AP1M2 Antibody

What is AP1M2 and why is it important in research?

AP1M2 (Adaptor-Related Protein Complex 1, mu 2 Subunit) is a protein encoded by the AP1M2 gene. It functions as part of the adaptor protein complex, which is involved in intracellular protein transport and endocytosis. Recent research has identified AP1M2 as differentially expressed in various cancer types, particularly showing high expression in invasive breast carcinoma . Its importance in research stems from its potential role as a tumor marker gene that could be valuable for early tumor screening and its correlation with patient outcomes and immune system functioning in cancer .

What types of AP1M2 antibodies are available for research?

Several types of AP1M2 antibodies are available for research purposes, including:

• N-Terminal targeting antibodies - These recognize epitopes at the N-terminal region of the AP1M2 protein, such as the synthetic peptide immunogen used in ABIN2784762 .

• Mid-region targeting antibodies - Antibodies targeting amino acids 164-423 of human AP1M2 .

• Full-length antibodies - Some antibodies target the complete protein (AA 1-423) .

These antibodies are predominantly available as rabbit polyclonal antibodies, though mouse polyclonal versions also exist . Most are unconjugated, making them versatile for various experimental applications.

What is the specificity and cross-reactivity profile of AP1M2 antibodies?

AP1M2 antibodies exhibit varying degrees of cross-species reactivity depending on their epitope targets. For example, the N-terminal targeting AP1M2 antibody (ABIN2784762) demonstrates predicted reactivity with multiple species:

This broad cross-reactivity is beneficial for comparative studies across different model organisms . Other AP1M2 antibodies may have more restricted reactivity profiles, such as those limited to human, mouse, and rat samples . When selecting an antibody for experimental use, researchers should carefully assess the specificity requirements for their particular study system.

What are the validated applications for AP1M2 antibodies?

AP1M2 antibodies have been validated for several experimental applications:

• Western Blotting (WB) - Most AP1M2 antibodies are validated for detecting the protein in Western blot applications, with recommended dilutions typically ranging from 1/500 to 1/2000 .

• Immunofluorescence/Immunocytochemistry (IF/ICC) - Some AP1M2 antibodies are suitable for cellular localization studies using IF/ICC techniques, with recommended dilutions of 1/50 to 1/200 .

• Enzyme-Linked Immunosorbent Assay (ELISA) - Select antibodies are validated for ELISA applications, with typical concentrations of 1 μg/ml .

• Immunohistochemistry (IHC) - Some antibodies can be used for tissue section analysis through immunohistochemistry .

The specific application suitability varies by antibody, and researchers should verify the validation status for their intended application before proceeding with experiments.

What are the recommended protocols for AP1M2 detection by Western Blotting?

For optimal detection of AP1M2 using Western Blotting, researchers should consider the following protocol guidelines:

• Sample preparation: Extract proteins from tissues or cells using standard lysis buffers containing protease inhibitors.

• Protein separation: Use SDS-PAGE with appropriate percentage gels (10-12% is typically suitable for the 48 kDa AP1M2 protein) .

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST.

• Primary antibody incubation: Dilute AP1M2 antibody according to manufacturer recommendations (typically 1/500 to 1/2000) and incubate overnight at 4°C.

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (anti-rabbit IgG for most AP1M2 antibodies).

• Detection: Visualize using enhanced chemiluminescence.

The expected molecular weight for AP1M2 is approximately 48 kDa, which should be confirmed during analysis . Optimization may be required based on specific experimental conditions and sample types.

How should AP1M2 antibodies be stored and handled for optimal performance?

For maximum stability and performance of AP1M2 antibodies, the following storage and handling recommendations should be followed:

• Storage temperature: Store at -20°C for long-term preservation .

• Format: Most AP1M2 antibodies are supplied in liquid form, typically in PBS (pH 7.3) containing 0.02% sodium azide and 50% glycerol .

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles that can degrade antibody quality .

• Thawing: Thaw antibodies gradually at cold temperatures before use.

• Working concentration: Dilute to working concentration immediately before use rather than storing diluted antibody.

• Contamination prevention: Use sterile techniques when handling antibodies to prevent microbial contamination.

Proper storage and handling are crucial for maintaining antibody specificity and sensitivity in experimental applications. Always check the manufacturer's specific recommendations for the particular antibody being used.

How is AP1M2 expression related to cancer development and progression?

Research using TCGA, GTEx, and CCLE databases has revealed that AP1M2 is abundantly expressed in various cancer types, with particularly high expression in invasive breast carcinoma . The relationship between AP1M2 expression and cancer development involves several key aspects:

These findings suggest that AP1M2 may play a role in cancer development and could potentially serve as a biomarker for cancer detection and prognosis, particularly in breast cancer.

What is the relationship between AP1M2 and immune infiltration in the tumor microenvironment?

Analysis of the relationship between AP1M2 expression and immune infiltration has revealed significant correlations that may have implications for cancer immunotherapy:

• Immune cell infiltration: AP1M2 expression in breast invasive carcinoma was found to be highly associated with infiltration levels of multiple immune cell types, including:

  • Macrophages

  • Dendritic cells

  • CD4+ T cells

  • CD8+ T cells

  • B cells

• Immune checkpoint correlation: Studies examined the relationship between AP1M2 expression and 47 immune checkpoint genes using Spearman correlation analysis .

• Immune and stromal scores: In breast cancer, AP1M2 expression levels were negatively correlated with immune system scores, stromal scores, and composite scores .

These findings suggest that AP1M2 may influence the tumor immune microenvironment, particularly in breast cancer, and could potentially impact the efficacy of immunotherapeutic approaches.

How does AP1M2 relate to tumor neoantigens and microsatellite instability?

Research has investigated the relationship between AP1M2 expression and key immunological features in cancer:

• Tumor neoantigens: AP1M2 expression was positively correlated with tumor immune neoantigens in breast invasive carcinoma . This is significant because tumor neoantigens can be recognized by specific immune cells and are generated by abnormal proteins resulting from genetic mutations .

• Microsatellite instability (MSI): A positive correlation was observed between AP1M2 expression and microsatellite instability in breast invasive carcinoma . MSI is known to increase tumor mutational burden and can influence response to immunotherapy.

• Mutational burden: The relationship between AP1M2 and tumor mutational burden (TMB) was investigated using Spearman correlation analysis .

These associations suggest that AP1M2 may be involved in processes that generate tumor-specific antigens and genetic instability, which can influence anti-tumor immune responses and potentially impact the efficacy of immunotherapies.

What methodologies are used to analyze AP1M2's role in cancer at the systems level?

Several sophisticated methodologies have been employed to investigate AP1M2's role in cancer at a systems level:

• Transcriptional data analysis: Researchers have utilized multiple databases (GTEx, CCLE, TCGA) to comprehensively analyze AP1M2 expression across various tissues and cancer types .

• Differential expression analysis: Comparison of AP1M2 expression between tumor and normal tissues using the UCSC Xena database with statistical analysis in RStudio .

• Survival analysis: Kaplan-Meier survival plots and Cox proportional hazards models to evaluate the impact of AP1M2 expression on clinical prognosis .

• Immune infiltration assessment:

  • CIBERSORT algorithm to calculate the relative proportions of immune cells in tumor samples

  • ESTIMATE package to determine immune and stromal scores in the tumor microenvironment

• Gene Set Enrichment Analysis (GSEA): To identify signaling pathways and functional categories associated with AP1M2 expression in various cancer types .

These methodologies provide complementary approaches to understanding the complex role of AP1M2 in cancer biology and could serve as a template for investigating other potential cancer biomarkers.

How can researchers effectively investigate the functional significance of AP1M2 in cancer models?

To investigate the functional significance of AP1M2 in cancer, researchers should consider the following methodological approaches:

• Gene expression modulation:

  • RNA interference (siRNA or shRNA) to knockdown AP1M2 expression

  • CRISPR-Cas9 gene editing to create knockout or mutant models

  • Overexpression systems to study gain-of-function effects

• Phenotypic assays following expression modulation:

  • Cell proliferation and viability assays

  • Migration and invasion assays

  • Colony formation assays

  • Apoptosis assays

• Protein interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Proximity ligation assays to confirm interactions in situ

  • Mass spectrometry to identify protein complexes

• In vivo models:

  • Xenograft models with AP1M2-modulated cancer cell lines

  • Patient-derived xenografts (PDXs)

  • Genetically engineered mouse models (GEMMs)

• Correlation with clinical parameters:

  • Analysis of AP1M2 expression in patient samples and correlation with clinical outcomes

  • Assessment of AP1M2's relationship with treatment response

These approaches can provide mechanistic insights into AP1M2's role in cancer development and progression, potentially identifying new therapeutic targets or biomarkers.

What are the current challenges and limitations in AP1M2 antibody-based research?

Despite the potential of AP1M2 as a cancer biomarker, several challenges and limitations exist in antibody-based research:

• Antibody specificity issues:

  • Cross-reactivity with related proteins or isoforms

  • Batch-to-batch variability in polyclonal antibodies

  • Limited availability of well-characterized monoclonal antibodies

• Methodological limitations:

  • Variability in tissue fixation and processing affecting epitope accessibility

  • Differences in antibody performance across applications (WB vs. IHC vs. IF)

  • Need for standardized protocols for quantitative analysis

• Biological complexity:

  • Heterogeneous AP1M2 expression within tumors

  • Context-dependent functions of AP1M2 in different cancer types

  • Limited understanding of AP1M2's molecular interactions and signaling pathways

• Clinical translation challenges:

  • Need for validation in large, diverse patient cohorts

  • Lack of standardized cutoff values for high vs. low expression

  • Integration with existing biomarkers and clinical parameters

Addressing these challenges requires rigorous validation of antibody specificity, development of standardized protocols, and integration of antibody-based studies with complementary approaches such as transcriptomics, proteomics, and functional assays.

How might AP1M2 serve as a biomarker for cancer diagnosis or prognosis?

Based on current research, AP1M2 shows considerable potential as a biomarker in several contexts:

These applications require further validation in larger patient cohorts and integration with other clinical and molecular parameters before adoption in clinical practice.

What is the potential of AP1M2 as a therapeutic target in cancer treatment?

While research on AP1M2 as a therapeutic target is still emerging, several aspects suggest its potential:

• Correlation with tumor biology:

  • AP1M2 expression influences activation of tumor-associated pathways in multiple cancer types

  • GSEA analysis has identified specific pathways affected by AP1M2 expression levels

• Immune microenvironment modulation:

  • AP1M2 expression correlates with immune cell infiltration in tumors

  • This connection suggests potential for combination with immunotherapeutic approaches

• Association with tumor neoantigens:

  • Positive correlation between AP1M2 expression and tumor neoantigens in breast cancer

  • May indicate mechanisms by which targeting AP1M2 could enhance immune recognition of tumors

• Therapeutic approaches to consider:

  • Small molecule inhibitors targeting AP1M2 protein function

  • Antibody-drug conjugates for targeted delivery to AP1M2-expressing cells

  • RNA interference approaches to downregulate AP1M2 expression

Further research is needed to elucidate the precise mechanisms by which AP1M2 contributes to cancer development and to develop effective therapeutic strategies targeting this protein.