Rna polymerase 2

Manufactured by Active Motif

RNA polymerase II is an enzyme responsible for the transcription of protein-coding genes in eukaryotic cells. It catalyzes the synthesis of messenger RNA (mRNA) from a DNA template, which is then used as the blueprint for the production of proteins.

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6 protocols using rna polymerase 2

Comprehensive Immunofluorescence Profiling of Spermatocytes

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Spreads of spermatocytes and immunofluorescence staining were prepared according to the previous references^{38 (link),39 (link)}. Briefly, seminiferous tubules were incubated in hypotonic extraction buffer (50 mM Sucrose, 17 mM Sodium citrate, 30 mM Tris (pH 8.2), 2.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (pH 8.3), and 5 mM EDTA) on ice for 20 min, minced in 100 mM sucrose, spread on slides, and fixed in 1% PFA with 0.1% Triton X-100. Slides were incubated in a humid chamber overnight, dried, and washed in phosphate-buffered saline (PBS) and water containing Photoflo (Kodak, NY, USA). Following blocking in 10% donkey serum and 3% bovine serum albumin, immunofluorescence staining was performed by incubating with the primary antibodies: γH2AX (1:1000; Novus, Cat# NB100–384), SYCP3 (1:100; Abcam, Cat# ab97672 or Novus, Cat# NB300–232), DMC1 (1:100, Santa Cruz, Cat# sc-22768), MLH1 (1:100, BD, Cat# 551092), SYCP1 (1:100, NOVAS, Cat# NB300–229), and RNA-Polymerase II (1:200, Active Motif, Cat# 39097), overnight at room temperature. Alexa 488 (1:400, Thermo Fisher) or Alexa 555 (1:200, Thermo Fisher) fluorescent secondary antibody was used. Slides were incubated with secondary antibodies at 37 °C for 1 h in dark, washed, and mounted with Vecta shield cover slips (Vector Laboratories, Cat# H-1000).

Pan H., Jiang N., Sun S., Jiang H., Xu J., Jiang X., Gao Q., Li L., Wu H., Zheng H., Qi Q., Li T., Zhang M., Zhang L., Wan X., Lin X., Wong J., Shi Q, & Li R. (2020). UHRF1-repressed 5’-hydroxymethylcytosine is essential for the male meiotic prophase I. Cell Death & Disease, 11(2), 142.

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Chromatin Immunoprecipitation (ChIP) Assay

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Chromatin immunoprecipitation (ChIP) assays were performed as described previously.38 (link), 39 (link) Antibodies for immunoprecipitation were from the following sources: V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A (RelA) (cat. 06-418; Millipore, Temecula, CA), histone H4 acetyl lysine 12 (H4K12ac, cat. 39165), histone deacetylase 3 (HDAC3; cat. 40968), and RNA polymerase II (cat. 39097; Active Motif, Carlsbad, CA). Precipitated DNA, SYBR Green Master Mix (Applied Biosystems, Foster City, CA), and primers specific to the human IL1B or rat IL1β gene promoter (Table 1) were used for real-time quantitative polymerase chain reaction (PCR). Fold differences in precipitated DNA were normalized against input.Table 1

Primers Specific to the Human IL1B and Rat II1b Promoters Used in ChIP–Quantitative PCR Assays

Species	Primer name	Primer sequence
Human
	IL1B-NF-κB-F	5’-TGGCCCTTCATTGTACCCAT-3’
	IL1B-NF-κB-R	5’-TCGTTGTGCAGTTGATGTCC-3’
	IL1B-core-F	5’-CTCAGTTTATTAGTCCCCTCCCC-3’
	IL1B-core-R	5’-CTCCCTCGCTGTTTTTATGGC-3’
Rat
	II1b-NF-κB-F	5’-GCTCCCTCAGCTTAAGTCCA-3’
	II1b-NF-κB-R	5’-CATTATTTCCCCCTGGACAA-3’
	II1b-core-F	5’-ATTCCCACCAAGCTTCTTCC-3’
	II1b-core-R	5’-TGGAGAGGATCCCAGATGAG-3’

F, forward; R, reverse.

Zhong X.S., Winston J.H., Luo X., Kline K.T., Nayeem S.Z., Cong Y., Savidge T.C., Dashwood R.H., Powell D.W, & Li Q. (2018). Neonatal Colonic Inflammation Epigenetically Aggravates Epithelial Inflammatory Responses to Injury in Adult Life. Cellular and Molecular Gastroenterology and Hepatology, 6(1), 65-78.

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ChIP-qPCR Analysis of Histone Modifications

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Sheared chromatin for ChIP analysis was generated using the ChIP-IT Express Kit (Active Motif, Carlsbad, CA, USA). Fifteen micrograms of chromatin were pre-cleared with Protein G magnetic beads (Cell Signaling) and isotype control antibodies, mouse IgG (clone G3A1, Cell Signaling) or rabbit IgG (clone DA1E, Cell Signaling), for 1 h at 4˚C. Immunoprecipitation with 5 μg of specified antibodies against NME1 (SC-NM301, Santa Cruz Biotechnologies), Histone 3 lysine 27 acetylation (H3K27ac, clone D5E4, Cell Signaling), Histone 3 lysine 4 trimethylation (H3K4, Cell Signaling), and RNA Polymerase II (Active Motif) was performed overnight at 4˚C on an end-to-end rotator. Eluted DNA was purified using the QiaQuick PCR Purification Kit (Qiagen) prior to qPCR analysis, using specified primer sequences found in Table I. Quantification was achieved by normalization to input DNA, and expressed as % input.

PAMIDIMUKKALA N.V., LEONARD M.K., SNYDER D., MCCORKLE J.R, & KAETZEL D.M. (2018). Metastasis Suppressor NME1 Directly Activates Transcription of the ALDOC Gene in Melanoma Cells. Anticancer research, 38(11), 6059-6068.

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MALAT1 Promoter ChIP Assay with AHR and RNAPII

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A chromatin immunoprecipitation (ChIP) assay was carried out with AHR (Enzo Life Sciences, Inc., Cat#: ALX-804-423-R100) and RNA polymerase II (Active Motif, Carlsbad, CA, Cat# 39097) antibodies using Panc-1 cell ChIP lysate treated with TCDD for 2 hour with the ChIP-IT Express Chromatin Immunoprecipitation Kit (Active Motif), according to the manufacturer’s protocol. The ChIP primer set that covers MALAT1 gene promoter proximal region (−442~−201): (forward): 5’-AGGAGAGAGGTGGGAAAGGAAG-3’ and (reverse) 5’-TGGTTCTAACCGGCTCTAGC-3’. All the ChIP-PCR reactions were carried out using a 7300HT Real-Time PCR system or a QuantStudio 3 Real Time PCR system with a 96-well block module (Applied Biosystems). The cycling conditions were 56°C for 30 min and 95°C for 10 min, followed by 48 cycles of 95°C for 25 s and 60°C for 60 s.

Lee J.E., Cho S.G., Ko S.G., Ahrmad S.A., Puga A, & Kim K. (2020). Regulation of a long noncoding RNA MALAT1 by Aryl Hydrocarbon Receptor in pancreatic cancer cells and tissues.. Biochemical and biophysical research communications, 532(4), 563-569.

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ChIP Assay of Mir155 Promoter

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ChIP assays were performed as described previously using ChIP-IT Express kit (Active Motif, Carlsbad, CA) (42 (link)). Antibodies for the immunoprecipitation are as follows: Histone H3 acetyl Lysine 9 (H3K9ac, Cat. # 39137), histone H4 acetyl Lysine 12 (H4K12ac, Cat. # 39165), and RNA polymerase II (Cat. # 39097, Active Motif, Carlsbad, CA). Precipitated DNA, PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA), and primers specific to the rat Mir155 gene promoter (forward: 5’-ggc ttg ctg aag gct gta tg-3’, reverse: 5’-aca ggt agg agt cag tca gag g-3’) were used for real-time polymerase chain reaction (qPCR). Fold differences in precipitated DNA were normalized against input.

Kline K.T., Lian H., Zhong X.S., Luo X., Winston J.H., Cong Y., Savidge T.C., Dashwood R.H., Powell D.W, & Li Q. (2019). Neonatal injury increases gut permeability by epigenetically suppressing E-cadherin in adulthood. Journal of immunology (Baltimore, Md. : 1950), 204(4), 980-989.

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Comprehensive Antibody Validation for Western Blot, Immunofluorescence, and ChIP-seq

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The antibodies used for western blot, immunofluorescence staining and ChIP-seq are obtained from different companies. Antibodies from ThermoFisher: GAP43 (Cat. # PA1-16729, 1:250 for immunofluorescence), goat anti-mouse IgG Alexa Fluor 594 (Cat. # A-11032, 1:250 for immunofluorescence staining). Antibodies from SigmaAldrich: FLAG (Cat. # F1804, 1:1000 for western blot) Antibodies from Santa Cruz: GAPDH (Cat. # sc-25778, 1:2000 for western blot), goat anti-mouse IgG HRP secondary antibody (Cat. # sc-2005, 1:2000), goat anti-rabbit IgG HRP secondary antibody (Cat. # sc-2004, 1:2000). Antibody from Abcam: H3K27ac (Cat. # ab4729, 4 µg/reaction for ChIP), HAND2 (Cat. # ab200040, 4 µg/reaction for ChIP). Antibody from Active Motif: RNA polymerase II (Cat. # 39097, 4 µg/reaction for ChIP). Antibody generated by collaborating with Rockland Immunochemicals Inc: CASZ1 (1:4000 for western blot, 4 µg/reaction for ChIP). Antibody from EMD Millipore: H3K27me3 (Cat. # 07-449, 4 µg/reaction for ChIP). Antibody from Cell Signaling Technology, H3K4me3 (Cat. # 9751, 4 µg/reaction for ChIP).

Liu Z., Zhang X., Xu M., Lei H., Shern J.F, & Thiele C.J. (2022). Loss of CASZ1 tumor suppressor linked to oncogenic subversion of neuroblastoma core regulatory circuitry. Cell Death & Disease, 13(10), 871.

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