Recombinant Anti-c-Myc antibody [Y69] - ChIP Grade - BSA and Azide free (ab168727)

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.

This data was developed using the same antibody clone in a different buffer formulation (ab32072).

Lanes 1 - 4: Merged signal (red and green). Green - ab32072 observed at 57 kDa. Red - loading control, ab8245 observed at 37 kDa.

 ab32072 was shown to react with MYC in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab256500 (knockout cell lysate ab263850) was used. Wild-type and MYC knockout samples were subjected to SDS-PAGE. ab32072 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.

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]

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM/TBST.

Clone Y69 (ab168727) has been successfully conjugated by Abcam. This image was generated using Anti-c-Myc antibody [Y69] (Alexa Fluor® 647). Please refer to ab190560 for protocol details.

ab190560 staining c-Myc in panc1 cells. The cells were fixed with 100% methanol (5 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab190560 at a working dilution of 1 in 100 (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal in 4% formaldehyde (10 min) fixed panc1 cells under the same testing conditions.

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

Clone Y69 (ab168727) has been successfully conjugated by Abcam. This image was generated using Anti-c-Myc antibody [Y69] (Alexa Fluor® 488). Please refer to ab190026 for protocol details.

ab190026 staining c-myc in Panc-1 cells. The cells were fixed with 4% formaldehyde (10 min), 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 ab190026 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

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

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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 was Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody at 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.  

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072). 

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

Photomicrographs of select tumors and reactive tissue stained for c-Myc (positive staining = 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling c-Myc with ab32072 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse spleen. 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) secondary antibody 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 (ab32072).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skin carcinoma tissue labelling c-Myc with unpurified ab32072 at 1/50.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

See Abreview

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

Immunohistochemistry (Formalin/PFA-fixed parffin-embedded sections) analysis of human urinary bladder transitional carcinoma tissue labelling c-Myc with unpurified ab32072.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32072).

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 10⁵ HeLa cells and 8 µg of ab32072 [Y69]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷HeLa cells and 4 µg 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. 

This data was developed using the same antibody clone in a different buffer formulation (ab32072).

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. 

This data was developed using the same antibody clone in a different buffer formulation (ab32072).