Anti-c-Myc 抗体 [Y69] - ChIP Grade

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (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 dilution. 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 dilution). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

ab32072 staining of Myc proto-oncogene protein in a HCT 116 cell spheroid. The cells were fixed with 4% paraformaldehyde (10 min), permeabilised with 2% Triton X-100 for 1h and then blocked with 1% BSA/10% normal goat serum/0.3M glycine 0.1% PBS-Tween overnight at room temperature. The spheroids were then incubated overnight at room temperature with ab32072 at 2 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 2 μg/ml. DAPI was used as nuclear counterstain (shown in blue). As secondary antibodies ab150081 Goat anti-Rabbit (Alexa Fluor^® 488) (shown in green) and ab150120 Goat anti-Mouse (Alexa Fluor^® 594) (shown in magenta) were used, incubated overnight at room temperature. All permeabilization, blocking and antibody incubation steps were performed using a rotary shaker.

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Similar results were observed using 100% Methanol (5min).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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

• ICC/IF

AbReview23419****

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

Image courtesy of Dr Vladimir Milenkovic by Abreview.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

ab32072 staining MYC in wild-type HEK293 cells (top panel) and MYC knockout HEK293 cells (ab256500) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then 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 with ab32072 at 5μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Immunocytochemistry/immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling c-Myc with purified ab32072 at 1/100 dilution. 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 dilution) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-c-Myc antibody [Y69] - ChIP Grade (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 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 >5000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 nm bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (Human embryonic kidney epithelial cell) transfected with His and Myc tagged cas9 construct cells labelling 6X His tag with ab15149 at 1/100 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution(Green).

Confocal image showing cytoplasmic staining in 293T cells transfected with His and Myc tagged mouse cas9 construct. The counterstain was observed in red. Nuclear DNA was labeled with DAPI (shown in blue).

ab32072 Anti-c-Myc rabbit monoclonal antibody was used to counterstain Myc at 1/500 followed by a secondary antibody ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at a 1/1000 dilution (Red).

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

Image from Simeone P et al. PLoS One. 2014;;9(7):e103030.; doi: 10.1371/journal.pone.0103030.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

Image from Kluk MJ et al. PLoS One. 2012;7(4):e33813. Fig 1.; doi: 10.1371/journal.pone.0033813.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

Image from Toon CW et al. PLoS One. 2014;9(2):e87456. Fig 1.; doi: 10.1371/journal.pone.0087456.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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 dilution)) 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°CIsotype 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

• IP

Unknown

Immunoprecipitation - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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]

All lanes:

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

Predicted band size: 48 kDa

true

Exposure time: 12min

• ChIP-seq

Lab

ChIP-sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 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.

• ChIP-seq

Lab

ChIP-sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa 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.

• ChIP-seq

Lab

ChIP-sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 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.

• ChIP-seq

Lab

ChIP-sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 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.

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

MYC CRISPR-Cas9 edited HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (<a href='/products/cell-lines/human-myc-c-myc-knockout-hek-293t-cell-line-ab256500'>ab256500</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 48 kDa

Observed band size: 45 kDa,57 kDa

false

• WB

Supplier Data

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T cell lysate at 20 µg

Lane 4:

MYC knockout HEK-293T cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

false

• WB

PubMed

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

Predicted band size: 48 kDa

false

This image is taken from Calabrese D. R. et al. Nat Commun. 2018; 9: 4229. Fig 3e doi: 10.1038/s41467-018-06315-w Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/.

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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 knockout cell line ab256500 (knockout cell lysate ab263850). The band observed in the knockout 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 knockout 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.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

MYC knockout HEK-293T cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 48 kDa

false

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (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.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate at 20 µg

Lane 3:

THP1 (Human acute monocytic leukemia cell line) Whole Cell Lysate at 20 µg

Lane 4:

A20 (Mouse B lymphoma cell line) Whole Cell Lysate at 20 µg

Lane 5:

RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 48 kDa

Observed band size: 57 kDa

true

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 45 kDa,57 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse brain cancer tissue lysate at 20 µg

Lane 3:

Mouse skin tissue lysate at 20 µg

Lane 4:

Mouse skin cancer tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 45 kDa

false

Exposure time: 60s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

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 found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates at 20 µg

Lane 3:

K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Lane 4:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 20 µg

Lane 5:

THP-1 (Human monocytic leukemia monocyte) whole cell lysates at 20 µg

Lane 6:

Rat spleen whole cell lysates at 20 µg

Lane 7:

L6 (Rat skeletal muscle myoblast) whole cell lysates at 20 µg

Lane 8:

Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysates at 20 µg

Lane 9:

RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 57 kDa,48 kDa,45 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST. Lanes 1 and 2 : 80 seconds exposure time. Lanes 3 to 5 : 5 seconds exposure time.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

Lane 1:

Rat pancreas lysates at 20 µg

Lane 2:

AR42J (Rat pancreatic tumor epithelial cell) whole cell lysates at 20 µg

Lane 3:

Rat-1 (Rat embryonic fibroblast) whole cell lysates at 20 µg

Lane 4:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

Lane 5:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 20000 µg/mL

Observed band size: 45 kDa,57 kDa

false

• WB

Lab

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

Different batches of ab32072 were tested on Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 0.2 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 57 kDa.

All lanes:

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

Predicted band size: 48 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.