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Ab5131

Manufactured by Merck Group
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The Ab5131 is a laboratory equipment product designed for performing various analytical and experimental tasks. It serves as a core function within the research and development process. The product specifications and technical details are available upon request.

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Lab products found in correlation

Ab5095, by Abcam (4 mentions) T5168, by Merck Group (2 mentions) Triptolide, by Merck Group (2 mentions) Bradford protein assay, by Bio-Rad (2 mentions) Ab290, by Abcam (1 mentions) Ab9050, by Abcam (1 mentions) α flag, by Merck Group (1 mentions) Ni nta column, by Bio-Rad (1 mentions) Anti p53, by Agilent Technologies (1 mentions) Ram hrp, by Agilent Technologies (1 mentions) Gar hrp, by Agilent Technologies (1 mentions) Amersham hybond ecl membrane, by GE Healthcare (1 mentions) Mab374, by Merck Group (1 mentions) Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Ab1791, by Merck Group (1 mentions) F3165, by Merck Group (1 mentions) Hiseq 2500 platform, by Illumina (1 mentions) Ab5095, by Merck Group (1 mentions) Ni sepharose 6 fast flow, by GE Healthcare (1 mentions)

7 protocols using ab5131

1

Chromatin Immunoprecipitation Protocol

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ChIP was performed as previously described (Zaratiegui, 2011 (link)). The following antibodies were used: Pol II pS2 - Abcam ab5095, Pol II pS5 - Abcam ab5131, H3K9me2 – Milipore 07-441, Myc - Invitrogen R950, GFP - Abcam ab290, RNA:DNA hybrids - S9.6.
Castel S.E., Ren J., Bhattacharjee S., Chang A.Y., Sánchez M., Valbuena A., Antequera F, & Martienssen R.A. (2014). Dicer promotes transcription termination at sites of replication stress to maintain genome stability. Cell, 159(3), 572-583.
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2

Purification and Characterization of dBigH1

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αdBigH1 antibodies are described in (6 (link)). αdH1 antibodies were a gift from Dr J. Kadonaga and are described in (16 (link)). αRpb3 antibodies were a gift from Dr J. Zeitlinger and are described in (17 (link)). The rest of antibodies used in these experiments were commercially available: αPol IIoser5 (Abcam, ab5131), αH3Kac (Millipore 06-599), αH3K36me3 (Cell Signaling, 9715), αH3 (Abcam, ab9050), αH4 (Abcam, ab10158) and αFLAG (Sigma F3165).
Recombinant His-tagged dBigH1 constructs were expressed in Escherichia coli BL21-LysS cells using pET30b(+) expression vectors (Novagen), and purified using Ni-NTA columns (BioRad) by conventional methods.
Climent-Cantó P., Carbonell A., Tatarski M., Reina O., Bujosa P., Font-Mateu J., Bernués J., Beato M, & Azorín F. (2020). The embryonic linker histone dBigH1 alters the functional state of active chromatin. Nucleic Acids Research, 48(8), 4147-4160.
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3

Cell Lysis and Western Blot Analysis

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U2OS cells were harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH 8.0; Sigma-Aldrich) supplemented with 1x PIC-C (Calbiochem), incubated on ice for an hour, then centrifuged (13,000 rpm at 4 °C for 5 minutes). The supernatant lysates were mixed with the same amount of 2x SDS loading buffer containing 5% β-mercaptoethanol (Sigma-Aldrich) and boiled for 5 minutes. The lysates were separated in SDS-PAGE, transferred to Amersham Hybond ECL-membrane (GE Healthcare) and incubated with the following primary antibodies: anti-p53 (Dako, IS616), anti-S15 P p53 (Cell signalling, 9284), 1BP7G5 anti-RPB1 (from L. Tora, IGBMC), anti-S2P RPB1 (Abcam, ab5095) and anti-S5P RPB1 (Abcam, ab5131), anti-GAPDH (Millipore, MAB374); then the following secondary antibodies: RAM-HRP (Dako, P0260) and GAR-HRP (Dako, P0448). Chemiluminescent detection was conducted using Immobilon Western Chemiluminescent HRP substrate (Millipore).
Borsos B.N., Huliák I., Majoros H., Ujfaludi Z., Gyenis Á., Pukler P., Boros I.M, & Pankotai T. (2017). Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage. Scientific Reports, 7, 40960.
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4

Cross-linking ChIP-seq and ChIP-qPCR

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After cross-linking, ChIP-qPCR and ChIP-seq were performed as described in Ribaud et al. (2012) (link) with the following modifications for ChIP-seq. A defined amount (5% of total chromatin) of cross-linked and sonicated chromatin from S. pombe was added to S. cerevisiae chromatin prior to the immunoprecipitation step. Following steps in the ChIP protocol were performed as described in Ribaud et al. (2012) (link). ChIP was performed using the following antibodies: for histone H3 ChIP, Abcam ab1791; for histone H3K9ac, Millipore 07-352; for histone H4ac, Millipore 06-866; for RNAPII, Abcam ab5131; for Flag, Sigma F3165; and for Myc, a homemade antibody (clone 9E10). DNA libraries where prepared using TruSeq Illumina kit according to the manufacturer's instructions and sequenced in single-end mode on the Illumina HiSeq 2500 platform at the Institute of Genetics and Genomics of Geneva (iGE3; http://www.ige3.unige.ch/genomics-platform.php). For ChIP-qPCR, primer sequences are available on request.
Bruzzone M.J., Grünberg S., Kubik S., Zentner G.E, & Shore D. (2018). Distinct patterns of histone acetyltransferase and Mediator deployment at yeast protein-coding genes. Genes & Development, 32(17-18), 1252-1265.
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5

Antibodies Generation and Validation

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Pol II S2P and Pol II S5P antibodies were purchased from Sigma-Aldrich (Ab5095 and Ab5131). For the ChIP-Seq analysis of CBP/Neijre, Rpb3, Spt5, NELF-A and NELF-E we used previously described antibodies2 ,3 (link). Antibodies against NELF-B (epitope corresponding to 150–594 amino acid residues) were generated in this study. The epitopes for antibody production were expressed as 6 × His-tagged fusion proteins in Escherichia coli, affinity-purified on Ni Sepharose 6 Fast Flow (GE Healthcare), according to the manufacturer’s protocol, and injected into rabbits, following the standard immunization procedure. Antibodies were affinity-purified using the same epitopes as were used for immunization. The specificity of antibodies against EcR, Spt5, NELF A and NELF B was characterized in the RNA interference experiments by the specific depletion of corresponding proteins (Figs. S1–S4). Antibodies production was performed according to procedures outlined in the NIH (USA) Guide for the Care and Use of Laboratory Animals. The protocol used was approved by the Committee on Bioethics of the Institute of Gene Biology of the Russian Academy of Sciences. All procedures were performed under conditions designed to minimize suffering.
Mazina M.Y., Kovalenko E.V, & Vorobyeva N.E. (2021). The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes. Scientific Reports, 11, 172.
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6

Transcriptional Regulation by Triptolide

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Scc1-mEGFP-AID cells were treated with 300 nM Triptolide (Sigma, T3652) for 3 hours, and cells were collected for western blot or Hi-C. For western blot, cells were resuspended in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate and 0.1% SDS), which additionally contained pepstatin, leupeptin and chymostatin (10 μg/ml each) and PMSF (1 mM). The protein concentration was determined with the Bradford Protein Assay (Bio-Rad Laboratories). SDS-PAGE and standard western blot technique were applied to detect individual proteins with specific antibodies: rabbit anti-RNA polymerase II phospho-Ser2 (Abcam, ab5095), rabbit anti-RNA polymerase II phospho-Ser5 (Abcam, ab5131) and mouse anti-tubulin (Sigma, T-5168).
Thiecke M.J., Wutz G., Muhar M., Tang W., Bevan S., Malysheva V., Stocsits R., Neumann T., Zuber J., Fraser P., Schoenfelder S., Peters J.M, & Spivakov M. (2020). Cohesin-Dependent and -Independent Mechanisms Mediate Chromosomal Contacts between Promoters and Enhancers. Cell Reports, 32(3), 107929.
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7

Triptolide treatment and Protein Analysis

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Scc1-EGFP-AID cells were treated with 300 nM Triptolide (Sigma, T3652) for 3 hours, and cells were collected for western blot or Hi-C. For western blot, cells were resuspended in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Nadeoxycholate and 0.1% SDS), which additionally contained pepstatin, leupeptin and chymostatin (10 µg/ml each) and PMSF (1 mM). The protein concentration was determined with the Bradford Protein Assay (Bio-Rad Laboratories). SDS-PAGE and standard Western blot technique were applied to detect individual proteins with specific antibodies. Rabbit anti-RNA polymerase II Ser2 (abcam, ab5095), rabbit anti-RNA polymerase II Ser5 (abcam, ab5131) and mouse anti-tubulin (Sigma, T-5168).
Thiecke M.J., Wutz G., Muhar M., Tang W., Bevan S., Malysheva V., Stocsits R.R., Neumann T., Zuber J., Fraser P., Schoenfelder S., Peters J., & Spivakov M. (2020). Cohesin-dependent and independent mechanisms support chromosomal contacts between promoters and enhancers.
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