Ab24758

Manufactured by Abcam

Sourced in United States

Ab24758 is a laboratory instrument used for the detection and measurement of specific molecules or analytes in a sample. It is a highly sensitive and accurate device designed for research and diagnostic applications. The core function of Ab24758 is to provide quantitative data on the presence and concentration of the target analyte within a sample.

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Lab products found in correlation

Ab817, by Abcam (3 mentions) Texas red conjugated anti rabbit antibody, by Jackson ImmunoResearch (3 mentions) Vectashield, by Vector Laboratories (3 mentions) Ab5131, by Abcam (3 mentions) Ab24759, by Abcam (2 mentions) Anti total pol 2, by Merck Group (2 mentions) Ma1 744, by Thermo Fisher Scientific (1 mentions) Pa5 72344, by Thermo Fisher Scientific (1 mentions) Pa5 13174, by Thermo Fisher Scientific (1 mentions) Anti rabbit a3687, by Merck Group (1 mentions) Ab5408, by Abcam (1 mentions) 4336 bpc 100, by Bio-Techne (1 mentions) Formaldehyde, by Merck Group (1 mentions) Glycine, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Magnify chromatin immunoprecipitation system, by Thermo Fisher Scientific (1 mentions) Sc 899x, by Santa Cruz Biotechnology (1 mentions) Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions) Rnapii, by Santa Cruz Biotechnology (1 mentions) Alexa flour 488, by Thermo Fisher Scientific (1 mentions)

12 protocols using ab24758

Protein Interaction and Regulation Analysis

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Western blot analysis: The following primary antibodies were used: PARP1 C terminal, rabbit polyclonal (#39561, Active Motif, Carlsbad, CA, USA); Actin, mouse monoclonal (MA1-744, Thermo Fisher Scientific, Waltham, MA); H5 (Ser2P), mouse monoclonal (ab24758, Abcam, Cambridge, MA, USA); IntS3, rabbit polyclonal (PA5-72344, Thermo Fisher Scientific, Waltham, MA, USA); AF9, rabbit polyclonal (PA5-13174, Thermo Fisher Scientific, Waltham, MA, USA), Cyclin T1, rabbit polyclonal (PA5-24163, Thermo Fisher Scientific, Waltham, MA, USA); PAR rabbit polyclonal (4336-BPC-100, Trevigen, Gaithersburg, MD, USA). Secondary antibodies used: anti-rabbit (A3687) and anti-mouse (A3562) IgG (whole molecule) alkaline phosphatase antibody (Sigma).
Co-immunoprecipitation and chromatin immunoprecipitation (ChIP): PARP1, rabbit polyclonal (#39561, Active Motif, Carlsbad, CA, USA); H5 (Ser2P), mouse monoclonal (ab24758, Abcam, Cambridge, MA, USA); 4H8, mouse monoclonal (ab5408, Abcam, Cambridge, MA, USA); 8WG16, mouse monoclonal (ab817, Abcam, Cambridge, MA, USA); IntS3, rabbit polyclonal (PA5-72344, Thermo Fisher Scientific, Waltham, MA, USA); AF9, rabbit polyclonal (PA5-13174, Thermo Fisher Scientific, Waltham, MA, USA).

Matveeva E.A., Dhahri H, & Fondufe-Mittendorf Y. (2022). PARP1′s Involvement in RNA Polymerase II Elongation: Pausing and Releasing Regulation through the Integrator and Super Elongation Complex. Cells, 11(20), 3202.

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Chromatin Immunoprecipitation Protocol for FLI1 and RNAPII

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2 × 10⁸ SK-N-MC and LAP-35 cells were cross-linked with 1% (w/v) formaldehyde (Sigma Aldrich) for 15 min at room temperature, and then formaldehyde was inactivated by the addition of 125 mM glycine (Sigma Aldrich). Cells were washed in cold PBS and lysed to isolate nuclei in a hypotonic buffer containing 5 mM PIPES (pH 8.0), 85 mM KCl, NP40 0.5%, 1 mM dithiothreitol, 10 mM β-glycerophosphate, 0.5 mM Na₃VO₄, and protease inhibitor cocktail (Sigma-Aldrich). Isolated nuclei were lysed in a buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 10 mM β-glycerophosphate, 0.5 mM Na₃VO₄, and protease inhibitor cocktail (Sigma-Aldrich) and sonicated with Bioruptor (Dyagenode) 2 fold for 6 min (30 sec sonication and 30 sec pause). Chromatin extracts containing DNA fragments (100 μg/sample) with an average size of 200 bp were pre-cleared overnight and then immunoprecipitated for 3 hours using 2 μg of anti-FLI1 (SAB2100822, Sigma Aldrich) or anti-RNAPII (N20, Santa Cruz Biotechnology) or anti RNAPII CTD repeat H14 and H5 (ab24759 and ab24758, Abcam) antibodies and Protein A/G Agarose/Salmon Sperm DNA (Merck Millipore). Precipitated DNA was extracted and analyzed by qPCR using primers listed in Supplementary Table 2.

Fidaleo M., Svetoni F., Volpe E., Miñana B., Caporossi D, & Paronetto M.P. (2015). Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9. Oncotarget, 6(31), 31740-31757.

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Immunofluorescent Localization of CENP-C and RNA Pol II

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Metaphase chromosome spreads derived from parental cells were rinsed in PBS, blocked in 5% horse serum then incubated overnight with anti-CENP-C antibody^{83 (link)} (non-commercial antibody provided by Stefania Purgato; 1:200) and anti-RNA pol II (1:1000, Abcam Ab24758) antibody. Secondary antibodies were FITC-conjugated anti-mouse and Texas Red-conjugated anti-rabbit antibodies (1:150, Jackson ImmunoResearch Laboratories). Slides were DAPI stained, mounted in Vectashield (Vector Laboratories, Cat No H-1000) and imaged on a Zeiss epifluorescence microscope using a 100x objective.

Naughton C., Huidobro C., Catacchio C.R., Buckle A., Grimes G.R., Nozawa R.S., Purgato S., Rocchi M, & Gilbert N. (2022). Human centromere repositioning activates transcription and opens chromatin fibre structure. Nature Communications, 13, 5609.

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Chromatin Immunoprecipitation Sequencing Protocol

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Samples for ChIP and ChIP-seq assays were prepared and analyzed according to Myers Lab ChIP-seq Protocol v041610 (http://myers.hudsonalpha.org/documents/, first accessed on 1 November 2011) by using MAGnify Chromatin Immunoprecipitation System protocol (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies used: MYC (sc-764Z, Santa Cruz Biotechnologies, Dallas, TX. USA), MAX (sc-197X, Santa Cruz, Dallas, TX. USA), RNAPII (sc-899X, Santa Cruz, Dallas, TX. USA), RNAPII phospho Ser5 (ab5131, Abcam, Cambridge, UK), RNA Pol II phospho Ser2 (ab24758, Abcam, Cambridge, UK and 3E19, Active Motif, Vinci-Biochem, FI, Italy), Flag (F1804, Sigma, St. Louis, Mo, USA). For RNA-seq, 2µg total RNA purified by PureLinkRNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used. ChIP-seq and RNA-seq libraries were prepared at Istituto di Genomica Applicata (IGA; www.appliedgenomics.org/ accessed on 13 December 2022) according to Illumina TruSeq DNA and TruSeq RNA Sample Preparation Guides. Samples were sequenced through Illumina HiSeq 2000 e 2500.

Scagnoli F., Palma A., Favia A., Scuoppo C., Illi B, & Nasi S. (2023). A New Insight into MYC Action: Control of RNA Polymerase II Methylation and Transcription Termination. Biomedicines, 11(2), 412.

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Investigating Bdnf Transcription in Morphine Withdrawal

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For each ChIP sample, VTA punches were pooled from two rats (4 punches) that were killed on day 28, after 14 days withdrawal from 14 days of prior morphine administration (n = 20 or 24). Anti-total-Pol II (mouse monoclonal, Millipore, 05-623), anti-phospho-Ser2-Pol II (antibody to Pol II phosphorylated at Ser2 of its C terminal domain) (mouse monoclonal, Abcam, ab24758), or anti-phospho-Ser5-Pol II (rabbit polyclonal, Abcam, ab5131) were used for this experiment. Primers for qChIP with Anti-total-Pol II and anti-phospho-Ser5-Pol II were designed to amplify 150–200 bp products that include CREB binding sites of Bdnf promoters. Primers for qChIP with anti-phospho-Ser2-Pol II were designed to amplify 150–200 bp products within the coding sequence of the Bdnf gene (Supplementary Table 3).

Koo J.W., Mazei-Robison M.S., LaPlant Q., Egervari G., Braunscheidel K.M., Adank D.N., Ferguson D., Feng J., Sun H., Scobie K.N., Damez-Werno D.M., Ribeiro E., Peña C.J., Walker D., Bagot R.C., Cahill M.E., Anderson S.A., Labonté B., Hodes G.E., Browne H., Chadwick B., Robison A.J., Vialou V.F., Dias C., Lorsch Z., Mouzon E., Lobo M.K., Dietz D.M., Russo S.J., Neve R.L., Hurd Y.L, & Nestler E.J. (2015). Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area. Nature neuroscience, 18(3), 415-422.

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Immunofluorescence Staining of Cellular Markers

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Cells were fixed in 4% ice-cold PFA for 10min, permeabilized with 1% Triton-X100 in PBS for 15 min and blocked with 10%NGS, 1% BSA in 0.1% PBT (0.1% Tween-20 in PBS) for 60 min. For digitonin experiments, cells were permeabilized in 0.003% digitonin (D141, Sigma) in PBS. Primary incubation was performed at 4°C overnight in 0.1% PBT containing 1% BSA. Secondary incubation was performed in primary incubation buffer for 45 min (RT) at a dilution of 1:500. Actin was counterstained with Phalloidin conjugated to either Texas Red-X for embryonic tissues and marker co-localization in vitro studies (Life Technologies), Alexa Flour 488 for cardiac sections of hypertrophic mice (Life Technologies) or CF405 for all other in vitro cultures (Biotium). Primary antibodies: H3K9me3 (ab8898, Abcam, 1:800), H3K27me3 (ab6002, Abcam, 1:200), RNA polymerase II CTD repeat YSPTSPS phospho S2 (ab24758, Abcam, 1:400), nesprin-1 (ab24742, Abcam, 1:500), DAPI (Invitrogen, 1:1000), and Emerin (30853S, CST,1:250).

Seelbinder B., Ghosh S., Schneider S.E., Scott A.K., Berman A.G., Goergen C.J., Margulies K.B., Bedi K., Casas E., Swearingen A.R., Brumbaugh J., Calve S, & Neu C.P. (2021). Nuclear Deformation Guides Chromatin Reorganization in Cardiac Development and Disease. Nature biomedical engineering, 5(12), 1500-1516.

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Investigating Bdnf Transcription in Morphine Withdrawal

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Immunofluorescence Microscopy of Mitotic Cells

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RPE-1 cells were grown to 80% confluency on coverslips and treated with colcemid for 2 h. Cells were incubated with hypotonic medium (60% medium, 40% water) for 3 min at 37°C and then centrifuged at 2500 rpm for 10 min. Cells were blocked in KCM buffer (120mM KCl, 20mM NaCl, 10mM Tris-HCl pH=7.7, 0.1% Triton-X-100, 0.5 mM EDTA) + 1% BSA for 30 min. Incubations with primary antibodies were conducted in blocking buffer for 1 h at room temperature using the following antibodies: ACA (1:500; 15-235-0001, Antibodies Incorporated), ATR (1:1000; A300-138A, Bethyl Laboratories) and RNA polymerase II S2P (1:1000; ab24758, Abcam). Samples were washed in KCM three times and then incubated with the respective secondary antibody (1:500) in blocking buffer for 45 min. Cells were washed in KCM three times and then fixed in KCM+4% formaldehyde for 10 min prior to DAPI staining and slide mounting. Images were acquired on a Fluorescent microscope DeltaVision Core system (Applied Precision) with 100X Olympus UPlanSApo 100 oil-immersion objective (NA 1.4), 250W Xenon light source equipped with a Photometrics CoolSNAP_HQ2 Camera. 4 µm Zstacks were acquired (Z step size: 0.2 µm).

Giunta S., Hervé S., White R.R., Wilhelm T., Dumont M., Scelfo A., Gamba R., Wong C.K., Rancati G., Smogorzewska A., Funabiki H., & Fachinetti D. (2020). CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy.

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Immunofluorescence of Mitotic Chromosomes

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Metaphase chromosome spreads derived from parental cells were rinsed in PBS, blocked in 5% horse serum then incubated overnight with anti-CENP-C antibody (1:200, gift from W. Earnshaw) and anti-RNA pol II (1:1000, Abcam Ab24758) antibody. Secondary antibodies were FITC-conjugated anti-mouse and Texas Red-conjugated anti-rabbit antibodies (1:150, Jackson ImmunoResearch Laboratories). Slides were DAPI stained, mounted in Vectashield (Vector Laboratories, Cat No H-1000) and imaged on a Zeiss epifluorescence microscope using a 100x objective.

Gilbert N., Naughton C., Huidobro C., Catacchio C.R., Buckle A., Grimes G.R., Nozawa R., Purgato S., & Rocchi M. (2022). Human centromere formation activates transcription and opens chromatin fibre structure.

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ChIP-Seq for Arabidopsis AGO1

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ChIP assays were performed as previously described (Fang et al., 2015) . Briefly, 10-day-old Col-0 and ago1-36 seedlings (about 1.5 g), without or with hormone and stress treatments, were cross-linked in 1% formaldehyde for 20 min. Then the nuclei were isolated and resuspended in high salt nuclear lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 4 mM MgCl 2 , 0.2% NP-40, 5 mM DTT, and Roche protease inhibitor cocktail). Chromatin was sonicated to an average size of 200-500 bp using the Bioruptor (Diagenode). After centrifugation at 16,000 g for 10 min, the supernatant was diluted 5-fold with dilution buffer (20 mM Tris-HCl, pH 7.5, 4 mM MgCl 2 , 0.2% NP-40). AGO1 (Qi et al., 2005) , myc (Abcam, ab32), Pol II (Abcam, ab817), Pol II Ser2P (Abcam, ab24758), or Pol II Ser5P antibody (Abcam, ab24759) was used to immunoprecipitate the protein-DNA complex. Rabbit IgG (Abmart, B30011) or mouse IgG (Abmart, B30010) was used as a negative antibody control for IP. After reverse cross-linking, digestion by proteinase K, the enriched DNA was purified for library construction for Illumina sequencing or for qPCR. The primers are listed in Table S6.

Liu C., Xin Y., Xu L., Cai Z., Xue Y., Liu Y., Xie D., Liu Y, & Qi Y. (2018). Arabidopsis ARGONAUTE 1 Binds Chromatin to Promote Gene Transcription in Response to Hormones and Stresses. Developmental cell, 44(3).

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