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Anti-c-Myc antibody [Y69] ab32072 is a rabbit monoclonal antibody that is used in c-Myc western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone Y69 has been tried and trusted by researchers since 2006 and is cited in >1650 publications
- Specificity confirmed with MYC knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation

New 20 ul size available


Images

Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072), expandable thumbnail
  • Immunoprecipitation - Anti-c-Myc antibody [Y69] - ChIP Grade (AB32072), expandable thumbnail

Publications

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IHC-PIPWBICC/IFChIC/CUT&RUN-seqChIP-seqFlow Cyt (Intra)
Human
Tested
Tested
Tested
Tested
Tested
Tested
Tested
Mouse
Expected
Expected
Tested
Expected
Expected
Expected
Expected
Rat
Expected
Expected
Tested
Expected
Expected
Expected
Expected

Tested
Tested

Species

Human

Dilution info

5 µg/mL

Notes

Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Human

Dilution info

5 µg/mL

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/1000

Notes

-

Species

Rat

Dilution info

1/1000

Notes

-

Species

Human

Dilution info

1/1000

Notes

-

Tested
Tested

Species

Human

Dilution info

5-10 µg/mL

Notes

1/100.

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Human

Dilution info

5 µg

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Human

Dilution info

8 µg for 107 Cells

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Human

Dilution info

1/76

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Associated Products

Select an associated product type

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Target data

Function

Transcription factor that binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3' (PubMed:24940000, PubMed:25956029). Activates the transcription of growth-related genes (PubMed:24940000, PubMed:25956029). Binds to the VEGFA promoter, promoting VEGFA production and subsequent sprouting angiogenesis (PubMed:24940000, PubMed:25956029). Regulator of somatic reprogramming, controls self-renewal of embryonic stem cells (By similarity). Functions with TAF6L to activate target gene expression through RNA polymerase II pause release (By similarity). Positively regulates transcription of HNRNPA1, HNRNPA2 and PTBP1 which in turn regulate splicing of pyruvate kinase PKM by binding repressively to sequences flanking PKM exon 9, inhibiting exon 9 inclusion and resulting in exon 10 inclusion and production of the PKM M2 isoform (PubMed:20010808).

Alternative names

Recommended products

Anti-c-Myc antibody [Y69] ab32072 is a rabbit monoclonal antibody that is used in c-Myc western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone Y69 has been tried and trusted by researchers since 2006 and is cited in >1650 publications
- Specificity confirmed with MYC knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation

New 20 ul size available

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

Y69

Purification technique

Affinity purification Protein A

Specificity

This antibody is specific for endogenous c-Myc. It does not detect Myc tag. Expression levels of the target protein vary with sample type and some optimization may be required.

Please see documentation for further information on positive controls (Chinese version)

Dissociation constant

3.8 x 10-12 M

Concentration
Loading...

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

The proto-oncogene MYC plays a role in human oncogenesis. For more information see here.

This recombinant rabbit monoclonal antibody (Y69) to c-Myc specifically detects endogenous c-Myc. ab32, the Mouse monoclonal antibody (9E10 clone) to the Myc tag however can be used to study Myc-tagged proteins. More information comparing the two antibodies can be found here.

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

The c-Myc protein also known as Myc or cMyc is a transcription factor with a molecular weight of approximately 49 kDa. It plays a mechanical role by binding to DNA where it regulates the expression of various genes. Researchers often detect c-Myc in the cell nucleus. It expresses in many cell types including embryonic stem cells and cancer cells. Due to its influential role in proliferation and growth scientists have highly studied c-Myc for its functions and implications.

Biological function summary

C-Myc influences cellular processes like cell cycle progression apoptosis and metabolism. It often forms a complex with its partner protein Max to bind target genes and activate transcription. This c-Myc/Max complex operates by manipulating gene expression patterns which leads to changes in cellular behavior. Through its activity c-Myc affects cell growth and division which remain critical for development and homeostasis.

Pathways

Many scientists understand that c-Myc serves major functions in the MYC signaling pathway and the PI3K/AKT signaling pathway. c-Myc modulates the expression of genes that govern cell cycle checkpoints and cellular metabolism through these pathways. In this context c-Myc interacts with proteins like TCF and cyclins allowing the fine-tuning of cell proliferation and growth signals. These pathway involvements highlight the protein's importance in fundamental cellular mechanisms and its regulation.

Associated diseases and disorders

Researchers have frequently associated c-Myc with cancer and cardiovascular disorders. In cancer c-Myc overexpression can drive tumorigenesis by promoting uncontrolled cell proliferation. This often involves interactions with other oncogenic proteins like Bcl-2. In cardiovascular disorders c-Myc dysregulation can disrupt normal cellular function and contribute to pathophysiological conditions. Studies continue to explore c-Myc's role in these diseases aiming to develop therapeutic strategies targeting its activity.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

32 product images

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail
    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/.

    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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail
    Image from Simeone P et al. PLoS One. 2014;;9(7):e103030.; doi: 10.1371/journal.pone.0103030.

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail
    Image from Toon CW et al. PLoS One. 2014;9(2):e87456. Fig 1.; doi: 10.1371/journal.pone.0087456.

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of colorectal carcinoma using rabbit anti-c-Myc antibody

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail
    Image from Kluk MJ et al. PLoS One. 2012;7(4):e33813. Fig 1.; doi: 10.1371/journal.pone.0033813.

    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.

  • Immunoprecipitation - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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)

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 48 kDa

    Exposure time: 12min

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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] (Anti-GAPDH antibody [6C5] - Loading Control 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 Human MYC (c-Myc) knockout HEK-293T cell line ab256500 (CRISPR-Cas9 edited cell lysate Human MYC (c-Myc) knockout HEK-293T 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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 3: Jurkat cell lysate at 20 µg

    Lane 4: SH-SY5Y cell lysate at 20 µg

    Performed under reducing conditions.

    Predicted band size: 48 kDa

    Observed band size: 45 kDa, 57 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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 (Human MYC (c-Myc) knockout HEK-293T cell line 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 Anti-alpha Tubulin antibody [DM1A] - Loading Control 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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).

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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

  • Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunofluorescence staining of HeLa cells using rabbit anti-c-Myc antibody

    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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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).

  • Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunofluorescence staining of HeLa cells using rabbit anti-c-Myc antibody

    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) (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 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) (1/500 dilution).

  • Flow Cytometry (Intracellular) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc flow cytometry staining of HeLa cells using rabbit anti-c-Myc antibody

    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 (Goat 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) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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.

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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 ECL Substrate Kit (High Sensitivity) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 48 kDa

    Observed band size: 57 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of human esophagus using rabbit anti-c-Myc antibody

    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail
    Image courtesy of Dr Vladimir Milenkovic by customer review.

    Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunofluorescence staining of HEK293 cells using rabbit anti-c-Myc antibody

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of human adenocarcinoa using rabbit anti-c-Myc antibody

    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.

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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] (Anti-GAPDH antibody [6C5] - Loading Control 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 Human MYC (c-Myc) knockout HEK-293T cell line ab256500 (knockout cell lysate Human MYC (c-Myc) knockout HEK-293T 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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

    Performed under reducing conditions.

    Predicted band size: 48 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of adenocarcinoma using rabbit anti-c-Myc antibody

    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) (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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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) (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.

  • Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunofluorescence staining of HeLa cells using rabbit anti-c-Myc antibody

    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 (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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of colon tissue using rabbit anti-c-Myc antibody

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

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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, Anti-GAPDH antibody [6C5] - 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 Human MYC (c-Myc) knockout HEK-293T cell line ab256500 (knockout cell lysate Human MYC (c-Myc) knockout HEK-293T cell lysate ab263850) was used. Wild-type and MYC knockout samples were subjected to SDS-PAGE. ab32072 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

    Lanes 1 - 4: Merged signal (red and green). Green - ab32072 observed at 57 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Human MYC (c-Myc) knockout HEK-293T cell line ab256500 (knockout cell lysate Human MYC (c-Myc) knockout HEK-293T cell lysate ab263850) was used. Wild-type and MYC knockout samples were subjected to SDS-PAGE. ab32072 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of lung adenocarcinoma using rabbit anti-c-Myc antibody

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

  • OI-RD Scanning - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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.

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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

    Exposure time: 3min

  • Immunocytochemistry/ Immunofluorescence - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 2 μg/ml. DAPI was used as nuclear counterstain (shown in blue). As secondary antibodies Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat anti-Rabbit (Alexa Fluor® 488) (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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).

  • Flow Cytometry (Intracellular) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc flow cytometry staining of c-MYC KO cells using rabbit anti-c-Myc antibody

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of human skin using rabbit anti-c-Myc antibody

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of human colon using rabbit anti-c-Myc antibody

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

    c-Myc immunohistochemistry staining of human colon using rabbit anti-c-Myc antibody

    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.

  • ChIC/CUT&RUN sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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 Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.

    Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.

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

  • Western blot - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 20000 µg/mL

    Observed band size: 45,57 kDa

  • ChIP-sequencing - Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072), expandable thumbnail

    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.

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