Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free

• ChIP

Unknown

ChIP - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

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
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175187).

• IP

Lab

Immunoprecipitation - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175187).

All lanes:

Immunoprecipitation - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (<a href='/en-us/products/primary-antibodies/suz12-antibody-epr5234n-chip-grade-ab175187'>ab175187</a>)

Predicted band size: 83 kDa

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• ChIP

Unknown

ChIP - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175187).

• WB

Lab

Western blot - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

This data was developed using the same antibody clone in a different buffer formulation (ab175187).

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.

All lanes:

Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (<a href='/en-us/products/primary-antibodies/suz12-antibody-epr5234n-chip-grade-ab175187'>ab175187</a>) 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

Lane 2:

Western blot - Human SUZ12 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-suz12-knockout-hela-cell-line-ab264983'>ab264983</a>)

Predicted band size: 83 kDa

Observed band size: 100 kDa

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• WB

Lab

Western blot - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

This data was developed using the same antibody clone in a different buffer formulation (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.

All lanes:

Western blot - Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (<a href='/en-us/products/primary-antibodies/suz12-antibody-epr5234n-chip-grade-ab175187'>ab175187</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

SUZ12 knockout HAP1 cell lysate at 20 µg

Predicted band size: 83 kDa

Observed band size: 90 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

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 ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods. This data was developed using the same antibody clone in a different buffer formulation (ab175187).

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

This data was developed using ab175187, the same antibody clone in a different buffer formulation.

CUT&RUN profiling with SUZ12 antibody demonstrates robust genome-wide enrichment in wild-type (WT) cells, which is markedly diminished in SUZ12 knockout (KO) cells. Heatmaps of genome-wide signal flanking annotated gene bodies (transcription start sites [TSS - 2kb] to transcription termination sites [TTS + 2 kb]) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with SUZ12 antibody (Abcam ab175187, 0.5 µg). 500,000 HeLa WT (Abcam ab255448) or KO (Abcam ab264983) cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. H3K4me3 and H3K27me3 antibodies were included as positive controls, showing enrichment at regions of active transcription or repressive chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramírez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to SUZ12 WT, with red indicating high localized enrichment and blue denoting background. Top panels show mean reads per kilobase per million mapped reads (RPKM) for each target. Validated antibodies show genome-wide enrichment above IgG background consistent with SUZ12 binding in WT cells and near complete loss of signal in KO cells.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (AB249844)

This data was developed using ab175187, the same antibody clone in a different buffer formulation.

CUT&RUN profiling with SUZ12 antibody reveals the expected genomic enrichment pattern in wild-type (WT) cells, which is substantially reduced in SUZ12 knockout (KO) cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with SUZ12 antibody (Abcam ab175187, 0.5 µg). 500,000 HeLa WT (Abcam ab255448) or KO (Abcam ab264983) cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).