dna seq kit, by Takara Bio (2 mentions)
Nextseq 500 system, by Illumina (2 mentions)
Ipfl00010, by Merck Group (2 mentions)
Imagej, by Bio-Rad (2 mentions)
Anti h2b antibody, by Abcam (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Cut tag it kit, by Active Motif (1 mentions)
Accutase, by Merck Group (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Anti β catenin 51067 2 ap, by Proteintech (1 mentions)
Anti h3k27me3 antibody, by Active Motif (1 mentions)
Epiquik chromatin immunoprecipitation kit, by Epigentek (1 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Protein a g dynabeads, by Thermo Fisher Scientific (1 mentions)
E220 focused ultrasonicator, by Covaris (1 mentions)
Anti h3k9ac antibody, by Active Motif (1 mentions)
9 protocols using anti h3k27ac antibody
1
ChIP Analysis of c-Myb Variants
2
ChIP-seq Protocol for CTCF and H3K27ac
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GSCs were lifted from plates using accutase (Sigma-Aldrich). Typically, 0.5 million cells were processed using the CUT&Tag-IT kit (Active Motif) and 1 μg of anti-CTCF (Active Motif #61311) or anti-H3K27ac antibody (Active Motif #9133) as per manufacturer’s instructions and the resulting libraries were sequenced on a NextSeq500 platform (Illumina) to obtain at least 107 reads. Read pairs were aligned to the human reference genome GRCh38 using Bowtie2 (v2.3.4.1), PCR duplicates were removed using the MarkDuplicates function in Picard tools (v2.20.7), and read coverage tracks (BigWig) were generated and normalized with the RPCG parameter using the bamCoverage function of deepTools2 (v3.5.1)75 (link). Peaks were called using SEACR (v1.3)76 (link) with an FDR cutoff of <0.01.
3
Profiling Active Chromatin by H3K27Ac ChIP-seq
4
SIRT6 Deacetylation Kinetics on Nucleosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type SIRT6 (500nM or 125nM) and SIRT6(R175A) (200nM) was incubated with nucleosomes (125nM) containing either acetylated H3K9, H3K56, or H3K27, and/or with truncated H2A (H2At) at 37 °C for varying time points in HDAC reaction buffer (25mM HEPES pH 7.3, 49 mM NaCl, 4.5mM MgCl2, 1mM DTT). Reactions were quenched with the addition of 2X SDS sample buffer, boiled, and then electrophoresed on a 12 % SDS-PAGE gel. Proteins on the gel were transferred to a PVDF membrane (Millipore IPFL 00010), which was first blocked with 10 % milk, and then probed with either 1:10,000 anti-H3K9ac antibody (Active Motif #39038), 1:1,000 anti-H3K56ac antibody (Active Motif #39133), or 1:3,000 anti-H3K27ac antibody (Active Motif #39082), and also probed with 1:3,000 anti-H2B antibody (Abcam #64039). Western blots were quantified using ImageJ (54 (link)) or Bio-Rad Image Lab.
5
ChIP-seq Profiling of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed using EpiQuik™ Chromatin Immunoprecipitation Kit (P-2002; Epigentek, Farmingdale, NY, USA) according to the manufacturer’s instructions. NSCLC cells were cross-linked and sonicated as previously described [24 (link)]. The sheared chromatin fragments (approximately 200–500 bp) were subjected to immunoprecipitation with ChIP-grade antibodies as follows: anti-SET1AD antibody (A300-289A; Bethyl Lab), anti-H3K4me3 antibody (ab8580; Abcam), anti-β-catenin (51067–2-AP; Proteintech), anti-WDR5 antibody (#13105; Cell Signaling Technology), anti-H3K27me3 antibody (39,157; Active Motif), anti-H3K27ac antibody (39,134; Active Motif) and normal Rabbit IgG (#2729; Cell Signaling Technology). The primers for ChIP-PCR were listed in Additional file 1 : Table S1.
6
Chromatin Immunoprecipitation Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 1% formaldehyde in PBS for 12 min, which was then quenched with glycine (final concentration 0.125 M). Fixed cells were washed and lysed as previously described (Latos et al., 2015 (link)). Chromatin was sonicated to an average size of 200–700 bp using a Bioruptor Pico (Diagenode). Immunoprecipitation was performed using 15 μg of chromatin and 2.5 μg of anti-H3K27ac antibody (Active Motif #39133). DNA purification was performed using the GeneJET PCR Purification Kit (Thermo Fisher) with DNA eluted in 80 μL of elution buffer. ChIP-seq libraries were prepared from 1 to 5 ng eluted DNA using NEBNext Ultra II DNA library Prep Kit (New England BioLabs). Libraries were sequenced on an Illumina NextSeq 500 with single-ended 75 bp reads at the Barts London Genome Centre.
7
Chromatin Immunoprecipitation Sequencing (ChIP-seq)
8
SIRT6 Deacetylation of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type SIRT6 (500 or 125 nM) and SIRT6(R175A) (200 nM) were incubated with nucleosomes (125 nM) containing either acetylated H3 K9, H3 K56, or H3 K27, and/or with C-terminally truncated H2A [H2A(2-119)] at 37°C for varying time points in HDAC reaction buffer [25 mM Hepes (pH 7.3), 49 mM NaCl, 4.5 mM MgCl2, and 1 mM DTT]. Reactions were quenched with the addition of 2× SDS sample buffer, boiled, and then electrophoresed on a 12% SDS-PAGE gel. Proteins on the gel were transferred to a polyvinylidene difluoride membrane (Millipore, IPFL 00010), which was first blocked with 10% milk, and then probed with either 1:10,000 anti-H3K9ac antibody (Active Motif, #39038), 1:1000 anti-H3K56ac antibody (Active Motif, #39133), or 1:3000 anti-H3K27ac antibody (Active Motif, #39082), and also probed with 1:3000 anti-H2B antibody (Abcam, #64039). Western blots were quantified using ImageJ (54 (link)) or Bio-Rad Image Lab. Control experiments show similar SIRT6 deacetylase activity using nucleosomes reconstituted from H3K9ac protein prepared in-house or purchased from the Histone Source.
9
Profiling Active Chromatin by H3K27Ac ChIP-seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Active status of chromatin was determined by histone 3 lysine 27 acetylation (H3K27Ac) levels using ChIP-seq. H3K27Ac ChIP assay was conducted with 5 μg of anti-H3K27Ac antibody (Active Motif, 39133) using the protocol described above. Sequencing libraries were prepared with 3 ng each of H3K27Ac ChIP DNA and input sample using SMARTer ThruPLEX DNA-seq kit (Takara, R400675). Libraries were sequenced with single-end (SE) 75 cycles on an Illumina Nextseq 500 system at the Broad Institute of Harvard and MIT and the reads were aligned to human reference genome hg19 using Burrows-Wheeler Alignment (BWA) tool44 (link). Genome-wide coverage was calculated after extending to 200 bases (approximate fragment size) and averaged over 25 bp windows using igvtools45 (link). Coverage was then normalized and scaled using RSeqC (http://rseqc.sourceforge.net/#normalize-bigwig-py ) . ChIP-seq peaks were called using MACS2 2.0.10.20120913.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!