Recombinant Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

Lanes 1 - 2: Merged signal (red and green). Green - ab175187 observed at 90 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab175187 was shown to react with SUZ12 in wild-type HAP1 cells in Western blot with loss of signal observed in SUZ12 knockout sample. Wild-type HAP1 and SUZ12 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab175187 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

ab175187 (purified) at 1/20 dilution (16 µg/mL) immunoprecipitating SUZ12 in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg.
Lane 1 (input): HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg
Lane 2 (+): ab175187 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab175187 in HeLa whole cell lysate
For western blotting, ab175187 at 1/500 dilution (0.636 µg/mL) and veriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution was used.

Blocking and diluting buffer: 5% NFDM /TBST.

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 10⁵ HeLa cells, and 5µg of ab175187 [EPR5234(N)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.

Additional screenshots of mapped reads can be downloaded here.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: SUZ12 knockout HAP1 cell lysate (20 µg)
Lane 3: Caco2 cell lysate (20 µg)
Lane 4: MCF7 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab175187 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab175187 was shown to specifically react with SUZ12 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when SUZ12 knockout samples were used. Wild-type and SUZ12 knockout samples were subjected to SDS-PAGE. ab175187 and ab8245 (loading control to GAPDH) were both 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1hr at room temperature before imaging.

Immunofluorescence analysis of MCF-7 cells labeling SUZ12 with ab175187 at a 1/50 dilution.

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SUZ12 (red) with purified ab175187 at a 1/2000 dilution (1ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue). 

Lanes 1- 2: Merged signal (red and green). Green - ab175187 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab175187 was shown to react with SUZ12 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264983 (CRISPR/Cas9 edited cell lysate ab257721) lane below 100kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and SUZ12 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab175187 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 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.

Western blot analysis on immunoprecipitation pellet from HeLa cell lysate using ab175187 at a 1/10 dilution.