ChIP on EP300 was performed as described previously (Jacobs et al. 2014 (link)), with an excess of EP300 antibody (C-20; sc-585 X; Lot B0211 and Lot E2610). For H3K27ac and H3K4me3 data, ChIP was based on Vermunt et al. (2014) (link), with 5 µg H3K27ac (Abcam ab4729, Lot GR3303561-2) and 6 µg H3K4me3 (Millipore 07-473, Lot 3394198) per sample. ChIP-seq data analysis on EP300 was performed as described previously (Jacobs et al. 2014 (link)). For the full procedure, see Supplemental Material .
H3k27ac
Manufactured by Abcam
Sourced in United States, United Kingdom
H3K27ac is a histone post-translational modification that is associated with the acetylation of lysine 27 on histone H3. This modification is generally found in the promoter regions of actively transcribed genes and is associated with active gene expression.
Automatically generated - may contain errors
Lab products found in correlation
H3k4me1, by Abcam (55 mentions)
H3k4me3, by Abcam (52 mentions)
H3k27me3, by Abcam (24 mentions)
H3k27me3, by Merck Group (24 mentions)
H3k4me3, by Merck Group (23 mentions)
H3k27me3, by Cell Signaling Technology (22 mentions)
H3k9ac, by Abcam (20 mentions)
H3k36me3, by Abcam (18 mentions)
H3k9me3, by Abcam (16 mentions)
Flag tag (flag), by Merck Group (13 mentions)
H3k4me3, by Active Motif (10 mentions)
H3k4me2, by Abcam (10 mentions)
H3k27me3, by Active Motif (8 mentions)
β actin, by Merck Group (8 mentions)
Bioruptor, by Diagenode (7 mentions)
Vinculin, by Merck Group (6 mentions)
Ab4729, by Abcam (6 mentions)
Rna polymerase 2, by Abcam (6 mentions)
H3k4me1, by Merck Group (6 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
231 protocols using h3k27ac
1
ChIP-seq for EP300, H3K27ac, and H3K4me3
2
Immunohistochemical Profiling of H3K27ac
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slides were baked overnight at 37°C, followed by deparaffinization in xylene and rehydration in ethanol. Heat-induced epitope retrieval was performed at pH 9 (Dako Target Retrieval Solution, S2367) for 30 minutes, pigmentation was removed using Pretreatment Solutions A and B (Polysciences), and endogenous peroxidase was removed with 3% H2O2. Serum-Free Protein Block (Dako, X909) was added for 30 minutes, followed by 30-minute incubation with the primary antibody H3K27ac (Abcam, 177178) diluted in Antibody Diluent with Background Reducing Components (Dako, S3022). Slides were incubated with the anti-rabbit secondary antibody for 30 minutes and streptavidin/HRP (Dako, P0397) for 30 minutes. Slides were developed using SignalStain DAB Chromagen. Images were taken on a Nikon Eclipse Ci microscope and analyzed using NIS-Elements BR Analysis software.
3
Chromatin Immunoprecipitation and Immunofluorescence Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used for ChIP were: H3 (Abcam ab1791), H3K27ac (Abcam ab4729), H3K27me3 (Millipore 07-499), H3K36me3 (Abcam ab9050), H3K4me3 (Abcam ab8580), and H3K9me3 (Abcam ab8898), and were validated by the company for specificity. The following primary antibodies were used for immunofluorescence: mouse anti-Nc82 (1∶20; Developmental Studies Hybridoma Bank) and rat anti-Fru M (1:200) (18) . The secondary antibodies used for immunofluorescence were Alexa Fluor goat anti-rat 488 (1:1000), goat anti-mouse 633 (1:500), rabbit anti-GFP 488 conjugate (1:600), and streptavidin 488 conjugate (1:4000) (Thermo Fisher).
4
Antibody Sources for AR and Associated Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sources for the antibodies and control IgGs were as following: AR (Santa Cruz, Cat. sc-816); pAR-S81 (EMD Millipore, Cat. 07–1375); pAR-S308 (Santa Cruz, Cat. sc-26406); pRNA Pol II Ser2 (Abcam, Cat., ab5095); pRNA Pol II Ser5 (Abcam, Cat. ab5131); CDK1 (Cell Signaling, Cat. 9112); pCDK1-T161 (Cell Signaling, Cat. 9114); CDK9 (Santa Cruz, Cat. sc-8338); Cyclin T1 (Santa Cruz, Cat. sc-10750); BRD4 (Bethyl, Cat. A301-985A); p300 (Santa Cruz, Cat. sc-585); Histone 3 (Abcam, Cat. ab1791); H3K27Ac (Abcam, Cat. ab4729); pH3-Ser10 (EMD Millipore, Cat. 06–570); FoxA1 (Abcam, Cat. Ab23738); PSA (Meridian Life Science, Cat. K92110R); Flag-M2 (Sigma-Aldrich, Cat. F3165); HA (Cell Signaling, Cat. 3724); β-Tubulin (EMD Millipore, Cat. MAB3408); β-Actin (Abcam, Cat. Ab6276); GAPDH (Abcam, Cat. Ab9485); Hsp90 (Santa Cruz, Cat. sc-69703), PP1 (Santa Cruz: Cat. sc-443; Cat. sc-6104; and Cat. sc-6105), and control IgGs (Santa Cruz: normal rabbit IgG, Cat. sc-2027; and normal goal IgG, Cat. sc-2028). Protein A and protein G was from Pierce (Cat. 20334 and Cat. 20399, respectively). The anti-flag M2 affinity gel was from Sigma-Aldrich (Cat. A2220). Western blots were developed using X-Ray film (Research Products International) and the Western Lightning Plus-ECL reagent (PerkinElmer). Images were acquired using a CanoScan LiDE 210 scanner.
5
ChIP-Seq Analysis of Retinal Epigenome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation (ChIP) assay was performed as previously described [22 (link)]. In summary, 6 pooled P14 C57BL/6 J wild-type or Crx−/− mouse retinas per sample were dissected and chromatin was cross-linked with 1% formaldehyde in PBS for 10 min at room temperature. Cross-linked cells were lysed and fragmented by sonication. Chromatin fragments were immunoprecipitated with the antibodies to H3K27ac (Abcam, Cambridge, UK; ab4729) and H3K4me3 (Millipore Sigma, Burlington, MA; 07-473), or normal rabbit/mouse IgG (Santa Cruz Biotechnology, Dallas, TX) bound to Protein A beads (Millipore, 16-125) or A/G beads (Santa Cruz Biotechnology). After extensive washing, the immunoprecipitated chromatin was eluted, heated to 67 °C to reverse the cross-links, and the DNA-purified by ethanol precipitation. Libraries were prepared using the DNA SMART ChIP-Seq Kit (Clonetech, Mountain View, CA). 10 ng of ChIP DNA was used as input for each sample.
6
ChIP-seq Antibody Validation Protocol
7
ChIP Assay for Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ChIP assay was conducted according to Dahl’s protocol [60 (link)]. Briefly, the cells were fixed with 1% formaldehyde and sonicated to shear the DNA. After centrifugation, the supernatants were incubated with H3K4Me3 (Abcam, ab8580), H3K27Me3 (Abcam, ab6002), H3K27Ac (Abcam, ab4729), H3K27Cro (Jingjie PTM Biolab, PTM-501), STAT3 Y705 (Abcam, ab76315) or RNAP II antibodies (Abcam, ab5131). Chromatin DNA was purified with protein G Dynabeads (Invitrogen, 10004D) and subjected to real-time PCR. The region-specific primers used are listed in Table S1.
8
ChIP-seq Antibody Validation and Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used for ChIP were: H3 (Abcam ab1791, lot no. GR177884-2), H3K27ac (Abcam ab4729, lot no. GR200563-1), H3K27me3 (Millipore 07–499, lot no. 2475696), H3K36me3 (Abcam ab9050, lot no. GR204353-1), H3K4me3 (Abcam ab8580, lot no. GR190202-1), and H3K9me3 (Abcam ab8898, lot no. GR186864-1), and were validated by the company for specificity. For the H3K4me3 antibody, abcam reports strong binding to H3K4me3 but some cross reactivity with H3K4me2 [103 ]. The following primary antibodies were used for immunofluorescence: mouse anti-Nc82 (1∶20; Developmental Studies Hybridoma Bank) and rat anti-FruM (1:200) [18 (link)]. The secondary antibodies used for immunofluorescence were Alexa Fluor goat anti-rat 488 (1:1000), goat anti-mouse 633 (1:500), rabbit anti-GFP 488 conjugate (1:600), and streptavidin 488 conjugate (1:4000) (Thermo Fisher).
9
ChIP-qPCR Protocol for Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (2 10 7 ) were cross-linked with 1% formaldehyde at 25°C for 10 min. Nuclei were digested with 100 units of MNase at 37°C for 15 min. Mono or dinucleosomes-sized chromatin was incubated with antibodies for 3 h at 4°C and recovered with protein A agarose beads. DNA was purified by phenol extraction and then analyzed by quantitative PCR. The sequences of primers for ChIP assay are presented in Supplementary Table S2 andS3.
Antibodies used in ChIP experiment are GATA-1 (sc-1233) from Santa Cruz Biotechnology, CTCF (07-729) from Millipore, Rad21 (ab992), H3 (ab1791) and H3K27ac (ab4729) from Abcam. Normal rabbit IgG (12-370) from Millipore was used as a negative control.
Antibodies used in ChIP experiment are GATA-1 (sc-1233) from Santa Cruz Biotechnology, CTCF (07-729) from Millipore, Rad21 (ab992), H3 (ab1791) and H3K27ac (ab4729) from Abcam. Normal rabbit IgG (12-370) from Millipore was used as a negative control.
10
Quantifying Histone Modifications in HOTAIR-Modulated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MGC-803 cells were transfected with the Lenti-HOTAIR and Lenti-HOTAIR si, NC and Lenti-NC as a control to detect the amounts of H3K27me3/ac in the nuclei, and images were captured using a microscope (Olympus IX81; Olympus Corporation). The antibodies used were as follows: H3K27me3 (ab6002; 5 µg/ml), H3K27ac (ab45173; 5 µg/ml) (both from Abcam) and E-cadherin (14472; Cell Signaling Technology; 5 µg/ml). DAPI was used to dye the nuclei into blue.
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!