Figure 1: c-Myc target protein structure.

c-Myc target introduction

Protein Function

• c-Myc is a widely acting transcription regulatory factor that plays a regulatory role in various cellular processes such as proliferation, differentiation, and apoptosis. Other family members include v-Myc, n-Myc, and l-Myc.
• c-Myc is one of the most common oncogenes, and its expression is frequently upregulated in tumors. It is generally believed that the activation of c-Myc can induce the occurrence of human tumors, so strict regulation of c-Myc is crucial. The c-Myc expression is induced by mitotic signals and suppressed by growth inhibitory signals.
• The primary regulatory function of c-Myc occurs through its binding with Max protein. Max is a relatively stable protein, but c-Myc is rapidly degraded with a half-life of 20-30 minutes.
• As a regulatory factor for somatic cell reprogramming, c-Myc, together with Sox2, Oct4, and KLF4, regulates the reprogramming of differentiated cells into an embryonic-like state.

Protein Expression

• As a transcription regulatory factor, c-Myc is tissue-specific and expressed highly in various tumor cells.

Protein Characteristics

• The expression level of c-Myc varies in different samples, so selecting appropriate samples for experiments is essential.
• c-Myc is an endogenous protein; please differentiate between c-Myc and Myc tags.

Protein Localization

• Cell nucleus.

Figure 2: c-Myc ICC experimental result image, Anti-c-Myc antibody [Y69] (ab32072). Green: c-Myc, Red: Tubulin, Blue: DAPI.

Isoforms & Post-translation modifications

Human (P01106): Isoforms 1-2: 49 kDa (predicted)
Mouse (P01108): 49 kDa (predicted)
Rat (P09416): 49 kDa (predicted)
c-Myc has multiple phosphorylation modifications at different sites
Acetylation modification
Ubiquitination modification
Glycosylation modification

WB experiment tips

Precautions

• The ab32072 antibody can only be used to detect endogenous c-Myc protein; it will not detect the Myc tag.
  c-Myc tissue specificity is low; the expression levels of proteins may vary in different samples, and some samples may show weak expression or no expression. Please select appropriate experimental samples and confirm the expression level of the target protein before detection. If the expression level is low, we recommend optimizing the experimental conditions, such as reducing dilution, increasing the amount of loading, using a high-sensitivity substrate, etc.
• We strongly recommend using positive controls to confirm that the experimental system works correctly.
• c-Myc undergoes various post-translational modifications, often resulting in the actual detected molecular weight not matching the predicted band size or the presence of two bands.

Positive controls

• Recombinant human c-Myc protein (ab169901)
• Jurkat, HeLa cell lines

Negative control

• Human MYC (c-Myc) knockout HEK-293T cell lysate (ab263850)

Example of results

Figure 3: Recombinant Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

Lane 1: 20 µg wild-type Jurkat cell lysate
Lane 2: 20 µg HeLa cell lysate
Lane 3: 20 µg wild-type HEK-293T cell lysate
Lane 4: 20 µg MYC knockout HEK-293T cell lysate

Primary antibody: Anti-c-Myc antibody [Y69] (ab32072), diluted at 1/1000 concentration
Predicted band size: 49 kDa
Detected band size: 57 kDa

Figure 4: Recombinant Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

Lane 1: MCF-7 (human breast cancer epithelial cells) whole cell lysate
Lane 2: Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate
Lane 3: K562 (human chronic myelogenous leukemia lymphocyte) whole cell lysate
Lane 4: Jurkat (human T cell leukemia T lymphocyte) whole cell lysate
Lane 5: THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 6: Rat spleen whole cell lysate
Lane 7: L6 (rat skeletal muscle myoblast) whole cell lysate
Lane 8: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate
Lane 9: RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Predicted band size: 49 kDa
Detected band sizes: 45, 57 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. To detect phosphorylated c-Myc protein, we recommend adding a complex phosphatase inhibitor to the lysis buffer.
3. Sonicate the cells to enrich the target protein.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.
2. We strongly recommend using a positive lysis buffer as a control when conducting new WB experiments.

Transfer:

1. It is recommended not to cut the membrane but to keep the whole or at least the membrane below 90 kDa for antibody incubation.
2. The PVDF membrane must be activated and thoroughly washed after activation to remove residual methanol on the membrane.
3. We strongly recommend using Coomassie Brilliant Blue staining after transfer to confirm the success of the transfer.

Antibody incubation:

1. Please choose the appropriate antibody working concentration according to the product manual. We recommend using fresh antibodies and not reusing antibodies.

IHC experiment tips

Precautions

• The expression level of c-Myc protein may vary in different samples, and some samples may show weak or no expression. Please choose appropriate experimental samples.
• We strongly recommend using a positive control to confirm that the experimental system is working properly.

Positive control

• Human colon adenocarcinoma tissue.

Example of results

Figure 5: Recombinant Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072).

Sample name: Human colon adenocarcinoma tissue.

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample fixation:

1. The time for sample fixation depends on the size of the tissue block and the type of tissue, but for most samples, such as fixation with 4% PFA, fixing at room temperature for 18-24 hours is more appropriate.

Blocking:

1. If using fluorescently conjugated secondary antibodies for the experiment, we recommend using a blocking solution containing 1% BSA and a final concentration of 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.
2. If subsequent detection is performed using HRP conjugates, please use 3% hydrogen peroxide to treat the sections for 10 minutes to block endogenous peroxidase.

Antigen retrieval:

1. When performing immunohistochemistry experiments on paraffin sections, please choose the appropriate antigen retrieval solution according to the instructions. We recommend using a pressure cooker for heat-induced antigen retrieval. You can try fixing the sections at 110°C for 15 minutes.

References

1. Chi V Dang. MYC on the path to cancer. Cell. 2012 Mar 30;149(1):22-35.
   doi: 10.1016/j.cell.2012.03.003.
2. Renumathy Dhanasekaran, Anja Deutzmann, Wadie D Mahauad-Fernandez, Aida S Hansen, Arvin M Gouw, Dean W Felsher. The MYC oncogene - the grand orchestrator of cancer growth and immune evasion. Nat Rev Clin Oncol. 2022 Jan;19(1):23-36. doi: 10.1038/s41571-021-00549-2.
3. Natalie Meyer, Linda Z Penn. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008 Dec;8(12):976-90. doi: 10.1038/nrc2231.
4. Stephanie C Casey, Ling Tong, Yulin Li, Rachel Do, Susanne Walz, Kelly N Fitzgerald, Arvin M Gouw, Virginie Baylot, Ines Gütgemann, Martin Eilers, Dean W Felsher. MYC regulates the antitumor immune response through CD47 and PD-L1. Science. 2016 Apr 8;352(6282):227-31. doi: 10.1126/science.aac9935. Epub 2016 Mar 10.