Anti-HDAC1 antibody - ChIP Grade (ab7028)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-Use 2 µg for 25 µg of chromatin. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use 2 µg for 25 µg of chromatin. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Publication protocol

ChIP was performed using the method described by Deliard et al (2013) with minor modifications. Briefly, 2 × 107 THP1 macrophages were cross‐linked with 1% formaldehyde for 10 min at room temperature and incubated with glycine for 5 min. Cells were collected by centrifugation, washed twice with PBS, lysed in lysis buffer, Dounce‐homogenized 10 times on ice, and incubated for 10 min. The lysates were centrifuged at 4,000 rpm for 5 min at 4°C, and the nuclear pellets were resuspended in SDS lysis buffer and sonicated for 15 cycles (30 s on, 30 s off). DNA concentrations were quantified, and 10 μg of chromatin DNA was used for each ChIP reaction. ChIP assays were performed with 4 μg of antibody and 40 μl of protein G magnetic beads. The samples were incubated overnight at 4°C, washed, and eluted. Bead‐bound DNA was reverse cross‐linked and purified. RT–qPCR analysis was performed to detect the presence of two MARCKS promoter region sequences or an ACTB2 sequence. ACTB2 primers were purchased from Active Motif (71005). The antibodies used in these experiments include α‐HDAC1 antibody (Abcam, ab7028), α‐APEX1 (Novus, NB100‐116), and α‐H3K27ac (Abcam, ab4726).

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Manufacturer protocol

Download the product protocol from Abcam for Anti-HDAC1 antibody - ChIP Grade (ab7028) below.

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