Anti-c-Myc 抗体 [Y69] - ChIP Grade (ab32072)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling c-Myc with ab32072 at a concentration of 1µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab32072 anti-c-Myc antibody [Y69] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling c-Myc with ab32072 at a concentration of 1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab32072 anti-c-Myc antibody [Y69] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

False colour image of Western blot: Anti-c-Myc antibody [Y69] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32072 was shown to bind specifically to c-Myc. A band was observed at 45/57 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in MYC CRISPR-Cas9 edited cell line ab256500 (CRISPR-Cas9 edited cell lysate ab263850). The band observed in the CRISPR-Cas9 edited lysate lane below 45/57 kDa is likely to represent a truncated form of c-Myc. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MYC CRISPR-Cas9 edited HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Human colorectal carcinoma (CRC) tissues stained for c-Myc using ab32072 at 1/100 dilution in immunohistochemical analysis.

Panel A: c-Myc positive IHC staining.

Panel B: c-Myc negative IHC staining.

For the full image see PMID 24503701.

Flow cytometry overlay histogram showing wild-type HEK293 (green line) and MYC knockout HEK293 stained with ab32072 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab32072) (1x 10⁶ in 100μl at 0.2 μg/ml (1/11500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type HEK293 - black line, MYC knockout HEK293 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HEK293 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5 µg of ab32072 [Y69]. 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 downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

Immunohistochemical analysis of Paraffin-embedded sections human skin tissue labelling c-Myc with ab32072 at 1/500 dilution, followed by a ready to use secondary Goat Anti-Rabbit IgG H&L (HRP). Counter stained with Haematoxylin. Secondary antibody only control: Used PBS instead of primary antibody

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Positive staining on human skin. The section was incubated with ab32072 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of Paraffin-embedded sections human cerebrum tissue labelling c-Myc with ab32072 at 1/500 dilution, followed by a ready to use secondary Goat Anti-Rabbit IgG H&L (HRP). Counter stained with Haematoxylin. Secondary antibody only control: Used PBS instead of primary antibody

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Negative control: no staining on human cerebrum. The section was incubated with ab32072 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Blocking buffer: 5% NFDM/TBST.

Blocking buffer: 5% NFDM/TBST.

c-Myc was immunoprecipitated using 0.5mg Jurkat (human T cell leukemia cell line from peripheral blood) whole cell extract, 5µg of unpurified rabbit monoclonal to c-Myc [Y69] and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with unpurified ab32072.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band: 57kDa; c-Myc [Y69]

Photomicrographs of select tumors and reactive tissue stained for c-Myc (positive staining  is the brown nuclei). Positive control (Burkitt lymphoma with a confirmed c-Myc translocation) revealed uniform, intense staining in >90% of tumor cells (Burkitt). In contrast, reactive lymphoid tissue revealed variable staining in only 10% of normal lymphocyte nuclei (Tonsil). Representative images from Diffuse large B cell lymphoma (DLBCL) cases and associated percent c-Myc+ tumor nuclei: Case 1, 90% MYC+; Case 7, 70% MYC+; and Cases 35 and 38, 30% c-Myc+. c-Myc staining was exclusively nuclear in all cases under the described staining conditions.

Blocking/Diluting buffer and concentation: 5% NFDM/TBST

Lanes 1 and 2: 80 seconds exposure time 

Lanes 3 to 5: 5 seconds exposure time 

Western blot of L363 MM cell and CA46 cells measuring expresison of c-Myc (using ab32072) and GAPDH as the dose of DC-34 increases. 

c-Myc protein levels are inhibited as a function of the dose of DC-34 in L363 cells; only the highest dose of DC-34 affected c-Myc in the more resistant CA46 Burkitt’s lymphoma cells.

ab32072 staining c-Myc in HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab32072 at 10μg/ml dilution (shown in green) and ab195889, mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of ab32072 staining c-Myc in human adenocarcinoma formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32072, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of ab32072 staining c-Myc in human esophagus formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32072, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the Secondary only control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Expression of c-Myc, as determined by immunohistochemical staining of glioblastoma sample (left) and low-grade glioma tumor (right) with ab32072. Representative samples are shown. Scale bars  = 20 µm. Nuclei were counterstained with hematoxylin (in blue).

For the full image see PMID 25050814.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human diffuse large B cell lymphoma tissue labelling c-Myc with purified ab32072 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarcinoma of the colon tissue labelling c-Myc with purified ab32072 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarinoma of colon tissue labelling c-Myc with unpurified ab32072.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarinoma tissue labelling c-Myc with unpurified ab32072.

The predicted molecular weight of c-Myc is 48 kDa (SwissProt), however we expect to observe a banding pattern at 57 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab32072 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP antibody, and visualised using ECL development solution ab133406.

Blocking and dilution buffer: 5% NFDM/TBST.

Immunocytochemistry/immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling c-Myc with purified ab32072 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (1/500).

Unpurified ab32072 staining c-Myc in HEK293 cells transfected with CACNB4-c-Myc by immunocytochemistry/ immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 then blocked using 5% serum for 20 minutes at 25°C. Samples were then incubated with ab32072 at a 1/250 dilution for 16 hours at 4°C. The secondary used was an Alexa Fluor^® 488 conjugated goat anti-rabbit polyclonal, used at a 1/500 dilution.

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ICC/IF image of unpurified ab32072 stained HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32072, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab32072 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32072, 1/76 dilution) for 30 min at 22°C. The secondary antibody used wasGoat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibodyat 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG [EPR25A] (monoclonal) (ab172730, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 nm bandpass filter. 

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ NCCIT cells and 8µg of ab32072 [Y69]. 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.