Cell pellets were collected for HCT-8 cells infected with C. parvum and transfected with the Full-Cdg2_FLc_0220 after 24 h, washed with PBS, cross-linked with 1% formaldehyde at 37°C for 10 min and quenched by 0.25 M glycine at room temperature for 5 min. Cell protein isolation and genomic DNA fragmentation were performed by using SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8.1) and sonication. 500 μg protein for each sample was used for ChIP analysis with Salmon Sperm DNA/Protein A Agarose (Millipore) and 2 μg normal mouse or rabbit IgG (Santa Cruz Biotechnology), rabbit polyclonal to Histone H3 (tri methyl K9) (Abcam), mouse monoclonal to Histone H3 (tri methyl K27) (Abcam), mouse polyclonal antibody to PRDM1 (Santa Cruz Biotechnology), and rabbit polyclonal antibody to G9α (Millipore) at 4°C overnight. Formaldehyde cross-linking complex was reversed at 65°C overnight. The methylation level for each selective DNA site was quantified by using Realtime PCR with primers listed in Table S1 , and the value was normalized to the input (1% of the starting chromatin).
Rabbit polyclonal to histone h3 tri methyl k9
Manufactured by Abcam
Rabbit polyclonal to Histone H3 (tri methyl K9) is a laboratory reagent used for the detection and quantification of histone H3 proteins that are tri-methylated at lysine 9 (H3K9me3). This antibody is raised in rabbits and recognizes the H3K9me3 modification, which is associated with transcriptional repression and heterochromatin formation.
Automatically generated - may contain errors
2 protocols using rabbit polyclonal to histone h3 tri methyl k9
1
Chromatin Immunoprecipitation of C. parvum Infected Cells
2
Visualizing Histone Modifications in Hermaphrodite Germlines
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One-day-old adult hermaphrodites were dissected in M9 with 0.05% Tetramizol to release gonads onto poly-L-lysine coated slides. Germlines were cracked by freeze/thawing in liquid nitrogen, then fixed in À20 C methanol for 1 min followed by 3.7% paraformaldehyde, 1xPBS, 0.08 M HEPES (pH 6.9), 1.6 mM MgSO 4 , 0.8 mM EGTA for 30 min. Primary antibodies used were rabbit polyclonal to Histone H3 (tri methyl K9) (Abcam) and mouse monoclonal to Histone H3 (di methyl K9) (Abcam), diluted 1:300 in 30% normal goat serum in PBS. Secondary antibodies used were goat anti-rabbit Alexa Fluor 555 and rabbit anti-mouse Alexa Fluor 555 (Invitrogen), diluted 1:1000 in 30% normal goat serum in PBS. DNA was visualized with DAPI. Fixing and staining was performed in parallel for all strains within a replicate. Two replicates were prepared independently. Comparisons between strains were made only within a replicate. Results from one replicate are presented and are representative of the second replicate.
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