MESP1 Antibody

What is MESP1 and what is its significance in developmental biology?

MESP1 functions as a master regulator of cardiovascular development in vertebrates. It represents the earliest marker of cardiovascular progenitors, tracing almost all cells of the heart including derivatives of both primary and second heart fields . Lineage tracing studies in mice have demonstrated that MESP1 is essential for early cardiac mesoderm formation and multipotent cardiovascular progenitor (MCP) migration . In primitive chordates, MESP also controls early cardiac progenitor specification and migration, suggesting this gene arose during chordate evolution specifically to regulate cardiovascular development .

What types of MESP1 antibodies are currently available for research applications?

Currently available MESP1 antibodies include monoclonal antibodies such as Mouse Anti-Human MESP1 Monoclonal Antibody (Clone #939826, Catalog #MAB92193), which represents the newer version of a previous antibody (Clone #939816, Catalog #MAB92192) . These antibodies are generated against E. coli-derived recombinant human MESP1 (Met1-Gln85) and recognize the N-terminal region of the protein . The antibodies have been validated for Western blot applications in stem cell research contexts.

How can I validate the specificity of a MESP1 antibody in my experimental system?

Validating MESP1 antibody specificity should include multiple approaches. Based on published research, an effective validation protocol includes Western blot analysis comparing samples with known MESP1 expression levels . For instance, comparing lysates from cells with inducible MESP1 expression before and after doxycycline treatment provides an excellent control system . The antibody should detect a specific band at approximately 45 kDa in transfected cells or 30 kDa in human embryonic stem cells, with band intensity correlating with MESP1 expression levels . Additionally, performing parallel experiments with MESP1 knockdown or knockout samples can further confirm antibody specificity .

How does MESP1 binding correlate with chromatin accessibility in cardiac development?

MESP1 binding patterns show significant correlation with chromatin accessibility in cardiac progenitor cells. ChIP-seq analysis of MESP1 binding sites coupled with single-cell ATAC-seq demonstrates that MESP1 peaks overlap with 1.3% (247/18,159) of cardiomyocyte/cardiac progenitor (CM/CP)-enriched accessible regions, compared to only 0.43% (92/21,211) of lateral plate mesoderm (LPM1)-enriched peaks . Motif analysis of these MESP1-dependent regions reveals enrichment for MESP1/bHLH and GATA factor binding sites in both CM/CP and LPM1 cells . This suggests MESP1 acts as a pioneer factor to establish chromatin accessibility at critical cardiac enhancers, particularly during the earliest stages of cardiac lineage specification.

What is the relationship between MESP1 expression and cancer progression?

MESP1 expression appears significantly upregulated in non-small cell lung cancer (NSCLC) compared to normal controls at both mRNA and protein levels . Functional studies demonstrate that high MESP1 expression promotes cell proliferation, migration, and invasion in NSCLC cell models . Mechanistically, knockdown of MESP1 activates the caspase-3/PARP1 signaling pathway, suggesting that MESP1 may support cancer progression by inhibiting apoptosis . These findings indicate MESP1 could serve as both a biomarker and potential therapeutic target in NSCLC, highlighting the importance of reliable MESP1 antibodies for cancer research applications.

How can MESP1 antibodies be used to track cardiovascular lineage differentiation?

MESP1 antibodies can be powerful tools for monitoring the earliest stages of cardiovascular differentiation. In embryonic stem cell (ESC) differentiation systems, MESP1 expression marks the specification of multipotent cardiovascular progenitors that give rise to cardiomyocytes, endothelial cells, and smooth muscle cells . Temporal tracking of MESP1 expression is critical, as it is transiently expressed during development . Researchers have demonstrated that MESP1 expression accelerates and enhances cardiac differentiation approximately 5-fold, as determined by cardiac troponin T (cTnT) and α-MHC expression . For optimal results, MESP1 antibody detection should be coupled with markers of differentiated cardiovascular lineages in time-course experiments.

What is the optimal protocol for MESP1 detection by Western blot?

The optimal Western blot protocol for MESP1 detection based on published research includes:

• Sample preparation: Lyse cells in appropriate buffer systems (Immunoblot Buffer Group 3 has been successfully used)

• Electrophoresis: Perform under reducing conditions

• Transfer: Use PVDF membrane for protein transfer

• Blocking: Follow standard protocols with appropriate blocking buffer

• Primary antibody: Incubate with Mouse Anti-Human MESP1 Monoclonal Antibody at 2 μg/mL concentration

• Secondary antibody: Use HRP-conjugated Anti-Mouse IgG Secondary Antibody

• Detection: Implement standard chemiluminescence detection methods

• Expected results: A specific band at approximately 45 kDa in transfected cells or 30 kDa in human embryonic stem cells

Note that molecular weight variations may reflect post-translational modifications or different MESP1 isoforms across cell types.

How can MESP1 ChIP-seq experiments be optimized for identifying direct target genes?

For optimal MESP1 ChIP-seq experiments, consider the following methodological approaches:

• Timing: Since MESP1 expression is transient during development, capturing the appropriate time points is critical. Studies have examined both early (12h) and late (24h) MESP1 binding events after induction

• Controls: Include appropriate input controls and IgG immunoprecipitation controls

• Antibody selection: Validate ChIP-grade MESP1 antibodies with preliminary ChIP-qPCR against known targets (e.g., GATA4)

• Crosslinking: Standard formaldehyde crosslinking protocols are effective for MESP1

• Data analysis: Integrate with other genomic datasets (RNA-seq, ATAC-seq) to correlate binding with gene expression and chromatin accessibility

• Motif analysis: Examine enriched motifs in MESP1-bound regions, which typically include MESP1/bHLH, ZIC, and GATA binding sites

Published studies have demonstrated that MESP1 binds directly upstream of cardiac regulators like GATA4, while some genes previously thought to be direct targets (e.g., NKX2-5) lack MESP1 binding in certain contexts .

What experimental systems best support functional studies of MESP1?

The most effective experimental systems for MESP1 functional studies include:

These systems have demonstrated that MESP1 promotes the differentiation of primary and second heart field derivatives, with approximately two-thirds of differentiated cells representing cardiovascular lineages following MESP1 expression in ESC models .

How can researchers address non-specific binding when using MESP1 antibodies?

When encountering non-specific binding with MESP1 antibodies, implement these troubleshooting strategies:

• Antibody titration: Optimize antibody concentration based on your specific sample type (2 μg/mL has been successfully used for Western blot)

• Blocking optimization: Test different blocking agents and increase blocking time

• Washing stringency: Increase wash steps and consider higher salt concentrations in wash buffers

• Antibody validation: Confirm specificity using positive controls (doxycycline-induced MESP1 expression) and negative controls (untreated cells)

• Clone selection: Different antibody clones may have different specificities; compare results between clone #939826 (MAB92193) and clone #939816 (MAB92192)

• Secondary antibody: Verify secondary antibody specificity and consider alternatives if cross-reactivity is suspected

What factors affect MESP1 detection in different developmental contexts?

Several factors can impact successful MESP1 detection across developmental contexts:

• Temporal expression: MESP1 is transiently expressed during early mesoderm specification, requiring precise timing for detection

• Protein modification: MESP1 may undergo post-translational modifications that affect antibody recognition, as evidenced by different apparent molecular weights across cell types (30 kDa vs. 45 kDa)

• Expression level: MESP1 expression may be low in some contexts, requiring sensitive detection methods

• Tissue fixation: For immunohistochemistry applications, fixation conditions must be optimized to preserve MESP1 epitopes

• Developmental stage: MESP1 expression patterns change dramatically across development, with early expression in cardiac progenitors and later expression in presomitic mesoderm

How can single-cell approaches be combined with MESP1 antibodies for heterogeneous populations?

For analyzing MESP1 expression in heterogeneous populations, consider these methodological approaches:

• Flow cytometry: Use fluorescently-labeled MESP1 antibodies in combination with other lineage markers to identify and isolate specific cell populations

• Mass cytometry: For higher-dimensional analysis, incorporate MESP1 antibodies into CyTOF panels for simultaneous detection of multiple markers

• Fluorescence-activated cell sorting (FACS): Isolate MESP1-positive cells for downstream analysis

• Single-cell RNA-seq integration: Correlate protein-level MESP1 detection with transcriptional profiles

• Imaging mass cytometry: For spatial context, use MESP1 antibodies in multiplexed tissue imaging

• Index sorting: Record single-cell MESP1 antibody signal intensity before sorting for clonal analysis

How might MESP1 antibodies contribute to regenerative medicine approaches?

MESP1 antibodies have significant potential in regenerative medicine applications by enabling:

• Identification and isolation of early cardiovascular progenitors for therapeutic transplantation

• Quality control assessment of cardiomyocyte differentiation protocols

• Monitoring the differentiation trajectories of pluripotent stem cells towards cardiovascular lineages

• Validation of direct reprogramming approaches targeting cardiac regeneration

• Development of engineered cardiac tissues with appropriate cellular composition

Since MESP1 expression massively promotes cardiovascular differentiation during embryonic development and pluripotent stem cell differentiation, antibodies against this marker can help standardize and optimize protocols for generating cardiovascular cells for cellular therapy, drug discovery, and toxicity screening .

What are the implications of MESP1 expression in cancer for therapeutic development?

The high expression of MESP1 in NSCLC and its correlation with increased cell proliferation, metastasis, and inhibition of apoptosis suggest several therapeutic implications:

• Diagnostic biomarker: MESP1 antibodies could help identify high-risk tumors with aggressive characteristics

• Therapeutic target: Strategies to inhibit MESP1 function may reduce tumor growth and metastasis

• Resistance mechanisms: MESP1 may contribute to treatment resistance by inhibiting apoptosis pathways

• Patient stratification: MESP1 expression levels could help identify patients likely to respond to specific treatments

• Monitoring: MESP1 antibodies could track treatment response and disease progression

Research has demonstrated that knocking down MESP1 activates the caspase-3/PARP1 signaling pathway, suggesting potential therapeutic approaches targeting this molecular mechanism .