Anti-DMAP1 antibody - ChIP Grade (ab2848)

  Immunocytochemistry/ Immunofluorescence - DMAP1 antibody (ab2848)

ab2848 staining DMAP1 in asynchronous HeLa cells in conjunction with a secondary goat anti-rabbit antibody conjugated to Cy3 ® (green). Cells were counterstained with DAPI (red) to show the nucleus. Please refer to abreview for further details.

This image was kindly submitted by Kirk McManus, as part of an abreview.

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  Western blot - DMAP1 antibody - ChIP Grade (ab2848)

Predicted band size : 52.9 kDa


The in vivo activity of endogenous DNMT1 was measured in HeLa cells infected with shRNA expressing lentivirus 2 days post-infection. The cells were pulsed with 10 μm of aza-dC for 1 hour and rapidly lysed with 1% sarkosyl followed by CsCl step gradient purification of genomic DNA. DNA fractions (1.7g/cc) were pooled, and the DNA concentration was measured by absorbance at 260 nm. Either 0.5, 1, or 2 μg of genomic DNA were applied to nitrocellulose membrane using the slot blot manifold. To analyze the protein expression, nuclear proteins were extracted. The cells were harvested and washed twice with a hypotonic buffer (10 mm Hepes, pH 7.6, 1.5 mm MgCl2, 10 mm KCl, 1 mm DTT) followed by a 10-minute incubation with hypotonic buffer on ice. The cells were homogenized by 10 strokes with a tight fitting Dounce homogenizer, and the nuclei were collected with centrifugation at 13,000 rpm, 4 °C for 30 seconds. The nuclei were then suspended in a salt extraction buffer (20 mm Hepes, pH 7.6, 1.5 mm MgCl2, 10% glycerol, 0.2 mm EDTA, 1 mm DTT, 0.5 mm PMSF, 420 mm NaCl) and incubated for 30 minutes on ice. The nuclear fraction was collected by centrifugation at 13,000 rpm, 4 °C for 30 minutes, and protein concentration was measured with Bio-Rad protein assay reagent. Typically, 5–10 μg of nuclear protein were run on SDS-PAGE, and after electrotransfer onto nitrocellulose. The membrane was then probed with ab2848.

Image from Lee GE, et al, J Biol Chem. 2010 Nov 26;285(48):37630-40. Epub 2010 Sep 23, Fig 2.

  Western blot - Anti-DMAP1 antibody - ChIP Grade (ab2848)

Anti-DMAP1 antibody - ChIP Grade (ab2848) at 1/500 dilution + whole cell lysate prepared from Xenopus laevis egg at 10 µg

Secondary
HRP-Protein A at 1/1000 dilution
developed using the ECL technique

Predicted band size : 52.9 kDa


Exposure time : 1 minute

Image courtesy of an anonymous Abreview.

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