Cells were lysed in lysed in 1% NP-40 lysis buffer (50 mM HEPES, 150 mM NaCl, 1% NP-40, 1 mM EDTA with protease inhibitor mixture and 1 mM PMSF). Lysates were incubated on ice for 30 minutes before clearing by centrifuging at 16,000 × g at 4°C for 20 min. Protein lysates were pre-cleared with Protein A/G plus agarose beads (SCBT, sc-2003) at 4°C for 1 hour. Then, 8% of cleared lysates were taken as input, the rest were incubated with either 5 µg FLAG M2 antibody (Millipore Sigma #F1804) or Mouse IgG control (BioLegend, 400101) on a rotor at 4°C for 2 hours. Then, Protein A/G plus agarose beads (SCBT, sc-2003) beads were added to each sample, incubated on a rotor at 4°C for 2 hours. Then, beads were washed in 1% NP-40 lysis buffer 3 times, 10 minutes each time at 4°C on a rotor. The immunoprecipitated proteins were then eluted in 1x Sample Buffer 2 (Invitrogen, 1981103) by boiling at 95°C for 10 minutes. And then, the samples were analyzed by immunoblot.
Protein a g plus agarose beads
Manufactured by SCBT
Protein A/G PLUS – agarose beads are a protein-based affinity resin designed for the purification of antibodies and immunoglobulins. The resin consists of recombinant Protein A and Protein G immobilized on cross-linked agarose beads, providing a versatile platform for the capture and separation of a wide range of immunoglobulin subtypes from various sample sources.
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3 protocols using protein a g plus agarose beads
1
Immunoprecipitation of FLAG-tagged Proteins
2
ChIP Assay Protocol for Protein-DNA Interactions
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ChIP assay was performed as described previously 8 (link). 2.5 X 106 (link) cells were taken for ChIP assay. The whole cell extract was pre–cleared using 60 μl of Protein A/G PLUS – agarose beads (SCBT; sc–2003) and incubated overnight with anti–P53, anti–H3K4me3, anti–Wdr5, anti–RbBP5, anti–Ash2L or control IgG antibodies. 1 μg of antibody was used for 100 μg of total lysate. Protein G agarose beads pre–blocked with salmon sperm DNA (to reduce non–specific DNA binding) was purchased from Millipore, Saint Charles, MO, USA (#16–2001) and 60 μl of this solution was used for each immunoprecipitation. 10 % of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (input). Both Immunoprecipitated and whole cell extract (input) were treated with RNaseA, proteinase K and DNA were purified using DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA; #69504). Quantitative real–time PCR was performed on this DNA to identify the amount of target sequence. The list of primers used is given in supplementary table .
3
ChIP Assay Protocol for Protein-DNA Interactions
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