重组Anti-Vitamin D Receptor抗体[EPR4552] - ChIP Grade (ab109234)

Western blot: Anti-VDR antibody [EPR4552] (ab109234) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab109234 was shown to bind specifically to VDR. A band was observed at 48 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in VDR knockout cell line. To generate this image, wild-type and VDR knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lanes 1-4: Merged signal (red and green). Green - ab109234 observed at 50 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab109234 Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade was shown to specifically react with Vitamin D Receptor in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265430 (knockout cell lysate ab257796) was used. Wild-type and Vitamin D Receptor knockout samples were subjected to SDS-PAGE. ab109234 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Chromatin was prepared from T-47D cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 5 µg of ab109234 (red), and 20 µL of protein A/G sepharose beads slurry (10 µL of sepharose A beads + 10 µL of sepharose G beads). 5 μg of rabbit normal IgG was added to the beads as a control sample (grey). The immunoprecipitated DNA was quantified by real time PCR (SYBR Green chemistry) with primers to c-Fos.

Blocking and diluting buffer: 5% NFDM/TBST.

Lane 1 (input): T-47D (Human ductal breast epithelial tumor epithelial cell) whole cell lysate, 10 μg
Lane 2(+): T-47D whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109234 in T-47D whole cell lysate

Ab109234 immunoprecipitating Vitamin D receptor in T-47D whole cell lysates. Capture antibody was used at a 1/30 dilution (2 μg in 0.35 mg lysates). For western blotting, primary antibody was used as ab109234 at 1/1000 dilution (0.62 μg/mL). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Blocking and diluting buffer: 5% NFDM/TBST.