Chromatin-enriched fraction purification was performed according to the yChEFs procedure (32 ). Briefly, 150 ml of exponentially growing yeast cells (OD600 ∼ 0.7–0.8) were harvested, at least in triplicate. The whole final purified chromatin-enriched fraction P3 was resuspended in 20 μl of 1× Tris-Glycine SDS Sample Buffer and incubated for 5 min at 100°C, followed by spinning at 10 000 rpm for 30 s. This chromatin pellet was used for SDS-PAGE, followed by western blotting with different antibodies: anti-C-Myc (9E10 Santa Cruz Biotechnology), anti-Rpb3 (anti-POLR2C; 1Y26, Abcam), anti-Histone H3 (ab1791; Abcam) or anti-phosphoglycerate kinase, Pgk1 (459250; Invitrogen). Histone H3 was used as a control of purified chromatin and Pgk1 as a control of cytoplasmic contamination.
Anti polr2c 1y26
Manufactured by Abcam
Sourced in United States
Anti-POLR2C;1Y26 is a lab equipment product. It is a monoclonal antibody that specifically recognizes the POLR2C protein. POLR2C is a subunit of RNA polymerase II, which is responsible for the transcription of protein-coding genes in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Pol 2 rpb4 2y14, by BioLegend (2 mentions)
Pgk1 22c5d8, by Thermo Fisher Scientific (2 mentions)
Glass bead, by Merck Group (2 mentions)
Dynabeads sheep anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti histone h3 ab1791, by Abcam (1 mentions)
Cfx384 real time pcr instrument, by Bio-Rad (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Anti h3 ab1791, by Abcam (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ab1791, by Abcam (1 mentions)
Anti ha 12ca5, by Roche (1 mentions)
6 protocols using anti polr2c 1y26
1
Chromatin Enrichment and Western Blotting
2
Chromatin Immunoprecipitation and Real-Time PCR Analysis
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The chromatin immunoprecipitation experiments were performed using anti-Rpb3 (anti-POLR2C;1Y26, Abcam) or anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend, San Diego, CA, USA) as previously described [62 (link)]. For real-time PCR, a 1:100 dilution was employed for the input DNA and a 1:4 dilution for the immunoprecipitated samples DNA. Genes were analyzed by quantitative real-time PCR in a CFX-384 Real-Time PCR instrument (BioRad) in triplicate with at least three independent biological replicates using SYBR premix EX Taq (Takara). Quantification was performed as indicated in the Figure Legends. The oligonucleotides utilized for the different PCRs are listed in Supplemental Table S3 .
3
Protein Immunodetection Workflow
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Protein electrophoresis and Western blot were carried out as described in [62 (link)].
For the Western blot analyzes, anti-CTD [63 (link)], anti-Rpb3 (anti-POLR2C;1Y26, Abcam), anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend, San Diego, CA, USA), anti-Rpb5 (a polyclonal antibody generated against S. cerevisiae Rpb5 in our lab), anti-Rpb6 (a gift from M. Werner), anti-Rpb7 (Rpb7 (yN-19); Santa Cruz Biotechnology, Dallas, TX, USA), antiphosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen), anti-H3 (ab1791; Abcam), antihemaglutinin (anti-HA; 12CA5; Roche) and PAP (Sigma) antibodies were used.
Intensities of immunoreactive bands on Western blots were quantified by densitometry using the software IMAGE STUDIO LITE from images acquired with an office scanner.
For the Western blot analyzes, anti-CTD [63 (link)], anti-Rpb3 (anti-POLR2C;1Y26, Abcam), anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend, San Diego, CA, USA), anti-Rpb5 (a polyclonal antibody generated against S. cerevisiae Rpb5 in our lab), anti-Rpb6 (a gift from M. Werner), anti-Rpb7 (Rpb7 (yN-19); Santa Cruz Biotechnology, Dallas, TX, USA), antiphosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen), anti-H3 (ab1791; Abcam), antihemaglutinin (anti-HA; 12CA5; Roche) and PAP (Sigma) antibodies were used.
Intensities of immunoreactive bands on Western blots were quantified by densitometry using the software IMAGE STUDIO LITE from images acquired with an office scanner.
4
Whole-Cell Protein Extraction and Immunoprecipitation
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Whole-cell protein extracts and protein immunoprecipitation were performed as described [62 (link)]. Briefly for protein extract preparation, 150 mL of cells growing exponentially (OD600 0.6–0.8) were centrifuged, pelleted, and resuspended in 0.3 mL of lysis buffer (50 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)) supplemented with 1X protease inhibitor cocktail (Complete; Roche; Basel, Switzerland), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, and 1 mM sodium fluoride. Cell disruption was carried out by vortexing (3 cycles, 5 min each) at 4 °C using 0.2 mL of glass beads (425–600 µm; Sigma, Darmstadt, Germany). For the Rpb3 immunopurification, 1 µg of anti-Rpb3 antibody (anti-POLR2C;1Y26, Abcam, Cambridge, UK) was coupled to 40 µL of Dynabeads Sheep-anti-Mouse IgG (Invitrogen, Waltham, MA, USA) per sample, and 2 mg of a whole-cell protein extract were used for each immunoprecipitation. The affinity-purified proteins were released from the beads by boiling for 10 min and were analyzed by Western blotting with different antibodies.
5
Protein Expression Analysis Pipeline
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Protein electrophoresis and western blot were carried out as described in [63] .
For the western blot analyses, anti-CTD [58] , anti-Rpb3 (anti-POLR2C;1Y26, Abcam), anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend), anti-Rpb5 (a polyclonal antibody generated against S. cerevisiae Rpb5 in our lab), anti-Rpb6 (a gift from M. Werner), anti-Rpb7 (Rpb7 (yN-19); Santa Cruz Biotechnology), anti-phosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen), anti-H3 (ab1791; Abcam), anti-hemaglutinin (anti-HA; 12CA5; Roche) and PAP (Sigma) antibodies were used.
For the western blot analyses, anti-CTD [58] , anti-Rpb3 (anti-POLR2C;1Y26, Abcam), anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend), anti-Rpb5 (a polyclonal antibody generated against S. cerevisiae Rpb5 in our lab), anti-Rpb6 (a gift from M. Werner), anti-Rpb7 (Rpb7 (yN-19); Santa Cruz Biotechnology), anti-phosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen), anti-H3 (ab1791; Abcam), anti-hemaglutinin (anti-HA; 12CA5; Roche) and PAP (Sigma) antibodies were used.
6
Protein Extraction and Immunoprecipitation Protocol
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Protein whole cell extracts and protein immunoprecipitation were performed as described [63] . Briefly for protein extract preparation, 150 ml of cells growing exponentially (OD 600 0.6-0.8) were centrifuged, pelleted and resuspended in 0.3 ml of lysis buffer (50 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)) supplemented with 1X protease inhibitor cocktail (Complete; Roche), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, and 1 mM sodium fluoride. Cell disruption was carried out by vortexing (3 cycles, 5 min each) at 4ºC using 0.2 ml of glass beads (425-600 µm; Sigma). For the Rpb3 immunopurification, 1µg of anti-Rpb3 antibody (anti-POLR2C;1Y26, Abcam) was coupled to 50 µl of Dynabeads Sheep-anti-Mouse IgG (Invitrogen) per sample, and 2 mg of a whole cell protein extract were used for each immunoprecipitation. A similar procedure was followed for TAP pull down, with 50 µl of Dynabeads-Pan Mouse (Invitrogen). The affinity-purified proteins were released from the beads by boiling for 10 min and were analysed by western blotting with different antibodies.
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