mycl1a Antibody

What is MYCL1/L-Myc and why is it significant in cancer research?

MYCL1 (L-Myc) is a member of the MYC family of proto-oncogene transcription factors belonging to the helix-loop-helix basic leucine zipper (HLH bzip) family. Like other MYC family members, MYCL1 heterodimerizes with partner proteins to bind to the DNA consensus site 5' CACGTG 3' . MYCL1 is implicated in controlling a wide range of cellular processes from cellular proliferation to apoptosis .

The gene was originally isolated from small cell lung carcinoma (SCLC) samples, and significant research has identified its involvement in this aggressive cancer type . Recent studies have found that MYCL1 amplification occurs in approximately 6.5% of SCLC cases, making it a potential biomarker and therapeutic target . Understanding MYCL1's role in oncogenesis is critical for developing targeted therapies, particularly for aggressive cancers like SCLC.

What are the primary applications for MYCL1 antibodies in experimental research?

Based on validated applications from multiple sources, MYCL1 antibodies demonstrate utility in several key research techniques:

Methodological approach: For optimal results in these applications, researchers should determine appropriate dilutions for each experimental setup. For Western blot, reducing conditions using Immunoblot Buffer Group 2 have been validated . For ICC, a protocol using 10 μg/mL primary antibody incubation for 3 hours at room temperature followed by appropriate secondary antibody has demonstrated specific staining .

How can researchers validate the specificity of MYCL1 antibodies?

Validating antibody specificity is critical for ensuring reliable experimental results. For MYCL1 antibodies, several methodological approaches are recommended:

• Positive control selection: Use nuclear extracts from validated cell lines showing endogenous MYCL1 expression (HeLa, A549, JEG-3, and NIH-3T3 have been confirmed)

• Molecular weight verification: Confirm detection at the expected molecular weight (~40 kDa for MYCL1)

• Cellular localization pattern: Verify appropriate nuclear and cytoplasmic distribution via immunocytochemistry

• Inducible expression systems: As demonstrated in published research, compare cells with doxycycline-inducible MYCL1 expression versus controls to confirm antibody specificity

• Cross-reactivity testing: Test antibody performance across species when using antibodies designed to detect both human and mouse MYCL1

What are the critical parameters for optimizing Western blot detection of MYCL1?

Successful Western blot detection of MYCL1 requires attention to several technical parameters:

• Sample preparation: Nuclear extracts yield better results than whole cell lysates due to the nuclear localization of MYCL1

• Buffer conditions: Immunoblot Buffer Group 2 under reducing conditions has been validated for MYCL1 detection

• Antibody concentration: 1 μg/mL of MYCL1 Antigen Affinity-purified Polyclonal Antibody has shown specific detection

• Secondary antibody selection: HRP-conjugated Anti-Goat IgG Secondary Antibody (for goat-derived primary antibodies) or appropriate species-matched secondary antibody

• Controls: Include both positive controls (cell lines with known MYCL1 expression) and negative controls (cell lines with low/no MYCL1 expression or siRNA knockdown samples)

For experimental troubleshooting, researchers should note that MYCL1 expression can be influenced by cellular conditions. For example, research has shown that doxycycline treatment to induce MYCL1 expression can be used to generate positive controls .

How should researchers store and handle MYCL1 antibodies to maintain optimal performance?

Proper storage and handling are essential for maintaining antibody activity:

• Storage temperature: Store at -20 to -70°C for long-term stability (up to 12 months from date of receipt)

• Short-term storage: For reconstituted antibodies, store at 2 to 8°C under sterile conditions for up to 1 month

• Medium-term storage: For reconstituted antibodies, maintain at -20 to -70°C under sterile conditions for up to 6 months

• Freeze-thaw cycles: Use a manual defrost freezer and avoid repeated freeze-thaw cycles which can degrade antibody performance

• Reconstitution: Follow manufacturer's specific reconstitution instructions as they may vary by antibody format

Methodological tip: Aliquot antibodies upon initial reconstitution to minimize freeze-thaw cycles and extend shelf life.

What is the significance of MYCL1 amplification and expression in SCLC and how can antibodies help characterize this?

MYCL1 amplification has emerged as an important molecular feature in SCLC:

• Prevalence: Research has identified MYCL1 amplification in approximately 6.5% of SCLC cases

• Fusion events: Some positive cases show MYCL1 gene amplification combined with fusion events, creating novel oncogenic drivers

• Protein expression: Studies have found that 6.5% of SCLC cases show positive L-Myc protein expression by immunohistochemistry

• Relationship to other MYC family members: Interestingly, no overlap was observed between cases with c-Myc protein expression (8.7% of cases) and those with L-Myc expression, suggesting distinct oncogenic pathways

• Therapeutic implications: MYCL1-positive cases may respond to specific therapies like alisertib, with one case report documenting near-complete response in a patient with MYCL1-JAZF1 fusion

Methodological approach for characterization: For comprehensive characterization of MYCL1 status in SCLC samples, researchers should employ a multi-modal approach combining FISH for gene amplification detection, IHC for protein expression analysis (using validated antibodies), and potentially RNA-seq for fusion transcript identification .

How can mass spectrometry complement antibody-based MYCL1 detection and characterization?

Mass spectrometry (MS) offers powerful complementary approaches to antibody-based detection:

• De novo sequencing: MS can provide direct protein sequence information, useful for confirming antibody specificity or identifying novel MYCL1 variants

• Post-translational modifications: MS can detect and characterize PTMs on MYCL1 that might not be detectable with available antibodies

• Quantitative analysis: MS provides absolute quantification that complements the semi-quantitative nature of antibody-based methods

• Low-abundance detection: Advanced MS approaches can detect MYCL1 at low abundance where antibody sensitivity might be insufficient

• Clinical applications: MS methods have been used to detect monoclonal antibodies directly from serum samples, suggesting potential for detecting MYCL1-related biomarkers in patient samples

Technical approaches: Multiple complementary proteases combined with dual fragmentation schemes (stepped HCD and EThcD) on all peptide precursors can achieve comprehensive sequence coverage . For protein detection from complex samples, both bottom-up (enzymatic digestion followed by LC-MS/MS) and top-down (intact protein analysis) approaches provide complementary information .

What considerations are important when using MYCL1 antibodies for immunohistochemistry in tissue samples?

IHC with MYCL1 antibodies requires specific methodological considerations:

• Antibody selection: Select antibodies validated for IHC applications with demonstrated specificity (e.g., anti-L-Myc antibody ab28739 has been validated for FFPE tissues)

• Sample preparation: Standard FFPE processing with 10% formalin fixation followed by deparaffinization and antigen retrieval

• Blocking and antibody incubation: Blocking with 1% BSA followed by overnight incubation at 4°C with primary antibody at optimized dilution (e.g., 1:200 for the above antibody)

• Detection system: Appropriate secondary antibody (e.g., Poly-HRP) followed by DAB visualization

• Interpretation criteria: Establish clear positivity thresholds (e.g., ≥10% cells showing brown color defined as "positive expression")

• Controls: Include both positive and negative controls, with PBS substitution for primary antibody serving as negative control

• Blinded evaluation: Have multiple pathologists evaluate slides independently to ensure reliable scoring

How should researchers design experiments to investigate MYCL1's role in metabolic regulation?

Research has revealed MYCL1's involvement in metabolic regulation, particularly in cancer cells. To effectively study this function:

• Model system selection: Use cell lines with inducible expression of MYC family members (MYC, MYCN, MYCL) to compare their differential effects

• Metabolic parameter measurement: Employ extracellular acidification rate (ECAR) measurements to assess glycolytic activity as influenced by MYCL1

• Experimental validation: Include oligomycin treatment to assess maximal glycolytic capacity in cells with altered MYCL1 expression

• Molecular pathway analysis: Investigate MYCL1's interaction with other transcription factors (e.g., NF-κB) in regulating metabolic genes

• Gene expression analysis: Examine changes in glycolytic gene expression and transporters like MCT1 following MYCL1 induction

Experimental approach: A comprehensive study would include Western blot confirmation of MYCL1 induction, ECAR measurements to assess functional impact on glycolysis, and molecular analysis of target gene expression using RT-qPCR or RNA-seq .

What are the key considerations when using MYCL1 antibodies in panels for autoantibody biomarker studies?

When incorporating MYCL1 antibodies in autoantibody panel studies:

• Panel composition: Include MYCL1 alongside other cancer-associated antigens such as p53, c-myc, HER2, NY-ESO-1, and others that have shown diagnostic potential

• Sample type selection: Use serum samples, being mindful of storage conditions to preserve antibody integrity

• Detection method optimization: Develop reliable ELISA or multiplex bead assays with optimized blocking to minimize background

• Statistical validation: Determine sensitivity and specificity of the panel for specific cancer types rather than relying on single autoantibody performance

• Population considerations: Account for demographics, cancer stage, and potential confounding conditions when establishing reference ranges

Methodological approach: Research has indicated that autoantibody panels show higher sensitivity than individual autoantibodies. For example, panels including multiple autoantibodies (p53, c-myc, HER2, NY-ESO-1, CAGE, MUC1, GBU4-5) have demonstrated high sensitivity for squamous cell lung cancer .

How can researchers effectively compare results from different MYCL1 antibody clones or suppliers?

To ensure valid comparisons between different MYCL1 antibodies:

• Side-by-side validation: Test multiple antibodies simultaneously on identical samples and under identical conditions

• Epitope mapping: Understand the specific epitopes recognized by each antibody (e.g., Gly16-Asn139 region of human MYCL1/L-Myc for R&D Systems antibodies)

• Isotype consideration: Note differences in antibody isotype and host species, as these affect secondary antibody selection and potential for cross-reactivity

• Application-specific validation: Validate each antibody specifically for the intended application rather than assuming cross-application performance

• Cross-referencing published literature: Review publications that have used specific antibody clones to verify performance characteristics

Experimental approach: Create a validation panel with known positive and negative controls, and systematically test each antibody across relevant applications while documenting sensitivity, specificity, optimal concentrations, and background levels.

What emerging applications might leverage MYCL1 antibodies for clinical diagnostics?

Several promising clinical applications for MYCL1 antibodies are emerging:

• Cancer subtyping: Identifying MYCL1-positive SCLC for potential targeted therapy approaches

• Liquid biopsy development: Detecting circulating MYCL1 or anti-MYCL1 autoantibodies as minimally invasive biomarkers

• Theranostic approaches: Using anti-MYCL1 antibodies to both identify patients and deliver therapeutic agents

• Immunotherapy stratification: Correlating MYCL1 status with immunotherapy response, particularly given observations of lymphocyte infiltration in MYCL1-positive cases

• Multi-marker panels: Incorporating MYCL1 detection in comprehensive molecular profiling panels for precision oncology

Research needs: Larger clinical studies are required to validate these applications. One study noted: "Although the sample size of our study on postoperative specimens for SCLC is relatively large, it is still insufficient and the MYCL1-and MYC-positive samples remain few." Future research should include larger patient cohorts with diverse demographics and disease stages.

What technical advancements might improve MYCL1 antibody performance in challenging applications?

The field continues to evolve with several promising technical advancements:

• Recombinant antibody technology: Development of recombinant MYCL1 antibodies with improved batch-to-batch consistency

• Single-domain antibodies: Engineered smaller antibody formats that may access epitopes unavailable to conventional antibodies

• MS-guided epitope selection: Using mass spectrometry to identify optimal epitopes for antibody development

• Multimodal detection systems: Combining antibody-based detection with orthogonal methods like MS or PCR for improved sensitivity and specificity

• Machine learning approaches: Applying computational methods to optimize antibody design and performance prediction

Future research should focus on developing antibodies specifically designed for challenging applications like fixed tissue IHC, where current MYCL1 antibodies may show limited performance or require extensive optimization.