Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling EHMT2/G9A with ab185050 at 1/150 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on tumor cells of the gastric adenocarcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on epithelial cells of the normal Human colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling EHMT2/G9A with ab185050 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing no staining on MCF7 cell line, as MCF7 cells have a very low level expression of EHMT2/G9A.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab185050 at 1/70 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling EHMT2/G9A with ab185050 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab185050 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

• IP

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Immunoprecipitation - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

EHMT2/G9A was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab185050 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab185050 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10ug (Input).

Lane 2 : ab185050 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab185050 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 30 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

All lanes:

Immunoprecipitation - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-antibody-epr18894-chip-grade-ab185050'>ab185050</a>)

Predicted band size: 132 kDa

Observed band size: 160 kDa,170 kDa

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on hepatocytes of the mouse liver is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on rat kidney is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

This data was developed using the same antibody clone in a different buffer formulation (ab185050).

Lanes 1-4 : Merged signal (red and green). Green - ab185050 observed at 160 kDa. Red - loading control ab8245 observed at 37 kDa.

ab185050 Anti-EHMT2/G9A antibody [EPR18894] 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. ab185050 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 [EPR18894] - ChIP Grade (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-antibody-epr18894-chip-grade-ab185050'>ab185050</a>) 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

false

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050)

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 ab185050, 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

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ChIC/CUT&RUN sequencing - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185050)

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 ab185050, 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.

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade - BSA and Azide free (AB240289)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL. 2.5X10^5 of Human wild-type HeLa cell line (ab255928) or EHMT2 (G9A) knockout HeLa cell line (ab265149) were used along with 5µg of ab185050 [EPR18894]. Assay Quality Control was conducted using 5µg Anti-CTCF (ab188408) on the same cell lines. 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 (ab185050).