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Rnapii

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RNAPII is a macromolecular complex that functions as the essential enzyme responsible for the transcription of DNA into messenger RNA (mRNA) in eukaryotic cells. It is a multi-subunit enzyme that plays a crucial role in gene expression.

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Lab products found in correlation

β actin, by Merck Group (3 mentions) Caspase glo 3 7 assay, by Promega (2 mentions) Biocoat matrigel invasion chamber, by Corning (2 mentions) Rneasy plus kit, by Qiagen (2 mentions) Celltiter blue cell viability assay, by Promega (2 mentions) P3xflag cmv 7, by Merck Group (2 mentions) Carboplatin, by Selleck Chemicals (2 mentions) Nup98, by Cell Signaling Technology (2 mentions) Migration chamber, by Ibidi (2 mentions) Pcas9 bb 2a puro px459 v2, by Addgene (2 mentions) Caspase 8, by Enzo Life Sciences (2 mentions) Annexin 5 and 7 aad, by BD (2 mentions) Viia 7 system, by Thermo Fisher Scientific (2 mentions) Nextseq 500 platform, by Illumina (2 mentions) Truseq chip sample preparation kit, by Illumina (2 mentions) Spriselect beads, by Beckman Coulter (2 mentions) Power sybr green kit, by Thermo Fisher Scientific (2 mentions) Igg from rabbit serum, by Merck Group (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Rad21, by Fortis Life Sciences (2 mentions)

Spelling variants of this product

Anti-RNAPII (2 citations) RNAPII-Ser5p (2 citations) RNAPII CTD S7 (2 citations)

7 protocols using rnapii

1

Comprehensive Protein Analysis in Cancer

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Antibodies and sources: CDK9 (1.1000), pCDK9 (Thr187) (1.1000), RNAPII (1.1000), phospho-RNAPII(Ser2) (1.1000), PARP (1.1000), and NUP98 (1.1000) (Cell Signaling Technology); HEXIM1 (1.1000), NELF-a (1.1000), LARP-7 (1.1000), c-Myc (1.1000), SPT5 (1.1000), and GAPDH (1.5000) (Santa Cruz Biotechnology, Dallas, TX, USA); BRD4 (1.1000) (Abcam, Waltham, MA, USA); Caspase-8 (1.1000) (Enzo Life Sciences, Farmingdale, NY, USA); β-Actin (1.10000), (Sigma-Aldrich, St. Louis, MO, USA); Cyclin T (1.1000) (Bethyl, Montgomery, TX, USA). Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assays (Promega, Madison, WI, USA); AnnexinV and 7AAD (BD); BAY1251152, BI894999, ABBV744, Paclitaxel and Carboplatin (Selleckchem, Houston, TX, USA); BioCoat Matrigel invasion chamber (Corning, Corning, NY, USA); Migration chamber (Ibidi, Gräfelfing, Germany); RNeasy Plus kit (Qiagen, Venlo, The Netherlands). The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene, Watertown, MA, USA); p3xFlag-CMV-7.1 (E7533, Sigma, Ronkonkoma, NY, USA).
Gasimli K., Raab M., Mandal R., Krämer A., Peña-Llopis S., Tahmasbi Rad M., Becker S., Strebhardt K, & Sanhaji M. (2023). Synergistic Sensitization of High-Grade Serous Ovarian Cancer Cells Lacking Caspase-8 Expression to Chemotherapeutics Using Combinations of Small-Molecule BRD4 and CDK9 Inhibitors. Cancers, 16(1), 107.
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2

Evaluation of THZ1 Signaling Pathways

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THZ1 (#M5228) was purchased from AbMole BioScience, Inc. (Houston, TX, USA). Antibodies against various proteins for Western blot analyses, such as CDK7, RNAPII, RNAPII pS5, RNAPII pS7, cleaved poly ADP ribose polymerase (PARP), cleaved caspase-3, cleaved caspase-7, VEGFR2, CD31, and VEGF, were obtained from Cell Signaling Technology (Danvers, MA, USA). The β-actin antibody was purchased from GeneTex (Irvine, CA, USA), and the α–tubulin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), Merck Millipore (Billerica, MA, USA), and Invitrogen (Carlsbad, CA, USA).
Shi C.S., Kuo K.L., Chen M.S., Chow P.M., Liu S.H., Chang Y.W., Lin W.C., Liao S.M., Hsu C.H., Hsu F.S., Chang H.C, & Huang K.H. (2019). Suppression of Angiogenesis by Targeting Cyclin-Dependent Kinase 7 in Human Umbilical Vein Endothelial Cells and Renal Cell Carcinoma: An In Vitro and In Vivo Study. Cells, 8(11), 1469.
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3

Comprehensive Protein Analysis Protocol

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Antibodies and sources: CDK9, pCDK9, RNAPII, phospho-RNAPII, TGM2, Cyclin B1, Cyclin E1, and NUP98 (Cell Signaling Technology); CDC37, SPT5, Stomatin, PLK1, Cyclin A1 (Santa Cruz Biotechnology); BRD4, Cyclin T1 and GFP (Abcam); Caspase-8 (Enzo Life Sciences); β-Actin, pCDK9, Vimentin, and Flag (Sigma-Aldrich).
Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assay (Promega); BrdU kit (Roche); Thymidine, 5-ethynyl uridine (EU), Azide-fluor 488 (Sigma-Aldrich); AnnexinV and 7AAD (BD); [γ-32P] ATP (3000Ci/mmol, Amersham Pharmacia); Trail, FasL (Enzo Life Sciences); PLA assay kit (Olink Biosciences); BAY1251152, Cisplatin, and Carboplatin (Selleckchem); BioCoat Matrigel invasion chamber (Corning); Migration chamber (Ibidi); RNeasy Plus kit (Qiagen), active GST-CDK9/Cyclin K (SRP5012, Sigma-Aldrich).
The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene); p3xFlag-CMV-7.1 (E7533, Sigma); pGEX-5X-3 (28–9545-55, GE Healthcare) and pEGFP-C2 (6083-1, Clontech). All siRNAs and primers were from Sigma-Aldrich.
Mandal R., Raab M., Rödel F., Krämer A., Kostova I., Peña-Llopis S., Medici G., Häupl B., Oellerich T., Gasimli K., Sanhaji M., Becker S, & Strebhardt K. (2022). The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer. Cellular and Molecular Life Sciences, 79(12), 597.
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4

ChIP-seq Protocol for Chromatin Binding

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Chromatin immunoprecipitation (ChIP) was performed as previously described (Letting et al., 2004 (link)). Antibodies include: CTCF (Millipore; 07-729), Rad21 (Bethyl Laboratories; A302-583A), mCherry (Abcam; Ab167453), RNAPII (Cell Signaling; Cat#14958), IgG from rabbit serum (Sigma; 15006). Quantitative polymerase chain reaction (qPCR) was performed using Power SYBR Green kit (Invitrogen; 4368577) with signals detected by ViiA7 System (Life Technologies). ChIP-seq libraries were prepared using Illumina’s TruSeq ChIP sample preparation kit (Illumina, Cat#IP-202-1012) according to manufacturer’s specifications, with the addition of size selection (left side at 0.9x, right side at 0.6x) using SPRIselect beads (Beckman Coulter, Cat#B23318). Library size was determined (average 351 bp, range 333-372 bp) using the Agilent Bioanalyzer 2100, followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems; Cat#KK4835). Libraries were then pooled and sequenced (1x75bp) on the Illumina NextSeq 500 platform according to manufacturer’s instructions. Bclfastq2 v 2.15.04 (default parameters) was used to convert reads to fastq.
Luan J., Xiang G., Gómez-García P.A., Tome J.M., Zhang Z., Vermunt M.W., Zhang H., Huang A., Keller C.A., Giardine B.M., Zhang Y., Lan Y., Lis J.T., Lakadamyali M., Hardison R.C, & Blobel G.A. (2021). Distinct properties and functions of CTCF revealed by a rapidly inducible degron system. Cell reports, 34(8), 108783.
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5

ChIP-qPCR protocol for histone and transcription factor analysis

Cited 1 time
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ChIP was performed by standard protocols [9 (link), 32 (link)]. Antibodies were specific for H3K27ac (Millipore 07–360), CTCF (07–729), or RNAPII (Cell Signaling Technology 14958S). ChIP-qPCR was performed using SYBR Green Master Mix (Thermo Fischer) on the QuantStudio 6 Flex System with results calculated using the percent input method with 2.5% starting chromatin input. Primer sequences are shown in Suppl.Table S1. All primers were previously published by the Harris group except for the mutagenesis primers and CRISPR-HDR primers/guides.
Paranjapye A., NandyMazumdar M, & Harris A. (2022). Krüppel-Like Factor 5 regulates CFTR expression through repression by maintaining chromatin architecture coupled with direct enhancer activation. Journal of molecular biology, 434(10), 167561.
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6

Comprehensive Antibody Inventory for Cellular Analyses

Cited 1 time
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The various primary antibodies, unless otherwise mentioned, were purchased from Sigma-Aldrich (USA), Cell Signalling Technology (USA) or Merck (India). In our experiments, BRG1 antibody (Sigma-Aldrich; Catalog #B8184), RNAPII (Cell Signalling Technology; Catalog 2629S), H3K9Ac (Sigma-Aldrich; Catalog #H0913); H3K9(Me)2 (Sigma-Aldrich; Catalog #D5567), γH2AX (Sigma-Aldrich; Catalog #H5912) and β-actin (Sigma-Aldrich; Catalog #A1978) were used. Anti-GFP antibody was purchased from Life Technologies (Catalog #A11120). Monoclonal antibodies against bovine SMARCAL1 were a kind gift from Dr. Joel W. Hockensmith, University of Virginia. The TRITC- and FITC-conjugated anti-rabbit (Merck; Catalog #RTC2) and anti-mouse (Merck; Catalog #FTC3) antibodies and the HRP-conjugated anti-mouse IgG (Merck; Catalog #HPO5) and anti-rabbit IgG (Merck; Catalog #HPO3) antibodies were obtained from Merck (India). Monoclonal anti-mouse Sp1 antibody (Millipore; Catalog #07-645) was a kind gift from Prof. B.N. Mallick, School of Life Sciences, JNU. Polyclonal antibody against SMARCAL1 was generated as explained previously19 (link).
Haokip D.T., Goel I., Arya V., Sharma T., Kumari R., Priya R., Singh M, & Muthuswami R. (2016). Transcriptional Regulation of Atp-Dependent Chromatin Remodeling Factors: Smarcal1 and Brg1 Mutually Co-Regulate Each Other. Scientific Reports, 6, 20532.
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7

ChIP-seq Protocol for Chromatin Binding

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Chromatin immunoprecipitation (ChIP) was performed as previously described (Letting et al., 2004 (link)). Antibodies include: CTCF (Millipore; 07-729), Rad21 (Bethyl Laboratories; A302-583A), mCherry (Abcam; Ab167453), RNAPII (Cell Signaling; Cat#14958), IgG from rabbit serum (Sigma; 15006). Quantitative polymerase chain reaction (qPCR) was performed using Power SYBR Green kit (Invitrogen; 4368577) with signals detected by ViiA7 System (Life Technologies). ChIP-seq libraries were prepared using Illumina’s TruSeq ChIP sample preparation kit (Illumina, Cat#IP-202-1012) according to manufacturer’s specifications, with the addition of size selection (left side at 0.9x, right side at 0.6x) using SPRIselect beads (Beckman Coulter, Cat#B23318). Library size was determined (average 351 bp, range 333-372 bp) using the Agilent Bioanalyzer 2100, followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems; Cat#KK4835). Libraries were then pooled and sequenced (1x75bp) on the Illumina NextSeq 500 platform according to manufacturer’s instructions. Bclfastq2 v 2.15.04 (default parameters) was used to convert reads to fastq.
Luan J., Xiang G., Gómez-García P.A., Tome J.M., Zhang Z., Vermunt M.W., Zhang H., Huang A., Keller C.A., Giardine B.M., Zhang Y., Lan Y., Lis J.T., Lakadamyali M., Hardison R.C, & Blobel G.A. (2021). Distinct properties and functions of CTCF revealed by a rapidly inducible degron system. Cell reports, 34(8), 108783.
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