Preparation of genomic DNA and Southern blotting was performed as previously described (Dvorin et al., 2010 (link)). Telomere Restriction Fragment Southern blots were performed as previously described (Merrick et al., 2010 (link)). Western blot lysates were probed with the following primary antibodies: αHA (1:1000) (clone 3F10, Roche Applied Science), αH3 (1:5000) (Upstate), αPfLDH (1:2000) (gift of Michael T. Makler), αRhopH3 (1:500) (gift of Jean-Claude Doury), αAcetyl-Lysine (1:1000) (#9441, Cell Signaling), αH3Ac (1:2000) (06-599, Millipore), αH3K9Ac (1:2000) (07-442, Millipore), αH3K14Ac (1:1000) (06-911, Millipore), αH3K9Me3 (1:2000) (07-442, Millipore). A previously described P. falciparum nuclear and cytoplasmic fractionation protocol (Voss et al., 2002 (link)) was adapted to assay Hda2-HA nuclear localization.
αh3ac
Manufactured by Merck Group
The αH3Ac is a laboratory equipment used for the detection and analysis of acetylated histone H3 proteins. It is a specific antibody that binds to the acetylated lysine residues on histone H3, allowing researchers to study epigenetic modifications and their impact on gene expression and cellular processes.
Automatically generated - may contain errors
3 protocols using αh3ac
1
Genomic DNA Extraction and Epigenetic Analysis
2
Western Blot Analysis of Toxoplasma Proteins
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Tachyzoites were collected, filtered, counted and lysed by 6 cycles of rapid freezing/defreeze in hypotonic buffer, and boiled with LB for 5 minutes. 0.5 to 1×107 parasites were loaded per well and resolved by 15% SDS-PAGE. Proteins were transferred to PVDF membrane for 1h at 100V. Western blot was then performed as described [90 (link)]. The primary antibodies: αrH2B.Z [22 (link)], αrH2A.Z [23 (link)], αrROP5 (rabbit) and αrROP18 (rabbit) were used at 1/5000, whereas α-c-Myc (rabbit, abCam ab9106) was used at 1/2000 and αrSag1 [87 (link)] 1/200 for 1 h at room temperature. αH3ac (rabbit, Millipore, 06–599B) 1/200, was incubated overnight. Appropriate secondary antibodies were used: phosphatase alkaline-conjugated goat anti-mouse or anti-rabbit (Sigma) along with the NBT and BCIP (Promega) detection system.
3
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were performed as previously described (12 (link)). The antibody used for ChIP was α-H3Ac (Millipore 06-599). The amount of immunoprecipitated chromatin was measured by qPCR with primers listed in Supplementary Table 2 . The 2−DDCT method (19 (link)) was used to determine the relative amounts of amplified products in samples. The value of each fragment was normalized to the respective input DNA (DNA isolated from chromatin that was cross-linked and fragmented under the same conditions as the immunoprecipitated DNA) and to Ubiquitin 10 (UBQ10) to obtain H3Ac enrichment.
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