Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade

• ChIP-seq

Lab

ChIP-sequencing - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 8 μg of ab133482 [EPR4019(2)]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here.

• WB

Lab

Western blot - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

Lanes 1 - 4 : Merged signal (red and green). Green - ab133482 observed at 170 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133482 was shown to recognize EHMT2/G9A when EHMT2/G9A knockout samples were used, along with additional cross-reactive bands. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab133482 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (ab133482) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

EHMT2/G9A knockout HAP1 cell lysate at 20 µg

Lane 3:

HEK293 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Predicted band size: 132 kDa

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• WB

Lab

Western blot - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

Lanes 1-4 : Merged signal (red and green). Green - ab133482 observed at 160 kDa. Red - loading control ab8245 observed at 37 kDa.

ab133482 Anti-EHMT2/G9A antibody [EPR4019(2)] was shown to specifically react with EHMT2/G9A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265149 (knockout cell lysate ab257080) was used. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab133482 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.

All lanes:

Western blot - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (ab133482) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

EHMT2/G9A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human EHMT2 (G9A) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ehmt2-g9a-knockout-hela-cell-line-ab265149'>ab265149</a>)

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 132 kDa

Observed band size: 160 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

CUT&RUN profiling with EHMT2 antibody reveals the expected genomic enrichment pattern in cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with EHMT2 antibody (Abcam ab133482, 0.5 µg). 500,000 HeLa 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).

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

CUT&RUN profiling with EHMT2 antibody demonstrates robust genome-wide enrichment in cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with EHMT2 antibody (Abcam ab133482, 0.5 µg). 500,000 HeLa cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. 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 ChAsE (Younesy et al., Bioinformatics 2016; PMID 27378294). Row-linked data are ranked by intensity relative to EHMT2, with red indicating high localized enrichment and blue denoting background.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

Immunohistochemical analysis of paraffin-embedded gastric adenocarcinoma tissue labelled with ab133482 at 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

Immunohistochemical analysis of paraffin-embedded lung squamous carcinoma tissue labelled with ab133482 at 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Unknown

Western blot - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (AB133482)

All lanes:

Western blot - Anti-EHMT2/G9A antibody [EPR4019(2)] - ChIP Grade (ab133482) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

NCCIT cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat-anti-rabbit at 1/2000 dilution

Predicted band size: 132 kDa

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