Anti-ELK1 antibody [E277]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ELK1 antibody [E277] (AB32106)

Immunocytochemistry/ Immunofluorescence analysis of A549 (human lung carcinoma epithelial cell) cells labeling ELK1 with purified ab32106 at 1/500 dilution (3.8 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ELK1 antibody [E277] (AB32106)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ELK1 with unpurified ab32106 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ELK1 antibody [E277] (AB32106)

Immunohistochemical analysis of paraffin embedded human breast carcinoma using ab32106 at a dilution of 1/50

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

• WB

Lab

Western blot - Anti-ELK1 antibody [E277] (AB32106)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : ELK1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa positive whole cell lysate (21 μg)
Lane 4 : MCF7 negative whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32106 observed at 55 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab32106 was shown to specifically react with ELK1 in wild-type HAP1 cells as signal was lost in ELK1 knockout cells. Wild-type and ELK1 knockout samples were subjected to SDS-PAGE. ab32106 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ELK1 antibody [E277] (ab32106)

Predicted band size: 45 kDa

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• WB

Lab

Western blot - Anti-ELK1 antibody [E277] (AB32106)

Lanes 1- 2 : Merged signal (red and green). Green - ab32106 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32106 was shown to react with ELK1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261764 (knockout cell lysate ab256904) was used. Wild-type HeLa and ELK1 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. ab32106 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 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-ELK1 antibody [E277] (ab32106) at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ELK1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ELK1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-elk1-knockout-hela-cell-line-ab261764'>ab261764</a>)

Predicted band size: 45 kDa

Observed band size: 55 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-ELK1 antibody [E277] (AB32106)

CUT&RUN profiling with ELK1 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 ELK1 antibody (Abcam ab32106, 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 ELK1, with red indicating high localized enrichment and blue denoting background.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-ELK1 antibody [E277] (AB32106)

CUT&RUN profiling with ELK1 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 ELK1 antibody (Abcam ab32106, 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).

• WB

Unknown

Western blot - Anti-ELK1 antibody [E277] (AB32106)

All lanes:

Western blot - Anti-ELK1 antibody [E277] (ab32106) at 1/500 dilution

Predicted band size: 45 kDa

Observed band size: 62 kDa

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