Ab4729

Manufactured by Cell Signaling Technology

Sourced in United States

Ab4729 is a primary antibody produced by Cell Signaling Technology. It is designed to detect specific targets using immunological techniques.

Automatically generated - may contain errors

Lab products found in correlation

Perfecta sybr green supermix, by Quanta Biosciences (1 mentions) Ab1791, by Abcam (1 mentions) Rotor gene rg3000 thermocycler, by Qiagen (1 mentions) Ab108341, by Abcam (1 mentions) Ab92513, by Abcam (1 mentions) Ab49900, by Abcam (1 mentions) Phospho pras40, by Cell Signaling Technology (1 mentions) Fkbp5, by Cell Signaling Technology (1 mentions) Phospho igf 1r, by Cell Signaling Technology (1 mentions) Irs 1, by Cell Signaling Technology (1 mentions) Phospho egfr, by Cell Signaling Technology (1 mentions) Phospho akt ser308, by Cell Signaling Technology (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) Ab10812, by Abcam (1 mentions) Anti h3k27me3, by Cell Signaling Technology (1 mentions) Anti histone 3, by Abcam (1 mentions) Anti h4ac, by Merck Group (1 mentions) Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions) Anti h3k9me2, by Active Motif (1 mentions) Anti h3k27ac, by Abcam (1 mentions)

9 protocols using ab4729

Chromatin Immunoprecipitation Assay on Drosophila Embryos

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Embryos were collected for 30 min and then aged at 25°C for 110 or 170 min to target nc13 and nc14b developmental stages. Embryos were then fixed and manually sorted as previously described (21 (link)). The following volumes of antibodies were used in ChIP experiments: Pc, 10 μl (47 (link)); Pho, 5 μl (48 (link)); E(z), 10 μl (49 (link)); Pcl, 10 μl (50 ); Hb, 5 μl; Cad, 5 μl (21 (link)); H3K27me3, 0.2 μl (Millipore #07-449); H3, 0.5 μl (Abcam #ab1791); mock, 0.5 μl (immunoglobulin G, Cell Signaling #2729); H3K27ac, 0.2 μl (Abcam #ab4729); and P-Rpb1 CTD (S5), 2 μl (Cell Signaling #D9N51). To approximately equalize the chromatin amount per ChIP using embryos at different developmental stages, we used a 2:1 ratio of nc13:nc14b as determined by total mass. Carefully staged embryos were then subjected to ChIP assays, and the enrichment values were calculated as previously described (21 (link)). Quanta Biosciences Perfecta SYBR green supermix was used for qPCR. All amplifications were performed in triplicate using Rotor Gene RG3000 thermocycler (Corbett Research). Sequences of PCR primers are listed in table S2.

Ghotbi E., Ye P., Ervin T., Kum A., Benes J, & Jones R.S. (2021). Polycomb-group recruitment to a Drosophila target gene is the default state that is inhibited by a transcriptional activator. Science Advances, 7(29), eabg1556.

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Immunohistochemical Analysis of OSCC Tissues

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Four-micrometer-thick sections from OSCC paraffin-embedded tissues were cut using a microtome and the sections were deposited on slides. Next, the slides were incubated at 60°C for 20min, deparaffinized in xylen and rehydrated in serial dilutions of ethanol (ethanol 100%, 96% and 70% for 2 min) and H2O which followed by incubation in PBS 3% hydrogen peroxide for 10min. The slides were then immersed in citrate buffer (pH = 6) and antigen retrieval was performed using microwave at 90°C for 15min. The sections were thereafter incubated with the primary antibodies, anti-H3K27ac (Abcam ab4729,1/200) and antiH3K27me3 (Cell signaling #9733, 1/200), for 1h at RT (For negative control PBS (phosphate buffered saline) was added instead of the primary antibody) which followed by three washes with PBS and then incubated with 100 ul of primary antibody amplifier master for 20min, after washing with PBS slides were incubated with 100 ul of secondary antibody for 30min, then washed 3 times in PBS. 1 drop of DAB chromogen concentrate was added to 1 drop of DAB substrate buffer (1:1) and 100 ul of it was used to develop the signal. After developing signal by DAB chromogen, the slides were dehydrated with serial dilutions of alcohol and mounted.

Shahhosseini A., Bourova-Flin E., Derakhshan S., Aminishakib P, & Goudarzi A. (2023). High levels of histone H3 K27 acetylation and tri-methylation are associated with shorter survival in oral squamous cell carcinoma patients. BioMedicine, 13(1), 22-38.

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ChIP-Seq with H3K27ac and H3K27me3 Antibodies

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The prepared chromatin was thawed on ice while 10ug of antibodies against either H3K27ac (Abcam #Ab4729) or H3K27me3 (Cell Signaling #CS9733S) was conjugated to a mix of magnetic Protein A and Protein G coupled beads (Invitrogen #100.02D and #100.04D, respectively) in the presence of 0.5% BSA in PBS with rotation at 4°C for 2 hours. Beads were washed 3 times with 0.5%BSA in PBS and either 5ug of chromatin was added to the H3K27ac ChIP or 10ug of chromatin was added to the H3K27me3 ChIP and rotated overnight at 4°C. The beads were washed 2 times with Tris-based RIPA buffer (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 7.4, 1mM EDTA pH 8.0, 0.1% Sodium Deoxycholate), 2 times with 0.3M NaCl RIPA (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 7.4, 1mM EDTA pH 8.0, 0.1% Sodium Deoxycholate, 0.3M NaCl), 2 times with LiCl Buffer (250mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 10mM Tris-HCl pH 8.0) and 2 times with TE buffer pH 7.6 (Fisher Scientific cat. no. BP2474-1). The beads were resuspended in 100uL of TE and RNAseA and PK digested/reverse crosslinked and purified as described in the chromatin prep. Chromatin libraries were prepared with 10–20ng of Input or ChIP’d DNA according to the ThruPLEX-FD Prep Kit (Rubicon #R40048) and sequenced by SE75 Next-Seq.

Zhang H., Qi J., Reyes J.M., Li L., Rao P.K., Li F., Lin C.Y., Perry J.A., Lawlor M.A., Federation A., De Raedt T., Li Y.Y., Liu Y., Duarte M.A., Zhang Y., Herter-Sprie G.S., Kikuchi E., Carretero J., Perou C.M., Reibel J.B., Paulk J., Bronson R.T., Watanabe H., Fillmore C.M., Kim C.F., Hammerman P.S., Brown M., Cichowski K., Long H., Bradner J.E, & Wong K.K. (2016). Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer. Cancer discovery, 6(9), 1006-1021.

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Comprehensive Protein Profiling Protocol

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The following antibodies were used for Western blotting and ChIP: AR (Abcam; ab108341; 1:1,000 for Western blotting), beta-actin (Abcam; ab49900; 1:50,000 for Western blotting), ERG (Abcam; ab92513; 1:1,000 for Western blotting), histone H3 (acetyl K27) (Abcam; ab4729), IRS-1 (Cell Signaling; 2390S; 1:1,000 for Western blotting), IRS-2 (Cell Signaling; 4502S; 1:1,000 for Western blotting), phospho-AKT (Ser308) (Cell Signaling; 13038S; 1:1,000 for Western blotting), phospho-AKT (Ser473) (Cell Signaling; 4060L; 1:1,000 for Western blotting), PTEN (Cell Signaling; 9559L; 1:1,000 for Western blotting), phospho-IGF1R (Cell Signaling; 3024S; 1:1,000 for Western blotting), FKBP5(Cell Signaling; 12210S; 1:1,000 for Western blotting), phospho-Pras40 (Cell Signaling; 2997s; 1:1,000 for Western blotting), phospho-EGFR (Cell Signaling; 3777S; 1:1,000 for Western blotting), phosphor-GSK3B (Cell Signaling; 9336S; 1:1,000 for Western blotting).

Mao N., Gao D., Hu W., Gadal S., Hieronymus H., Wang S., Lee Y.S., Sullivan P., Zhang Z., Choi D., Rosen N., Sawyers C.L., Gopalan A., Chen Y, & Carver B.S. (2020). Oncogenic ERG represses PI3K signaling through down-regulation of IRS2. Cancer research, 80(7), 1428-1437.

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Immunostaining of Drosophila Embryos

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Embryos were collected on grape agar plates, dechorionated for 2 min in 50% bleach, and fixed in methanol-heptane (1:1) for 5 min. The fixed embryos were stored in methanol at −20°C. Before immunostaining, the embryos were first rehydrated gradually (5 min each in 1:3, 1:1, and 3:1 PTA:methanol and then 10 min in PTA). PTA consisted of PBS supplemented with 0.1% Triton X-100, and 0.02% azide. The embryos were then blocked in PBTA (PTA plus 1% BSA) for 30 min and incubated with primary antibodies (1:100 in PBTA) for 1 h at room temperature or overnight at 4°C. The following primary antibodies were used: anti-Histone 3 (Abcam, ab1791), anti-H3K9me1 (Active Motif, 39249), anti-H3K9me2 (Active Motif, 39683 and 39375), anti-H3K9me3 (Active Motif, 39765; Millipore, clone CMA308), anti-H3K9ac (Abcam, ab10812), anti-H3K27ac (Abcam, ab4729), anti-H3K27me3 (Cell Signaling, 9733), and anti-H4ac (Millipore, 06-598). The embryos were washed three times for 5 min each in PBTA and incubated with the appropriate fluorescently labeled secondary antibodies (Molecular Probes) for 1 h in the dark at room temperature. They were then washed four times for 5 min each in PBTA and mounted in VectaShield mounting medium with DAPI (Vector Laboratories). For TALE-light stainings, GFP- or mCherry-tagged purified TALE-light protein (1:500) was included during the incubation with secondary antibodies.

Yuan K, & O'Farrell P.H. (2016). TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos. Genes & Development, 30(5), 579-593.

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CUT&RUN Profiling of ILC3s

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CUT&RUN was performed on ∼13,000 Nkp46⁺ ILC3s from Maf^+/+Il7r^Cre and Maf^fl/flIl7r^Cre SILP as previously described (Skene et al., 2018 (link); see RNA-seq methods for gating strategy). In short, after sorting, the cells were permeabilized with 0.005% digitonin freshly made, bound to concanavalin A beads, incubated with H3K27ac, H3K27me3, or IgG antibodies overnight at 4°C with rotation. The remaining steps were performed as described in the original protocol. The following antibodies were used: H3K27ac (Abcam; ab4729), H3K27me3 (Cell Signaling; 9733S), and rabbit anti-mouse IgG (Abcam; ab46540). CUT&RUN libraries were submitted to the Duke Sequencing and Genomic Technologies Shared Resource facility for sequencing on an Illumina NextSeq500 using a 42-bp paired-end mode.

Parker M.E., Barrera A., Wheaton J.D., Zuberbuehler M.K., Allan D.S., Carlyle J.R., Reddy T.E, & Ciofani M. (2019). c-Maf regulates the plasticity of group 3 innate lymphoid cells by restraining the type 1 program. The Journal of Experimental Medicine, 217(1), e20191030.

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Western Blot Analysis of Cellular Proteins

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The harvested cells were lysed and centrifuged for supernatant collection. Then, 25 μg of protein was loaded into each well of a 10% or 15% SDS-PAGE gel. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane, followed by 1 h room temperature blocking with 5% non-fat milk. The membrane was then incubated with primary antibody (TRIM27: Abcam, USA, ab154931; p16: Abcam, ab51243; cleaved caspase-3: Abcam, ab2302; β-gal: Santa Cruz Biotechnology, sc-377257; p53: Santa Cruz Biotechnology, sc-393031; p21: Santa Cruz Biotechnology, sc-6246; H3K27ac: Abcam, ab4729; GAPDH: Cell Signaling Technology, #5174) overnight at 4°C. The membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody (Beyotime Biotechnology: A0208 or A0216) for 1 h at 37°C. The proteins were visualized by exposing the membrane to a chemiluminescence detection kit (ECL, Millipore, USA) for image scanning. GAPDH was utilized as the loading control.

Xing L., Tang X., Wu K., Huang X., Yi Y, & Huan J. (2020). TRIM27 Functions as a Novel Oncogene in Non-Triple-Negative Breast Cancer by Blocking Cellular Senescence through p21 Ubiquitination. Molecular Therapy. Nucleic Acids, 22, 910-923.

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Immunostaining of Drosophila Ovaries

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Ovaries were dissected in ice-cold PBS solution. Dissected ovaries were fixed in 4% paraformaldehyde in 1x PBS for 15 minutes at room temperature. Primary antibodies were added and then incubated for overnight at 4°C. Antibodies used in this study are rabbit anti-GFP (Invitrogen #A11122, 1:1000), mouse anti-GFP (Invitrogen #A11120, 1:1000), rabbit anti-H3K4Me2 (Millipore #07-030, 1:1000), rabbit anti-H3K27Ac (Abcam #ab4729, 1:1000), rabbit anti-H3K27Me3 (Cell signaling #9733, 1:1000), mouse anti-Lsd1 (1:2500,²⁵ (link) mouse anti-Hnt (DSHB #1G9, 1:20) and mouse anti-Cut (DSHB #2B10, 1:25). Secondary antibodies used are goat anti-rabbit 488 (Invitrogen #A11008, 1:500), goat anti-mouse 488 (Invitrogen #A11001, 1:500), goat anti-mouse 568 (Invitrogen #A11004, 1:500) and goat anti-rabbit 568 (Invitrogen #A11011, 1:500). Stained ovaries were mounted in mounting medium with DAPI (EMS#17989-20) on glass slides. Images were taken on Zeiss Axio Imager 2/Apotome.2 microscope and processed with ImageJ software. As previously described, the number of follicle cells was quantified by taking pictures of stage 10 egg chambers and counting the number of nuclei (based on DAPI signals) per side using ImageJ software.²⁵ (link)

Lin C.T., Ting R.T., Ou Y.H., Shao T.L, & Lee M.C. (2024). Protein degradation of Lsd1 is mediated by Bre1 yet opposed by Lsd1-interacting lncRNAs during fly follicle development. iScience, 27(5), 109683.

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Profiling Chromatin Modifications in Mecp2 KO Mice

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Cortices from adult Mecp2 KO and littermate wild type controls, or adult Nestin-cre; Dnmt3a^fl/fl and littermate wild type controls (Dnmt3a^fl/fl) were collected and homogenized with a dounce-homogenizer and tight pestle 15 times in 5 ml. Buffer HB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH7.8, 1mM DTT, 0.15 mM spermine, 0.5 mM spermidine, Roche protease inhibitor tablets, 10mM Sodium Butyrate, 1mM RNase Inhibitor when doing downstream RNA-seq). IGEPAL CA-630 (Sigma) was added to a final concentration of 0.3% to the homogenate, followed by douncing 5 more times with tight pestle. Homogenized cortices were filtered through a 40um strainer, and centrifuged at 500g at 4°C. The nuclei were resuspended in CUT&RUN wash buffer, and CUT&RUN experiments were performed as described above using anti-MECP2 antibodies (Chen et al., 2003 (link)), anti-H3K27ac antibodies (Abeam ab4729, RRID:AB_2118291) and IgG (Cell Signaling #2729S, RRID:AB_1031062).

Stroud H., Yang M.G., Tsitohay Y.N., Davis C.P., Sherman M., Hrvatin S., Ling E, & Greenberg M.E. (2020). An activity-mediated transition in transcription in early postnatal neurons. Neuron, 107(5), 874-890.e8.

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