SK-N-BE2 cells were fixed with 1% paraformaldehyde at room temperature for 10 min, then quenched with 0.125 M glycine for 5 min and lysed in SDS lysis buffer. Cell lysate was subjected to a Bioruptor Pico Sonifier to shear chromatin DNA to a size range of 500–1000 bp. Precleared chromatin was immunoprecipitated with antibody against MYCN or mouse IgG for 16 h at 4 °C. Antibody–chromatin complexes were pulled down with protein G agarose/salmon sperm DNA beads (Roche) (1 h, 4 °C). The eluted DNA was purified and quantified by qPCR using specific primers listed in Supplementary Table 2 .
Protein g agarose salmon sperm dna beads
Manufactured by Roche
Protein G agarose/salmon sperm DNA beads are an affinity chromatography resin used for the purification of antibodies from biological samples. The beads are composed of agarose matrix crosslinked with protein G, a bacterial protein that binds to the Fc region of immunoglobulins. The salmon sperm DNA component is used to reduce non-specific binding. This resin can be used in batch or column chromatography procedures to isolate and concentrate antibodies from complex mixtures.
Automatically generated - may contain errors
4 protocols using protein g agarose salmon sperm dna beads
1
ChIP-qPCR analysis of MYCN in SK-N-BE2 cells
2
ChIP-qPCR Profiling of Notch1 in T-ALL
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ChIP was performed using human T-ALL SIL-ALL cells10 (link),49 (link). These cells were treated with dimethyl sulfoxide (DMSO) or Compound E (1 μM, Merck) for 24 h, then fixed with 1% paraformaldehyde at room temperature for 10 min. Cells were subjected to a Bioruptor Pico Sonifier to shear chromatin DNA to a size range of 500–1000 bp. Precleared chromatin was immunoprecipitated with antiserum against intracellular NOTCH1 or rabbit IgG (sc-3888, Santa Cruz Biotechnology) for 16 h at 4 °C. Antibody–chromatin complexes were pulled down with protein G agarose/salmon sperm DNA beads (Roche) (1 h, 4 °C). The eluted material was reverse-cross-linked and treated with proteinase K (40 μg ml−1). Immunoprecipitated DNA was purified by phenol/chloroform extraction, eluted by distilled H2O, and quantified by CFX Connect Real-Time PCR System (Bio-Rad) using specific primers listed in Supplementary Table 1 .
3
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were performed using two-week-old plants grown on MS-agar plates, as described previously [52 (link)]. Whole plants were vacuum-infiltrated with 1% (v/v) formaldehyde for cross-linking and ground in liquid nitrogen after quenching the cross-linking process. Chromatin preparations were sonicated into 0.4- to 0.7-kb fragments and precleared with salmon sperm DNA/Protein G agarose beads (Roche, Indianapolis, IN), and an anti-MYC antibody (Millipore, Billerica, MA) was added to the mixture. The precipitates were eluted from the beads, and cross-links were reversed. Residual proteins were removed by incubation with proteinase K. DNA was then recovered using a SV minicolumn (Promega). Quantitative PCR was performed to determine the amounts of genomic DNA enriched in the chromatin preparations.
4
ChIP Assay for Transcription Factor Binding
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ChIP assays were performed, essentially as described previously [59 (link)], in biological triplicates using three independent plant materials grown under identical conditions. Seven-day-old 35S:MYC-CCA1 and 35S:LHY-MYC transgenic plants grown on MS-agar plates were vacuum-infiltrated with 1 % (v/v) formaldehyde for cross-linking and ground in liquid nitrogen after quenching the cross-linking process. Chromatin preparations were sonicated into 0.5- to 1-kb fragments. An anti-MYC antibody (Millipore, Billerica, MA) was added to the chromatin solution, which was precleared with salmon sperm DNA/protein G agarose beads (Roche, Indianapolis, IN). The precipitates were eluted from the beads. Cross-links were reversed, and residual proteins were removed by incubation with proteinase K. DNA was recovered using the QIAquick PCR purification kit (Qiagen, Valencia, CA). Quantitative PCR was performed to determine the amounts of genomic DNA enriched in the chromatin preparations. The primers used are listed in Additional file 10 .
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