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

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5µg of ab175187 [EPR5234(N)]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

Additional screenshots of mapped reads can be downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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.

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.