Di-methylated H3K79 ChIP assays were performed as described previously (13 (link)). For ChIP with anti-Flag and anti-CRM1 antibodies, formaldehyde-fixed cells were lysed with a mild lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mm EDTA, 1% Triton-X) and sonicated to an average fragment size of 1.5 kb. Immunoprecipitation was performed with anti-FLAG M2 Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) or anti-CRM1 (Santa Cruz, Dallas, TX or Bethyl, Montgomery, TX) antibodies incubated at 4oC for 3–5 hr or overnight, respectively. Salmon sperm-conjugated protein G sepharose beads (35 µL, Millipore, Billerica, MA, USA) were added to anti-CRM1 ChIPs and rocked at 4°C for an additional 3 hr. Following RNAse A and proteinase K treatment, input and ChIP DNA were purified with a PCR purification kit (Qiagen) and amplified by real time RT-PCR. Amplification values were normalized to input. Primer sequences used to amplify genomic DNA are listed in Supplementary Table 2 .
Salmon sperm conjugated protein g sepharose beads
Manufactured by Merck Group
Sourced in United States
Salmon sperm-conjugated protein G sepharose beads are a type of affinity chromatography resin. They are composed of sepharose beads conjugated with protein G, which is a bacterial protein that binds to the Fc region of antibodies. The salmon sperm DNA is used as a blocking agent to reduce non-specific binding. This product is commonly used for the purification of antibodies from complex samples.
Automatically generated - may contain errors
2 protocols using salmon sperm conjugated protein g sepharose beads
1
ChIP Assay for H3K79 Dimethylation
2
ChIP Assay for H3K79 Dimethylation
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Di-methylated H3K79 ChIP assays were performed as described previously (13 (link)). For ChIP with anti-Flag and anti-CRM1 antibodies, formaldehyde-fixed cells were lysed with a mild lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mm EDTA, 1% Triton-X) and sonicated to an average fragment size of 1.5 kb. Immunoprecipitation was performed with anti-FLAG M2 Affinity Gel (Sigma-Aldrich, St. Louis, MO, USA) or anti-CRM1 (Santa Cruz, Dallas, TX or Bethyl, Montgomery, TX) antibodies incubated at 4oC for 3–5 hr or overnight, respectively. Salmon sperm-conjugated protein G sepharose beads (35 µL, Millipore, Billerica, MA, USA) were added to anti-CRM1 ChIPs and rocked at 4°C for an additional 3 hr. Following RNAse A and proteinase K treatment, input and ChIP DNA were purified with a PCR purification kit (Qiagen) and amplified by real time RT-PCR. Amplification values were normalized to input. Primer sequences used to amplify genomic DNA are listed in Supplementary Table 2 .
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