Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

This ICC/IF data was generated using the same anti-phospho S62 c-Myc antibody clone, EPR17924, in a different buffer formulation (cat# ab185656).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling c-Myc (phospho S62) with ab185656 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Confocal image showing nuclear staining on HeLa cells.
The staining decreased after blocking with phospho peptide (100μg/ml) overnight.

The control peptide is a non-phospho peptide.

The nuclear counter stain 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 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab185656 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 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 1/500 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling c-Myc (phospho S62) with purified ab185656 at 1/150 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185656).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

This IHC data was generated using the same anti-phospho S62 c-Myc antibody clone, EPR17924, in a different buffer formulation (cat# ab185656).

Immunohistochemical analysis of paraffin-embedded Human endometrium cancer tissue labeling c-Myc (phospho S62) with ab185656 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Nuclear staining on cancer cells of Human endometrial cancer 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.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

c-Myc (phospho S62) was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with 200nM TPA for 10 minutes with ab185656 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab185656 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1500 dilution.

Lane 1 : HeLa whole cell lysate treated with 200nM TPA for 10 minutes,10 μg (Input).

Lane 2 : ab185656 IP in HeLa whole cell lysate treated with 200nM TPA for 10 minutes.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab185656 in HeLa whole cell lysate treated with 200nM TPA for 10 minutes.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185656).

All lanes:

Immunoprecipitation - Anti-c-Myc (phospho S62) antibody [EPR17924] (<a href='/en-us/products/primary-antibodies/c-myc-phospho-s62-antibody-epr17924-ab185656'>ab185656</a>)

Predicted band size: 48 kDa

Observed band size: 58 kDa

false

Exposure time: 5s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling c-Myc (phospho S62) with ab185656 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Nuclear staining on rat testis 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 (ab185656).

Heat mediated antigen retrieval was performed 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-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling c-Myc (phospho S62) with ab185656 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Nuclear and cytoplasmic staining on mouse spleen 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 (ab185656).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185656).

CUT&RUN profiling with c-Myc antibody reveals the expected genomic enrichment pattern in wild-type (WT) cells, which is substantially reduced in MYC knockout (KO) cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with c-Myc antibody (Abcam ab185656, 0.5 µg). 500,000 HEK293T WT or KO (Abcam ab256500) 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-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185656).

CUT&RUN profiling with c-Myc antibody demonstrates robust genome-wide enrichment in wild-type (WT) cells, which is markedly diminished in MYC knockout (KO) 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 c-Myc antibody (Abcam ab185656, 0.5 µg). 500,000 HEK293T WT or KO (Abcam ab256500) 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 deepTools (Ramírez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to c-Myc WT, with red indicating high localized enrichment and blue denoting background. Validated antibodies show genome-wide enrichment above IgG background consistent with c-Myc binding in WT cells and near complete loss of signal in KO cells.

• Dot

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Dot Blot - Anti-c-Myc (phospho S62) antibody [EPR17924] - BSA and Azide free (AB232691)

Dot blot analysis of c -Myc (phospho T58) peptide (Lane 1), c-Myc non-phospho peptide (a control peptide for c-Myc phospho T58) (Lane 2), c-Myc (phospho S62) peptide (Lane 3), and c-Myc non-phospho peptide (a control peptide for c-Myc phospho S62) (Lane 4), labeled using ab185656 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer : 5% NFDM/TBST.

Lanes 1, 2 and 4 are control peptides, lane 3 contains the immunogen peptide.

Exposure time=3 minutes.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185656).