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

13 product images

• 

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

  Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab175187 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  Primers and probes are located in the first kb of the transcribed region.
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

• 

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

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution was used.
  
  Blocking and diluting buffer: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

  Predicted band size: 83 kDa

• 

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

  Lanes 1- 2: Merged signal (red and green). Green - ab175187 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (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 knockout cell line Human SUZ12 knockout HeLa cell line ab264983 (knockout cell lysate Human SUZ12 knockout HeLa cell lysate ab257721) lane below 100kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and SUZ12 knockout 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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.

  All lanes: Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: SUZ12 knockout HeLa cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 83 kDa

  Observed band size: 100 kDa

• 

  Western blot - 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 Anti-alpha Tubulin antibody [DM1A] - 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 Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  All lanes: Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187) at 1/1000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: SUZ12 knockout HAP1 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 83 kDa

  Observed band size: 90 kDa

• 

  ChIC/CUT&RUN sequencing - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

  CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ChIC/CUT&RUN pAG-MNase ab285373, 2.5 x 10^5 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.

• 

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

  Chromatin was prepared from F9 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab175187 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci).
  Primers and probes are located in the first kb of the transcribed region.

• 

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

  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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

  All lanes: Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

  Predicted band size: 83 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

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

• 

  Flow Cytometry (Intracellular) - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).
  

• 

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

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

  All lanes: Immunoprecipitation - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

  Predicted band size: 83 kDa

• 

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

  Lanes 1- 2: Merged signal (red and green). Green - ab175187 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (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 Human SUZ12 knockout HeLa cell line ab264983 (CRISPR/Cas9 edited cell lysate Human SUZ12 knockout HeLa 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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.

  All lanes: Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187) at 1/1000 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: SUZ12 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 83 kDa

  Observed band size: 100 kDa

• 

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

  All lanes: Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187) at 1/1000 dilution

  Lane 1: SW480 cell lysates at 10 µg

  Lane 2: HeLa cell lysates at 10 µg

  Lane 3: MCF-7 cell lysates at 10 µg

  Lane 4: 293T cell lysates at 10 µg

  Predicted band size: 83 kDa

• 

  ChIC/CUT&RUN sequencing - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)

  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 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 Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.