LEMD2 Antibody

What is LEMD2 and why is it important to study?

LEMD2 is a nuclear envelope protein with a reported length of 503 amino acid residues and a mass of 57 kDa in humans. It contains the characteristic LEM (LAP2, Emerin, MAN1) domain and localizes primarily to the nuclear envelope, with additional presence in the cytoplasm. LEMD2 plays critical roles in skeletal muscle cell differentiation, genome stability, and cardiac function . Studying LEMD2 is important because mutations in this protein are associated with severe cardiomyopathy and other envelopathies—devastating genetic diseases that primarily affect heart and skeletal muscle tissues . Additionally, the LEMD2 gene has been linked to cataracts, expanding its clinical significance beyond muscle-related disorders .

What are the common applications for LEMD2 antibodies in research?

LEMD2 antibodies are utilized across multiple experimental techniques:

These applications have established LEMD2's expression patterns across various tissues and facilitated investigation of its roles in nuclear envelope integrity and cellular function .

How specific are commercial LEMD2 antibodies, and how should specificity be validated?

Commercial LEMD2 antibodies target either the N-terminus or C-terminus of the protein, each with distinct specificity profiles. Proper validation is essential and should include:

• Positive controls: Using cells/tissues known to express LEMD2 (widely expressed across tissue types)

• Negative controls: Using knockdown/knockout models or tissues with naturally low expression

• Transfection validation: Overexpressing tagged LEMD2 in cell lines like HeLa to confirm antibody recognition

• Western blot analysis: Confirming the detected protein is of the expected molecular weight (57 kDa)

• Cross-reactivity assessment: Testing against homologous proteins, particularly other LEM domain-containing proteins

When selecting antibodies, researchers should note that N-terminal specific antibodies (like HPA017340 Sigma, dilution 1:100) and C-terminal specific antibodies (like ab89866 Abcam, dilution 1:50) may yield different labeling patterns depending on experimental context and potential protein isoforms .

How can LEMD2 antibodies be used to investigate nuclear envelope pathologies in cardiomyopathy models?

LEMD2 antibodies are instrumental in investigating nuclear envelope abnormalities associated with cardiomyopathies through multi-level approaches:

• Immunohistochemical analysis: LEMD2 antibodies can reveal nuclear envelope morphological changes in cardiac tissue sections from disease models. In knockin mice carrying the human c.T38>G Lemd2 mutation, immunolabeling shows disorganized heterochromatin patterns at the nuclear periphery of cardiomyocytes .

• Co-localization studies: Combining LEMD2 antibodies with markers for heterochromatin (H3K9me3), DNA damage (γH2AX), or other nuclear envelope proteins (Emerin, Lamin A/C) helps characterize the molecular basis of envelope disruption.

• Temporal expression analysis: Following LEMD2 expression during disease progression using quantitative immunoblotting with standardized loading controls.

• Gene therapy validation: Using LEMD2 antibodies to confirm successful protein restoration following therapeutic interventions, such as adeno-associated virus-mediated gene therapy that has shown rescue of cardiac function in mouse models .

When designing these experiments, researchers should select antibodies validated for the specific species being studied, as LEMD2 orthologs have been confirmed in mouse, rat, bovine, chimpanzee, and chicken models .

What experimental considerations are important when investigating LEMD2 post-translational modifications?

LEMD2 undergoes phosphorylation and potentially other post-translational modifications that may regulate its function . When investigating these modifications:

• Phospho-specific antibodies: Consider using phospho-specific antibodies if available, or combine general LEMD2 antibodies with phosphorylation detection methods.

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation states

  • For mass spectrometry analysis, enrich for phosphopeptides using titanium dioxide or IMAC

  • Consider comparing samples treated with phosphatases versus untreated controls

• 2D gel electrophoresis: This technique can separate LEMD2 isoforms with different phosphorylation states before western blotting.

• Kinase prediction and validation: Use bioinformatic tools to predict potential kinases for LEMD2, then validate with kinase inhibitors or knockdowns followed by immunodetection.

• Functional correlation: Design experiments that correlate phosphorylation status with functional outcomes, such as protein-protein interactions, localization changes, or stability alterations.

When interpreting results, be aware that phosphorylation patterns may differ between cell types and physiological or pathological states.

How can chromatin association of LEMD2 be analyzed using antibody-based techniques?

LEMD2's role in organizing heterochromatin at the nuclear envelope can be investigated through several antibody-dependent approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use LEMD2 antibodies to pull down protein-DNA complexes

  • Analyze associated DNA sequences through qPCR or sequencing

  • Focus on regions known to associate with the nuclear lamina

• Proximity Ligation Assay (PLA):

  • Combine LEMD2 antibodies with antibodies against heterochromatin markers

  • Visualize and quantify protein-protein interactions within 40nm distance

  • Particularly useful for analyzing LEMD2's association with specific chromatin regulators

• ChIP-re-ChIP:

  • Sequential immunoprecipitation with LEMD2 antibodies followed by antibodies against chromatin proteins

  • Identifies genomic regions where both proteins co-occupy

• Immuno-FISH:

  • Combine LEMD2 immunofluorescence with fluorescence in situ hybridization

  • Directly visualize LEMD2's spatial relationship with specific genomic loci

• DamID-based approaches:

  • Create LEMD2-Dam methyltransferase fusion proteins

  • Identify DNA sequences proximal to LEMD2 through methylation mapping

  • Validate findings with LEMD2 antibody-based ChIP

These approaches have revealed that LEMD2 particularly associates with transcriptionally silent heterochromatin regions, which become disorganized in cardiomyocytes from disease models carrying LEMD2 mutations .

What fixation and permeabilization methods provide optimal results for LEMD2 immunofluorescence studies?

Optimal detection of LEMD2 at the nuclear envelope requires careful attention to fixation and permeabilization procedures:

Permeabilization recommendations:

• Triton X-100 (0.1-0.5%) for 5-10 minutes works well for most nuclear envelope proteins

• Digitonin (50-100 μg/mL) provides gentler permeabilization that better preserves nuclear envelope structure

• NP-40 (0.1-0.3%) offers an alternative when Triton X-100 results are suboptimal

For optimal results, researchers should perform a fixation/permeabilization matrix experiment to determine ideal conditions for their specific antibody and cell type. When using rabbit anti-LEMD2 antibodies (such as HPA017340 Sigma or ab89866 Abcam), paraformaldehyde fixation followed by Triton X-100 permeabilization has yielded successful nuclear envelope labeling in both somatic cells and germ cells in disaggregated testes .

How should researchers optimize western blot protocols for LEMD2 detection?

LEMD2 western blotting requires specific optimization due to its membrane association and relatively moderate expression levels:

• Sample preparation:

  • Nuclear fraction enrichment improves detection sensitivity

  • Include protease inhibitors to prevent degradation

  • For membrane proteins, avoid boiling samples (heat at 70°C for 10 minutes instead)

• Gel selection and transfer:

  • 10% SDS-PAGE gels typically provide good resolution for the 57 kDa LEMD2 protein

  • Semi-dry transfer at lower voltage (10-15V) for longer duration (45-60 min) improves transfer efficiency

  • PVDF membranes may provide better results than nitrocellulose for nuclear envelope proteins

• Blocking and antibody incubation:

  • 5% non-fat dry milk in TBST is generally effective

  • For phospho-specific detection, use 5% BSA instead

  • Primary antibody incubation at 4°C overnight at dilutions between 1:500-1:2000

  • Consider using signal enhancers for low abundance samples

• Controls and validation:

  • Include positive control lysates from cells with known LEMD2 expression

  • Consider running a LEMD2 knockdown/knockout sample as negative control

  • Verify antibody specificity with peptide competition assays

Western blot is the most widely used application for LEMD2 antibodies, making these optimization steps particularly important for generating reliable, reproducible results .

What are the best approaches for dual immunolabeling with LEMD2 and other nuclear envelope proteins?

Studying LEMD2 in relation to other nuclear envelope components requires careful planning of dual immunolabeling experiments:

• Antibody species selection:

  • Choose primary antibodies raised in different species (e.g., rabbit anti-LEMD2 with mouse anti-Emerin)

  • If using same-species antibodies, consider directly conjugated antibodies or sequential immunostaining protocols

• Cross-reactivity prevention:

  • Test each primary and secondary antibody individually before combining

  • Include controls omitting each primary antibody to verify secondary antibody specificity

  • Use highly cross-adsorbed secondary antibodies to minimize non-specific binding

• Sequential staining protocol:

  • First round: Incubate with first primary antibody, wash, apply first secondary antibody, wash

  • Blocking step: Apply additional blocking with serum matching second primary antibody's species

  • Second round: Incubate with second primary, wash, apply second secondary, wash

• Special considerations for nuclear envelope proteins:

  • Some epitopes may be masked by protein-protein interactions at the nuclear envelope

  • Consider mild extraction methods to expose hidden epitopes (brief Triton X-100 treatment before fixation)

  • Optimize fixation conditions that work compatibly for both target proteins

Successfully combining rabbit anti-LEMD2 (specific to N-terminus, dilution 1:100, HPA017340 Sigma) with antibodies against other nuclear envelope components like Emerin (mouse anti-EMERIN, dilution 1:100, NCL-EMERIN Leica) has been demonstrated in previous studies examining nuclear envelope protein distribution .

How can researchers resolve weak or absent LEMD2 immunofluorescence signal?

When LEMD2 antibodies yield weak or no signal in immunofluorescence experiments, consider these systematic troubleshooting approaches:

• Epitope accessibility issues:

  • Try alternative fixation methods (switch from PFA to methanol or vice versa)

  • Implement antigen retrieval techniques (citrate buffer pH 6.0, heat-mediated)

  • Test mild extraction with 0.1% Triton X-100 before fixation to expose nuclear envelope epitopes

• Antibody-specific considerations:

  • Increase antibody concentration (try a dilution series: 1:50, 1:100, 1:200)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Switch between N-terminal and C-terminal targeting antibodies

  • Verify antibody activity with a simple dot blot test

• Signal amplification options:

  • Implement tyramide signal amplification (TSA)

  • Use biotin-streptavidin amplification systems

  • Consider brighter fluorophores or higher-sensitivity detection systems

• Expression verification:

  • Confirm LEMD2 expression in your sample via RT-PCR or western blot

  • Some cell types or developmental stages may have naturally low LEMD2 expression

  • Check if experimental conditions might downregulate LEMD2 expression

If basic troubleshooting fails, consider testing antibodies from different suppliers or epitope regions, as rabbit anti-LEMD2 specific to N-terminus (HPA017340 Sigma) and rabbit anti-LEMD2 specific to C-terminus (ab89866 Abcam) may perform differently in certain experimental systems .

What are common sources of non-specific binding with LEMD2 antibodies and how can they be minimized?

Non-specific binding can significantly impact LEMD2 antibody experiments. Here are common sources and mitigation strategies:

• Cross-reactivity with other LEM-domain proteins:

  • LEMD2 shares structural similarities with other LEM-domain proteins (LEMD1, LEMD3, Emerin)

  • Validate specificity using knockout/knockdown controls

  • Consider peptide competition assays to confirm binding specificity

  • Use antibodies raised against unique regions of LEMD2

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to blocking solution for better penetration

• Secondary antibody issues:

  • Use highly cross-adsorbed secondary antibodies

  • Include controls with secondary antibody only

  • Consider directly conjugated primary antibodies to eliminate secondary antibody problems

• Sample-specific background:

  • Some tissues (especially muscle, brain) may have inherent autofluorescence

  • Pretreat sections with Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence

  • Use confocal microscopy with spectral unmixing for challenging samples

• Antibody quality control:

  • Store antibodies according to manufacturer recommendations

  • Avoid repeated freeze-thaw cycles

  • Centrifuge antibody solutions before use to remove aggregates

  • Consider adding 0.05% sodium azide for long-term storage

Carefully optimized protocols have demonstrated specific nuclear envelope labeling in both somatic cells and various cell types in disaggregated testes using validated LEMD2 antibodies .

How can researchers interpret conflicting results when using different LEMD2 antibodies?

When different LEMD2 antibodies yield conflicting results, systematic investigation is necessary:

• Epitope mapping analysis:

  • Document exactly which region each antibody targets

  • N-terminal antibodies (like HPA017340 Sigma) versus C-terminal antibodies (like ab89866 Abcam) may detect different isoforms or modified versions

  • Consider if post-translational modifications might mask specific epitopes

• Isoform-specific detection:

  • LEMD2 has two reported isoforms

  • Design RT-PCR experiments to determine which isoforms are expressed in your system

  • Select antibodies that can distinguish between isoforms if relevant to your research question

• Validation with orthogonal techniques:

  • Confirm findings with non-antibody methods (fluorescent protein tagging, RNA-level detection)

  • Use mass spectrometry to definitively identify protein presence

  • Implement CRISPR-Cas9 tagging of endogenous LEMD2 for verification

• Experimental condition variations:

  • Document all experimental variables (fixation, permeabilization, blocking, incubation times)

  • Standardize protocols when comparing different antibodies

  • Consider if different antibodies require different optimal conditions

• Antibody validation status:

  • Review literature citations for each antibody

  • Check manufacturer validation data

  • Consider additional validation experiments (overexpression, knockdown)

When interpreting conflicting results, remember that apparently contradictory findings may represent biologically meaningful differences in protein conformation, complex formation, or subcellular localization rather than technical artifacts.

How have LEMD2 antibodies contributed to understanding cardiomyopathy mechanisms?

LEMD2 antibodies have been instrumental in elucidating the molecular mechanisms underlying LEMD2-associated cardiomyopathy:

• Structural abnormalities visualization:

  • Immunofluorescence with LEMD2 antibodies revealed nuclear envelope deformations in cardiomyocytes lacking LEMD2

  • These studies demonstrated disorganization of heterochromatin associated with the nuclear envelope in knockin mice carrying the human c.T38>G Lemd2 mutation (p.L13>R in the LEM domain)

• Pathway identification:

  • Co-immunostaining with LEMD2 and p53 pathway component antibodies uncovered a mechanistic link between LEMD2 deficiency and p53-mediated apoptosis

  • This approach identified extensive DNA damage in cardiomyocytes lacking functional LEMD2

• Therapeutic intervention validation:

  • LEMD2 antibodies confirmed successful protein expression following gene therapy approaches

  • Immunodetection verified cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus, which successfully rescued cardiac function in mouse models

• Disease progression monitoring:

  • Quantitative immunoblotting with LEMD2 antibodies tracked protein expression changes during disease development

  • This approach helps establish temporal relationships between LEMD2 loss and onset of pathological changes

These findings collectively demonstrate that LEMD2 is essential for genome stability and cardiac function, providing critical insights into the pathomechanisms of nuclear envelopathies affecting cardiac tissue .

What are the emerging applications of LEMD2 antibodies in developmental biology research?

LEMD2 antibodies are increasingly being utilized to investigate developmental processes:

• Muscle differentiation studies:

  • LEMD2 is involved in skeletal muscle cell differentiation

  • Antibody labeling during myoblast differentiation reveals dynamic changes in LEMD2 expression and localization

  • Co-labeling with differentiation markers helps establish temporal relationships between LEMD2 function and muscle development

• Nuclear envelope assembly dynamics:

  • LEMD2 antibodies enable tracking of nuclear envelope reformation during mitosis

  • Time-course immunofluorescence studies reveal the sequence of nuclear envelope protein recruitment

  • Super-resolution microscopy with LEMD2 antibodies provides nanoscale insights into envelope assembly

• Tissue-specific expression patterns:

  • Immunohistochemistry across developmental stages reveals tissue-specific LEMD2 expression patterns

  • Particularly important for understanding why mutations cause tissue-specific pathologies despite broad expression

• Cross-species developmental comparisons:

  • LEMD2 orthologs have been identified in mouse, rat, bovine, chimpanzee, and chicken

  • Comparative studies using species-appropriate antibodies reveal evolutionarily conserved functions

  • Helps distinguish fundamental roles from species-specific adaptations

• Stem cell differentiation:

  • LEMD2 antibodies help track nuclear envelope remodeling during stem cell differentiation

  • May provide insights into nuclear reprogramming mechanisms during development

These applications highlight LEMD2's broader roles beyond pathology, establishing its importance in normal development and cell differentiation processes.

How can researchers best implement LEMD2 antibodies in genome organization studies?

LEMD2's role in organizing chromatin at the nuclear periphery makes it valuable for genome architecture research:

These approaches have demonstrated that cardiomyocytes from LEMD2 mutant models exhibit disorganization of transcriptionally silent heterochromatin associated with the nuclear envelope, providing mechanistic insights into disease pathology .