MEF2A Antibody

What is MEF2A and why is it an important research target?

MEF2A (myocyte enhancer factor 2A) is a transcription factor that binds specifically to the MEF2 element (5'-YTA[AT]₄TAR-3') found in numerous muscle-specific genes . It plays diverse roles in:

• Skeletal and cardiac muscle development

• Neuronal differentiation and survival

• Cell growth, survival, and apoptotic processes

• Regulation of vascular endothelial function

• Metabolic regulation, particularly in glucose transport

MEF2A is highly expressed in heart tissue, followed by skin and brain tissue . It belongs to the MEF2 family of transcription factors, which includes MEF2A, MEF2B, MEF2C, and MEF2D, with MEF2A having irreplaceable functions despite some potential redundancy among family members .

What applications are MEF2A antibodies commonly used for?

MEF2A antibodies are validated for multiple experimental applications:

Importantly, optimal dilutions should be determined empirically for each specific antibody and experimental system to obtain reliable results .

What are the key differences between monoclonal and polyclonal MEF2A antibodies?

Monoclonal MEF2A Antibodies:

• Recognize a single epitope on the MEF2A protein

• Provide high specificity and consistency between lots

• Example: Mouse monoclonal MEF2A antibody [B-4] recognizes MEF2A of mouse, rat, and human origin

• Typically generate less background but may be more sensitive to epitope masking

Polyclonal MEF2A Antibodies:

• Recognize multiple epitopes on the MEF2A protein

• Provide higher sensitivity but potentially more cross-reactivity

• Example: Rabbit polyclonal antibodies targeting specific regions (N-terminal, C-terminal, or middle regions)

• Better tolerance for minor protein denaturation/modifications

The choice between monoclonal and polyclonal should be based on specific experimental needs, with monoclonals preferred for applications requiring high specificity and polyclonals for applications needing higher sensitivity or when the epitope structure might be compromised .

How can I validate the specificity of MEF2A antibodies?

Validating antibody specificity is crucial for reliable research results. For MEF2A antibodies, consider these strategies:

• Positive and negative controls:

  • Use tissues/cells known to express MEF2A (heart, brain tissues) as positive controls

  • Use MEF2A knockout/knockdown cells as negative controls

  • Include MEF2A-transfected cells compared to non-transfected cells

• Cross-reactivity assessment:

  • Check cross-reactivity with other MEF2 family members (MEF2B, MEF2C, MEF2D)

  • Some antibodies are specifically validated to not cross-react with related family members

• Molecular weight verification:

  • Confirm the observed molecular weight matches the expected size (54-55 kDa for full-length MEF2A)

  • Multiple bands may represent isoforms (MEF2A has 8 identified isoforms)

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • Verify signal disappearance in Western blot or immunostaining

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of MEF2A

  • Consistent results with multiple antibodies increase confidence

What are the optimal sample preparation methods for detecting MEF2A in different applications?

For Western Blotting:

• Cell lysis: Use RIPA buffer containing protease inhibitors

• Nuclear extraction recommended (MEF2A is predominantly nuclear)

• Protein denaturation: Heat samples at 95°C for 5 minutes in reducing SDS buffer

• Loading: 20-50 μg of total protein per lane

• Transfer: Use PVDF membrane for optimal protein binding

For Immunohistochemistry:

• Fixation: 10% neutral buffered formalin

• Antigen retrieval: Epitope retrieval with citrate buffer pH 6.0 for FFPE tissue sections

• Alternative: TE buffer pH 9.0 for enhanced retrieval

• Blocking: 5% normal serum from the same species as the secondary antibody

• Primary antibody incubation: Overnight at 4°C for optimal signal

For Immunoprecipitation:

• Cell lysis: Use gentler lysis buffers (e.g., NP-40 buffer)

• Pre-clearing: Incubate lysate with protein A/G beads to reduce non-specific binding

• Antibody incubation: 2-5 μg antibody per 500 μg-1 mg of protein lysate

• Capture: Protein A/G magnetic beads for efficient complex isolation

• Washing: Multiple stringent washes to reduce background

How do I troubleshoot weak or absent MEF2A signals in Western blot?

When facing weak or absent MEF2A signals, consider these methodological solutions:

• Antibody concentration:

  • Increase primary antibody concentration (try 1:500 instead of 1:2000)

  • Extend incubation time (overnight at 4°C)

• Protein denaturation:

  • Ensure complete denaturation (check reducing agent freshness)

  • Consider non-reducing conditions if epitope is sensitive to reduction

• Sample preparation:

  • Enrich nuclear fraction (where MEF2A predominantly localizes)

  • Use protease inhibitors to prevent degradation

  • Check protein extraction efficiency and loading amount

• Detection system:

  • Use more sensitive detection systems (enhanced chemiluminescence)

  • Consider signal amplification methods

• Epitope masking:

  • Post-translational modifications may mask epitopes

  • Try antibodies targeting different regions of MEF2A

  • Consider phosphatase treatment if phosphorylation affects recognition

• Expression levels:

  • Confirm MEF2A expression in your specific sample

  • Consider using positive control samples (heart, brain tissue)

How can MEF2A antibodies be used to study protein-protein interactions?

MEF2A participates in complex protein interaction networks that modulate its function. Antibodies are crucial tools for studying these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-MEF2A antibodies to pull down MEF2A and its binding partners

  • Reciprocal IP can confirm interactions (e.g., STAT3-MEF2A interaction)

  • Example protocol: Use 2-5 μg antibody per 500 μg-1 mg protein lysate with protein A/G magnetic beads

• Proximity ligation assay (PLA):

  • Detect in situ protein interactions with high sensitivity

  • Combine MEF2A antibody with antibodies against suspected interacting partners

  • Particularly useful for detecting transient or weak interactions

• ChIP-seq analysis:

  • Study MEF2A interaction with chromatin and DNA binding sites

  • Identify MEF2A transcriptional targets genome-wide

  • MEF2A has been shown to associate with the ZNF16 promoter

• Pull-down assays coupled with mass spectrometry:

  • Systematic unbiased screening of MEF2A interactome

  • Research has revealed MEF2A interactions with proteins involved in cell death regulation, inflammatory responses, actin dynamics, and stress signaling

  • Example finding: "MEF2A interaction network in cardiomyocytes revealed protein networks involved in the regulation of programmed cell death, inflammatory responses, actin dynamics and stress signaling"

• Affinity purification-mass spectrometry approach:

  • Flag-tagged MEF2A can be expressed and purified from target cells

  • iTRAQ peptide labeling enables quantitative proteomics analysis

  • Validated interactions include those with RBPMS and STAT3

How do MEF2A antibodies help elucidate tissue-specific functions of MEF2A?

MEF2A exhibits tissue-specific expression patterns and functions. Antibodies enable researchers to investigate these varied roles:

• Cardiac functions:

  • Immunohistochemistry shows high MEF2A expression in heart tissue

  • MEF2A antibodies reveal altered expression in cardiomyopathies

  • MEF2A knockout studies show cardiac defects and mitochondrial abnormalities

  • Co-IP studies identified MEF2A interactions with cardiac-specific proteins

• Neuronal development and function:

  • MEF2A antibodies detect high expression in brain tissues, particularly in cerebellar granule neurons

  • Immunofluorescence reveals subcellular localization in neurons

  • MEF2A is linked to neurodegenerative diseases through promoter hypermethylation or genetic variants

  • Knockout studies using anti-MEF2A antibodies for validation show behaviors related to autism and drug addiction

• Vascular endothelial studies:

  • Antibodies detect MEF2A in vascular endothelial cells

  • MEF2A has been shown to maintain normal physiological function of vascular endothelial cells

  • Knockout studies validated with antibodies show that loss of MEF2A promotes vascular inflammation and atherosclerosis

• Skeletal muscle research:

  • Detection of MEF2A expression changes during myoblast differentiation

  • Studies show MEF2A is essential for correct differentiation of myoblasts

  • ChIP assays using MEF2A antibodies identify muscle-specific gene targets

What are the best practices for studying MEF2A phosphorylation and other post-translational modifications?

MEF2A undergoes various post-translational modifications that regulate its activity. Studying these modifications requires specialized approaches:

• Phosphorylation-specific antibodies:

  • Use phospho-specific antibodies targeting known sites (e.g., S454)

  • Apply λ-phosphatase treatment as a negative control

  • Use kinase activators/inhibitors to validate specificity

  • Example: "Magic™ Anti-MEF2A (Phospho S454) polyclonal antibody" for detecting phosphorylated MEF2A

• Sequential immunoprecipitation:

  • First IP with general MEF2A antibody

  • Second IP with modification-specific antibodies

  • Western blot analysis to determine modification levels

• 2D gel electrophoresis:

  • Separate MEF2A isoforms by charge and mass

  • Detect with MEF2A antibodies

  • Identify post-translationally modified forms

• Mass spectrometry analysis:

  • Immunoprecipitate MEF2A with validated antibodies

  • Perform MS analysis to identify all modifications

  • Quantify modification stoichiometry

• SUMOylation and ubiquitination analysis:

  • In cerebellar granule neurons, phosphorylated and SUMOylated MEF2A represses transcription of NUR77

  • Co-IP with SUMO or ubiquitin antibodies

  • Use proteasome inhibitors to accumulate ubiquitinated forms

• Functional correlation:

  • Correlate modification state with functional outcomes

  • For example, "Halting the degradation of ubiquitinated MEF2A by neurotoxins induces the characteristics of Parkinson's disease"

How can MEF2A antibodies be used to study cardiovascular diseases?

MEF2A has been implicated in various cardiovascular pathologies. Antibodies enable detailed investigations:

• Genetic variants analysis:

  • Detect altered MEF2A protein levels or truncations in patient samples

  • Some MEF2A mutations have been associated with coronary artery disease/myocardial infarction in familial studies

  • Immunoblotting can detect abnormal protein products from mutant alleles

• Expression analysis in diseased tissues:

  • Compare MEF2A expression between normal and diseased cardiac tissue

  • Quantitative analysis using immunohistochemistry or Western blotting

  • "In the diabetes model of insulin deficiency induced by streptozotocin, the expression of MEF2A in heart and skeletal muscle decreases significantly"

• Cell type-specific changes:

  • Perform dual immunofluorescence with cell type markers

  • Track MEF2A changes in specific cardiac cell populations

  • MEF2A plays roles in both cardiomyocytes and vascular endothelial cells

• Functional studies:

  • Monitor MEF2A localization and activity in response to stress

  • Use phospho-specific antibodies to track activation state

  • MEF2A is involved in cardiomyocyte survival pathways

• Drug response studies:

  • Track MEF2A expression/modification changes in response to cardiac medications

  • Example: "The protective effect of some drugs on cerebral ischemia injury was shown to depend on an increase in MEF2A activity"

What methodological approaches can be used to study MEF2A in neurodegenerative diseases?

MEF2A has been implicated in several neurodegenerative conditions. Specialized approaches include:

• Brain region-specific expression analysis:

  • Use immunohistochemistry to map MEF2A expression across brain regions

  • Compare expression patterns between normal and diseased brains

  • MEF2A is especially abundant in granule neurons of the cerebellar cortex

• Post-mortem tissue analysis:

  • Analyze MEF2A levels in post-mortem brain samples from patients with neurodegenerative diseases

  • Compare with age-matched controls

  • Hypermethylation of the MEF2A promoter or genetic variants have been linked to Alzheimer's disease

• Alternative splicing detection:

  • Design experiments to detect MEF2A splice variants

  • "Significant differences have been reported in the MEF2A mRNA splices observed in muscle tissues from patients with myotonic dystrophy (DM) and neuromuscular disorder (NMD)"

  • Verify splice variants at protein level using specific antibodies

• Disease model validation:

  • Use MEF2A antibodies to validate animal models of neurodegeneration

  • "Deletion of Mef2a can lead to behaviors related to autism and drug addiction"

  • Verify protein changes mirror human pathology

• Neuroprotective mechanisms:

  • Study MEF2A in neuroprotective pathways

  • Example finding: "Dexmedetomidine combined with netrin-1 can reduce cerebral ischemic injury by inhibiting endoplasmic reticulum stress via activation of the ERK5/MEF2A pathway"

How do researchers investigate MEF2A's dual roles in cancer progression and suppression?

MEF2A exhibits context-dependent roles in cancer, acting as either a tumor promoter or suppressor. Antibodies help elucidate these complex functions:

• Expression correlation studies:

  • Compare MEF2A levels between tumor and adjacent normal tissues

  • Correlate expression with clinical outcomes

  • "MEF2A expression is thought to promote the progression of multiple myeloma and of colorectal cancer, with higher expression correlating with poor prognosis"

• Cell line-specific functional analysis:

  • Track MEF2A expression and localization in different cancer cell lines

  • Correlate with invasive/metastatic potential

  • Example: "P38 MAPK activation in the gastric cancer cell line MKN45 up-regulates GLUT-4 in a MEF2A-dependent manner and promotes glucose uptake and cell growth"

• Co-factor dependent activity:

  • Study MEF2A interactions with different co-factors in cancer

  • "MEF2 can support oncogenic or tumor suppressive activity, depending on its interactions with co-activators or with co-suppressor chaperones"

  • Example: "In leiomyosarcoma (LMS), the expression level of class II HDACs determines whether MEF2 plays a tumor inhibitory or promoting role"

• Transcriptional target analysis:

  • Identify MEF2A targets in cancer cells using ChIP-seq

  • Example finding: "MEF2A transcriptionally activates expression of the lncRNA HCP5, thus inhibiting the progression of gastric cancer"

• Therapeutic response prediction:

  • Monitor MEF2A as a biomarker for treatment response

  • Example: "Epigalocatechin-3-gallate (EGCG) promotes the expression of KLF4 via MEF2A, thereby inhibiting the growth of gastric cancer cells"

What are the best strategies for multiplexing MEF2A detection with other proteins?

Multiplexing enables simultaneous detection of MEF2A and other proteins, providing valuable contextual information:

• Multicolor immunofluorescence:

  • Use spectrally distinct fluorophores for different targets

  • Select antibodies from different host species to avoid cross-reactivity

  • Example: Detect MEF2A (rabbit antibody) alongside tissue-specific markers (mouse antibody)

  • Useful for studying MEF2A in specific cell populations

• Sequential immunostaining:

  • Perform complete staining for one target

  • Strip or quench

  • Stain for second target

  • Particularly useful for antibodies from same species

• Multiplex Western blotting:

  • Use differently sized targets that can be distinguished on same blot

  • Apply antibodies from different species

  • Utilize different reporter systems (HRP, fluorescent secondaries)

  • Example: MEF2A (54 kDa) can be detected alongside structural proteins or signaling molecules

• Proximity ligation assay (PLA):

  • Detect MEF2A interactions with specific partners

  • Signals only appear when proteins are within 40 nm

  • Quantifiable interaction signals in situ

  • Example application: Detecting MEF2A-STAT3 interactions in cardiomyocytes

• Mass cytometry (CyTOF):

  • Label antibodies with isotopically pure metals

  • Allow highly multiplexed protein detection

  • Requires specialized equipment but enables detection of >40 proteins simultaneously

How can ChIP-seq and other genomic approaches be optimized with MEF2A antibodies?

Chromatin immunoprecipitation sequencing (ChIP-seq) with MEF2A antibodies reveals genome-wide binding patterns:

• Antibody selection for ChIP:

  • Use antibodies specifically validated for ChIP applications

  • Test multiple antibodies targeting different epitopes

  • Monoclonal antibodies often provide higher specificity

• Chromatin preparation:

  • Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes)

  • Ensure appropriate sonication to generate 200-500 bp fragments

  • Verify fragment size distribution by gel electrophoresis

• ChIP protocol optimization:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5 μg antibody per IP reaction

  • Include appropriate controls (IgG, input)

  • Validate enrichment at known MEF2A target genes

• Data analysis considerations:

  • Use appropriate peak calling algorithms

  • Identify MEF2A consensus motifs within peaks

  • Integrate with expression data to identify functional targets

  • MEF2A binds the consensus sequence 5'-YTA[AT]₄TAR-3' in numerous muscle-specific genes

• ChIP-seq integration with other approaches:

  • Combine with RNA-seq to correlate binding with gene expression

  • Integrate with ATAC-seq for chromatin accessibility analysis

  • Example application: "MEF2A associates with chromatin to the ZNF16 promoter"

What are the considerations for studying MEF2A in different experimental models?

MEF2A functions vary across species and experimental systems. Consider these methodological aspects:

• Species-specific antibody validation:

  • Confirm antibody reactivity with your species of interest

  • Available antibodies show reactivity patterns with:

    • Human MEF2A

    • Mouse MEF2A

    • Rat MEF2A

    • Drosophila Mef2

    • Zebrafish Mef2

    • Saccharomyces MEF2

• Model system considerations:

  • Cell lines: Verify endogenous MEF2A expression levels

  • Primary cells: Consider tissue-specific expression patterns

  • Animal models: Account for developmental and tissue-specific expression

  • Positive control samples include: SW480 cells, mouse skeletal muscle, heart tissue

• Knockout/knockdown validation:

  • Use MEF2A antibodies to confirm knockout/knockdown efficiency

  • Account for potential compensation by other MEF2 family members

  • "In light of the apparent functional redundancy of MEF2, an interesting question is whether other MEF2 members have high expression levels in individuals who carry loss-of-function mutations in MEF2A"

• Developmental timing:

  • Consider developmental stage-specific expression patterns

  • MEF2A plays crucial roles during embryonic development

  • "Expression of different MEF2 members overlaps in distinct patterns during embryogenesis and in adult tissues"

• Transgenic models:

  • Use antibodies to validate transgene expression

  • Distinguish between endogenous and exogenous protein

  • Example approach: Flag-tagged MEF2A expression followed by validation with both anti-Flag and anti-MEF2A antibodies