LIG4 Antibody

What is the biological function of LIG4 and why is it important in research?

LIG4 is a DNA ligase involved in DNA non-homologous end joining (NHEJ) that is essential for double-strand break (DSB) repair and V(D)J recombination. It catalyzes the final ligation step during DSB repair by resealing DNA breaks after gap filling is completed . LIG4 functions by joining single-strand breaks in double-stranded DNA through an ATP-dependent reaction . This protein is critically important in research because it represents a key component of the cellular DNA damage response machinery. LIG4 deficiency has been linked to immunodeficiency, developmental abnormalities, and increased cancer susceptibility, making it a valuable target for studies in genomic stability, DNA repair mechanisms, and related pathologies .

What are the key characteristics of the LIG4 protein that researchers should be aware of?

The human LIG4 protein has several important characteristics that researchers should consider when designing experiments:

• Full protein length: 911 amino acid residues

• Calculated molecular weight: 104 kDa

• Observed molecular weight in experiments: 100-104 kDa

• Subcellular localization: Primarily nuclear

• Protein family: Member of ATP-dependent DNA ligase family

• Gene ID (NCBI): 3981

• UniProt ID: P49917

The protein forms a functionally important subcomplex with XRCC4, which enhances its joining activity. This LIG4-XRCC4 complex is responsible for the NHEJ ligation step . Notably, LIG4 demonstrates mechanistic flexibility, as it can ligate nicks and compatible DNA overhangs alone, while in the presence of XRCC4, it can ligate ends with 2-nucleotide microhomology and 1-nucleotide gaps .

What experimental applications are LIG4 antibodies suitable for?

LIG4 antibodies have been validated for several standard experimental techniques:

LIG4 antibodies have been successfully used with various human samples and cell lines, including PC-3, HeLa, HepG2, Jurkat, and Ramos cells, as well as human testis tissue . For optimal results, researchers should titrate antibodies in their specific testing systems, as experimental conditions can significantly impact performance .

How should LIG4 antibodies be stored and handled to maintain reactivity?

For maximum stability and performance, LIG4 antibodies should be:

• Stored at -20°C, where they typically remain stable for one year after shipment

• Maintained in appropriate storage buffer (commonly PBS with 0.02% sodium azide and 50% glycerol at pH 7.3)

• Aliquoted for antibodies not containing glycerol to prevent repeated freeze-thaw cycles

• Brought to room temperature before opening to prevent condensation that could impact antibody stability

Some commercial antibodies may contain small amounts of BSA (0.1%) in smaller size formats, which should be considered when designing experiments sensitive to bovine proteins .

How can I validate LIG4 antibody specificity for my experimental system?

Validating antibody specificity is crucial for generating reliable data. For LIG4 antibodies, consider implementing these validation strategies:

• Positive and negative control samples: Use cell lines known to express LIG4 (like HeLa, HepG2) as positive controls, and consider using LIG4-knockout or siRNA-mediated knockdown samples as negative controls.

• Molecular weight verification: Confirm that the observed band in Western blot corresponds to the expected molecular weight of LIG4 (100-104 kDa).

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding, which should eliminate or significantly reduce the specific signal.

• Multiple antibody validation: Use antibodies from different sources or those recognizing different epitopes of LIG4 to confirm findings.

• Genetic models: Leverage LIG4-deficient patient samples or engineered cell lines with known mutations (like the p.R278L and p.R278H mutations studied in expression systems ) to verify antibody specificity.

• Subcellular localization: Confirm that the staining pattern in IF experiments is consistent with the known nuclear localization of LIG4 .

The gold standard validation would include detection in samples with normal LIG4 expression versus those with genetically verified LIG4 deficiency or knockdown.

What are the key considerations when optimizing immunofluorescence protocols for LIG4 detection?

Optimizing immunofluorescence protocols for LIG4 requires attention to several critical factors:

• Fixation method: LIG4 is a nuclear protein involved in DNA repair. For optimal detection, 4% paraformaldehyde fixation for 15-20 minutes followed by permeabilization with 0.2-0.5% Triton X-100 is often effective.

• Antigen retrieval: For some tissues, especially in IHC-P, heat-induced epitope retrieval may be necessary to expose the epitope. Citrate buffer (pH 6.0) is commonly used.

• Background reduction: Use appropriate blocking (3-5% BSA or serum from the species of the secondary antibody) for at least 1 hour at room temperature.

• Antibody titration: Start with the manufacturer's recommended dilution range (typically 1:50-1:500 for IF/ICC) and optimize based on signal-to-noise ratio .

• Counterstains: Include DAPI to identify nuclei, as LIG4 should show nuclear localization. Consider co-staining with other proteins in the NHEJ pathway like XRCC4 to confirm functional localization.

• Positive controls: HepG2 cells have been validated for positive LIG4 immunoreactivity in IF experiments .

• Confocal imaging: Due to the nuclear localization and involvement in repair foci, confocal microscopy may provide better resolution of LIG4 distribution patterns than standard fluorescence microscopy.

How can I use LIG4 antibodies to study DNA damage response pathways?

LIG4 antibodies can be powerful tools for investigating DNA damage response pathways through several strategic approaches:

• DNA damage induction and repair kinetics: Treat cells with DNA-damaging agents (ionizing radiation, etoposide, or bleomycin) and use LIG4 antibodies to track the recruitment of LIG4 to damage sites at different time points post-treatment.

• Co-localization studies: Perform dual immunofluorescence with LIG4 antibodies and other NHEJ components (DNA-PKcs, Ku70/80, XRCC4, XLF) to analyze their spatial and temporal relationships during repair.

• Chromatin immunoprecipitation (ChIP): Use LIG4 antibodies to identify genomic regions where LIG4 is recruited following DNA damage or during V(D)J recombination.

• Proximity ligation assay (PLA): Apply PLA using LIG4 antibodies and antibodies against interaction partners like XRCC4 to visualize and quantify protein-protein interactions in situ at damage sites.

• Immunoprecipitation (IP): Use LIG4 antibodies to pull down LIG4 complexes and identify associated proteins through mass spectrometry or Western blotting.

• Cell cycle analysis: Combine LIG4 immunostaining with cell cycle markers to examine how LIG4 activity and localization change throughout different cell cycle phases.

• Functional compensation: In LIG4-deficient or mutant backgrounds, examine how other DNA repair pathways (homologous recombination) may be upregulated as compensatory mechanisms.

What are the common pitfalls when interpreting LIG4 expression data from patient samples?

Interpreting LIG4 expression data from patient samples requires careful consideration of several potential confounding factors:

• Heterozygous vs. homozygous mutations: Monoallelic (heterozygous) LIG4 mutations may cause human immune dysregulation via haploinsufficiency , while biallelic mutations cause more severe LIG4 syndrome. Antibody detection may not distinguish between partial and complete loss of function.

• Post-translational modifications: DNA damage-induced modifications of LIG4 may affect antibody recognition, potentially leading to false-negative results.

• Tissue-specific expression: LIG4 expression varies across tissues, with particularly critical roles in developing neurons and immune cells . Reference ranges should be tissue-specific.

• Protein stability vs. functionality: Some pathogenic mutations affect LIG4 activity without altering protein stability or expression levels. Western blot data alone may not reflect functional deficits.

• Copy number variations: As seen in patient P13 from the referenced study, copy number variations in the LIG4 gene may complicate expression analysis . Combining antibody-based detection with genetic analysis is recommended.

• Technical variability: Fixation methods, sample processing, and antibody batches can introduce variability. Use standardized protocols and include positive and negative controls in each experiment.

• Alternative splice variants: Consider the possibility of alternative LIG4 isoforms that may or may not be recognized by your antibody of choice.

What are the optimal protocols for detecting LIG4 via Western blotting?

For optimal detection of LIG4 via Western blotting, consider the following protocol recommendations:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying DNA damage response

  • Sonicate briefly to ensure complete nuclear protein extraction

• Gel selection:

  • Use 6-8% SDS-PAGE gels to properly resolve the 100-104 kDa LIG4 protein

  • Consider gradient gels (4-12%) when analyzing LIG4 along with its interaction partners

• Transfer conditions:

  • Transfer to PVDF membrane at 100V for 90-120 minutes or overnight at 30V (4°C)

  • Use transfer buffer containing 10-20% methanol

• Blocking:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • For phospho-specific detection, 5% BSA in TBST is preferred

• Antibody incubation:

  • Primary antibody: Dilute LIG4 antibody 1:1000-1:4000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Secondary antibody: HRP-conjugated anti-rabbit or anti-mouse (depending on the primary antibody host) at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • For low expression samples, consider more sensitive ECL substrates or longer exposure times

• Controls and normalization:

  • Include loading controls (β-actin, GAPDH, or nuclear proteins like Lamin B1)

  • Consider positive controls from cell lines with known LIG4 expression (HeLa, HepG2)

• Troubleshooting tips:

  • If signal is weak: Increase antibody concentration, extend incubation time, or use signal enhancement systems

  • If background is high: Increase washing steps, decrease antibody concentration, or try alternative blocking agents

How can I distinguish between wild-type and mutant LIG4 using antibody-based approaches?

Distinguishing between wild-type and mutant LIG4 using antibody-based approaches presents challenges but can be achieved through several strategies:

• Mutation-specific antibodies: For common LIG4 mutations, consider developing or sourcing mutation-specific antibodies that selectively recognize the mutant protein. This approach is particularly useful for mutations that cause significant conformational changes.

• Expression system comparison: As demonstrated in research with p.R278L and p.R278H mutations, overexpression systems using HEK293T cells transfected with wild-type and mutant LIG4 cDNAs in a tagged vector (like p3xFlag-CMV-7.1) can provide comparative expression data . Western blot with either anti-LIG4 or anti-tag antibodies allows direct comparison.

• Functional readouts: Combine antibody detection with functional assays that can distinguish between wild-type and mutant LIG4 activity. For example:

  • Immunofluorescence to analyze formation of repair foci after DNA damage

  • Co-immunoprecipitation to assess interaction with XRCC4 or other partners

  • Proximity ligation assays to quantify protein-protein interactions

• Size-based differentiation: Some mutations may result in truncated proteins that can be distinguished by molecular weight in Western blots.

• Subcellular localization: Certain mutations may affect nuclear localization, which can be visualized by immunofluorescence microscopy.

• Quantitative approaches: Combine antibody-based detection with quantitative PCR to correlate protein levels with gene expression.

• Reconstitution experiments: As referenced in the LIG4 knockout Jurkat T cell reconstitution experiments, express wild-type versus mutant LIG4 in a null background to assess functional consequences .

The choice of approach depends on the specific mutation being studied and the available resources.

What controls should be included when conducting immunohistochemistry with LIG4 antibodies?

For rigorous immunohistochemistry experiments with LIG4 antibodies, include the following controls:

• Positive tissue controls:

  • Human testis tissue has been validated for positive LIG4 expression

  • Tissues with high rates of V(D)J recombination (thymus, tonsils, lymph nodes)

  • Embryonic or developing neural tissues (given LIG4's role in neuronal development)

• Negative controls:

  • Isotype control: Use the same species and isotype as the primary antibody (e.g., Mouse IgG2a for 66705-1-Ig) at the same concentration

  • No primary antibody control: Perform the staining protocol omitting only the primary antibody

  • Peptide competition: Pre-incubate primary antibody with the immunizing peptide

• Genetic controls (gold standard):

  • Tissues from LIG4-deficient patients or animal models (when available)

  • Tissues with siRNA-mediated LIG4 knockdown

  • CRISPR/Cas9-engineered cell lines with LIG4 knockout

• Technical controls:

  • Dilution series of primary antibody to establish optimal signal-to-noise ratio

  • Different antigen retrieval methods to optimize epitope exposure

  • Multiple chromogenic or fluorescent detection systems to confirm specificity

• Validation controls:

  • Sequential tissue sections stained with different LIG4 antibodies recognizing different epitopes

  • Correlation with mRNA expression (RNA in situ hybridization or RT-PCR from microdissected regions)

• Counterstains:

  • Nuclear counterstain (hematoxylin or DAPI) to confirm nuclear localization of LIG4

  • Cell type-specific markers to identify which cells express LIG4 within heterogeneous tissues

These comprehensive controls ensure the reliability and specificity of LIG4 detection in tissue samples.

How should I troubleshoot non-specific bands in Western blots using LIG4 antibodies?

Non-specific bands in LIG4 Western blots can complicate data interpretation. Follow this systematic troubleshooting approach:

• Antibody validation and selection:

  • Verify that the antibody has been validated for Western blot applications

  • Consider using recombinant monoclonal antibodies (like EPR16531 ) which typically show higher specificity than polyclonal antibodies

  • Review literature or manufacturer data for reported non-specific bands

• Optimizing blocking conditions:

  • Test different blocking agents (5% milk, 5% BSA, commercial blockers)

  • Extend blocking time to 2 hours or overnight at 4°C

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Antibody dilution and incubation:

  • Increase antibody dilution (try 1:4000 if currently using 1:1000)

  • Reduce incubation time or temperature

  • Dilute antibody in the same buffer used for blocking

• Washing optimization:

  • Increase number and duration of wash steps

  • Use higher concentrations of Tween-20 in wash buffer (0.1-0.5%)

  • Consider adding low salt concentration (50mM NaCl) to wash buffer

• Sample preparation improvements:

  • Use fresh protease inhibitors

  • Optimize lysis conditions for nuclear proteins

  • Centrifuge lysates at high speed to remove insoluble material

  • Consider nuclear/cytoplasmic fractionation to enrich for LIG4

• Band identification strategies:

  • Compare with positive control samples (HeLa, HepG2 cells)

  • Analyze molecular weight carefully - LIG4 should appear at 100-104 kDa

  • Run a LIG4-depleted sample (siRNA knockdown) alongside as negative control

  • Consider peptide competition to identify specific bands

• Technical modifications:

  • Use freshly prepared buffers

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Reduce transfer time or voltage if high MW proteins are concerning

If a particular non-specific band persists, document its molecular weight and pattern across samples, as this information can help distinguish it from the specific LIG4 signal in future experiments.

How can LIG4 antibodies be used to study its role in neuronal development and related disorders?

LIG4 antibodies can provide valuable insights into neuronal development and related disorders through several strategic research approaches:

• Developmental expression profiling:

  • Use LIG4 antibodies for immunohistochemistry on brain tissue sections across developmental stages

  • Quantify LIG4 expression levels in different brain regions during neurogenesis

  • Correlate with neuronal differentiation markers to establish temporal relationships

• Microcephaly models:

  • Given the association between LIG4 deficiency and microcephaly , use antibodies to study LIG4 expression in:

    • Patient-derived brain organoids

    • Animal models of microcephaly

    • Neural progenitor cells with LIG4 mutations

• DNA damage response in neurons:

  • Study neuronal response to genotoxic stress using LIG4 antibodies to track repair processes

  • Combine with markers of apoptosis to understand how LIG4 deficiency contributes to neuronal death

  • Assess DNA damage accumulation in LIG4-deficient neurons using double immunostaining with γH2AX

• Neurodevelopmental pathway integration:

  • Use co-immunoprecipitation with LIG4 antibodies to identify neuron-specific interaction partners

  • Perform immunofluorescence co-localization studies with developmental signaling pathway components

  • Analyze post-translational modifications of LIG4 in neural tissues

• Therapeutic monitoring:

  • Evaluate potential therapeutic approaches (e.g., gene therapy) by monitoring restoration of LIG4 expression

  • Use quantitative immunohistochemistry to assess dose-dependent effects of interventions

• Advanced imaging applications:

  • Apply super-resolution microscopy with LIG4 antibodies to visualize its distribution at DNA damage sites in neurons

  • Use live-cell imaging with fluorescently tagged antibody fragments to track LIG4 dynamics during neuronal differentiation

These approaches can significantly advance our understanding of how LIG4 dysfunction contributes to neurodevelopmental disorders and potentially inform therapeutic strategies.

What techniques can be used to study the interaction between LIG4 and XRCC4 using antibodies?

The interaction between LIG4 and XRCC4 is crucial for NHEJ function and can be studied using several antibody-based techniques:

• Co-immunoprecipitation (Co-IP):

  • Precipitate LIG4 using anti-LIG4 antibodies and detect XRCC4 in the precipitate by Western blot

  • Perform reciprocal experiments using anti-XRCC4 antibodies for precipitation

  • Include controls for antibody specificity and non-specific binding

• Proximity Ligation Assay (PLA):

  • Use paired antibodies against LIG4 and XRCC4

  • This technique generates fluorescent signals only when the proteins are in close proximity (<40 nm)

  • Quantify interaction frequency in different cellular contexts or following DNA damage

• Bimolecular Fluorescence Complementation (BiFC):

  • Express LIG4 and XRCC4 fused to complementary fragments of a fluorescent protein

  • Use antibodies against tag epitopes to confirm expression levels

  • Fluorescence occurs only when the proteins interact

• Förster Resonance Energy Transfer (FRET):

  • Label LIG4 and XRCC4 antibodies with compatible fluorophores for FRET

  • FRET signal indicates close proximity (<10 nm) between proteins

  • Particularly useful for studying interaction dynamics in living cells

• Chromatin Immunoprecipitation (ChIP):

  • Perform sequential ChIP (re-ChIP) using antibodies against LIG4 followed by XRCC4

  • Identify genomic regions where both proteins co-localize

  • Correlate with sites of DNA damage or V(D)J recombination

• Immunofluorescence co-localization:

  • Use different fluorescently-labeled antibodies against LIG4 and XRCC4

  • Analyze co-localization patterns before and after DNA damage induction

  • Quantify using Pearson's correlation coefficient or Manders' overlap coefficient

• Protein complementation assays:

  • Express LIG4 and XRCC4 fused to complementary fragments of a reporter enzyme

  • Use antibodies to confirm expression levels

  • Enzymatic activity indicates protein-protein interaction

• Mass spectrometry following immunoprecipitation:

  • Precipitate LIG4-XRCC4 complex using specific antibodies

  • Identify interaction domains and post-translational modifications

  • Map the interaction interface through cross-linking mass spectrometry

These techniques provide complementary information about the LIG4-XRCC4 interaction in different biological contexts.

How can I use LIG4 antibodies to study the relationships between DNA repair defects and immunodeficiency?

LIG4 antibodies provide powerful tools for investigating the mechanistic links between DNA repair defects and immunodeficiency:

• Patient-derived sample analysis:

  • Compare LIG4 expression in samples from patients with immunodeficiency versus healthy controls

  • Correlate LIG4 protein levels with clinical parameters of immune function

  • Study both monoallelic and biallelic LIG4 mutation carriers to understand dose-dependent effects

• V(D)J recombination assessment:

  • Use chromatin immunoprecipitation with LIG4 antibodies to identify binding at recombination signal sequences

  • Perform immunofluorescence to visualize LIG4 recruitment during lymphocyte development

  • Correlate with RAG1/2 and recombination efficiency

• Lymphocyte development tracking:

  • Analyze LIG4 expression across developmental stages of B and T cells using flow cytometry

  • Compare receptor diversity in LIG4-deficient versus normal lymphocytes

  • Use immunohistochemistry to examine LIG4 expression in primary and secondary lymphoid organs

• DNA damage response in immune cells:

  • Quantify LIG4 recruitment to DNA damage sites in lymphocytes following radiation

  • Compare repair kinetics between different immune cell subsets

  • Correlate with cell survival and genomic stability

• Class switch recombination (CSR) studies:

  • Analyze LIG4 involvement in CSR using chromatin immunoprecipitation

  • Correlate CSR efficiency with LIG4 expression levels

  • Examine the impact of LIG4 mutations on antibody diversity

• Bone marrow reconstitution models:

  • Use LIG4 antibodies to monitor protein expression in reconstitution experiments

  • Track restoration of immune function in relation to LIG4 levels

  • Compare wild-type versus mutant LIG4 in rescue experiments

• Autoimmunity connections:

  • Study the paradoxical relationship between immunodeficiency and autoimmunity in LIG4 mutations

  • Use immunohistochemistry to examine LIG4 expression in tissues with immune infiltration

  • Correlate with markers of autoimmunity like tissue-specific autoantibodies

• Therapeutic development:

  • Monitor LIG4 expression as a biomarker during therapeutic interventions

  • Use antibodies to assess restoration of normal protein function in gene therapy approaches

This multifaceted approach can uncover the molecular mechanisms linking LIG4 deficiency to both immunodeficiency and autoimmunity.

What are the best practices for using LIG4 antibodies in high-throughput screening applications?

Implementing LIG4 antibodies in high-throughput screening requires careful optimization and standardization:

• Antibody selection and validation:

  • Choose antibodies with demonstrated specificity and sensitivity (e.g., rabbit recombinant monoclonal EPR16531)

  • Validate across multiple platforms (Western blot, ELISA, immunofluorescence)

  • Consider using directly conjugated antibodies to eliminate secondary antibody steps

• Assay miniaturization and automation:

  • Optimize antibody concentrations in reduced volumes (96/384-well formats)

  • Establish reproducible automated protocols for liquid handling

  • Develop robust fixation and staining procedures compatible with automation

• Readout optimization:

  • For high-content screening:

    • Optimize nuclear segmentation parameters

    • Define quantitative metrics for LIG4 nuclear localization

    • Establish thresholds for foci formation following DNA damage

  • For ELISA-based screening:

    • Optimize coating, blocking, and detection conditions

    • Establish standard curves using recombinant LIG4 protein

    • Evaluate detection limits and dynamic range

• Quality control measures:

  • Include positive and negative controls on each plate

  • Use internal normalization controls (housekeeping proteins)

  • Calculate Z'-factor to assess assay robustness: aim for Z' > 0.5

  • Implement outlier detection algorithms

• Multiplexing strategies:

  • Combine LIG4 antibodies with antibodies against other NHEJ components

  • Use spectrally distinct fluorophores for simultaneous detection

  • Include DNA damage markers (γH2AX) and cell cycle indicators

• Data analysis pipelines:

  • Develop automated image analysis workflows for high-content screening

  • Implement machine learning algorithms for pattern recognition

  • Establish dose-response relationship analysis tools

• Hit validation strategy:

  • Define secondary confirmation assays using orthogonal methods

  • Establish threshold criteria for hit selection

  • Include counter-screens to eliminate false positives

• Technical considerations:

  • Standardize fixation conditions (4% paraformaldehyde generally works well)

  • Optimize permeabilization to ensure nuclear access

  • Use appropriate blocking to minimize background (3-5% BSA in PBS)

  • Consider batch effects in plate-to-plate variations

These best practices will help ensure reliable and reproducible results when using LIG4 antibodies in high-throughput screening applications.

How are LIG4 antibodies being used to study connections between DNA repair defects and cancer?

LIG4 antibodies are enabling researchers to explore the complex relationship between DNA repair defects and cancer through several innovative approaches:

• Cancer predisposition in LIG4 syndrome:

  • Using immunohistochemistry to analyze LIG4 expression in tumors from LIG4 syndrome patients

  • Comparing DNA repair capacity between patient-derived and normal cells using LIG4 antibodies

  • Tracking chromosomal instability in LIG4-deficient cells with immunofluorescence

• Prognostic biomarker development:

  • Analyzing LIG4 protein levels in tumor tissue microarrays across cancer types

  • Correlating expression with patient outcomes and therapy response

  • Developing quantitative immunohistochemistry protocols for clinical application

• Therapeutic resistance mechanisms:

  • Studying LIG4 expression changes in response to radiation and chemotherapy

  • Using antibodies to track upregulation of NHEJ in treatment-resistant tumors

  • Developing combination therapies targeting LIG4-dependent repair pathways

• Synthetic lethality approaches:

  • Identifying cancers with defects in complementary repair pathways

  • Using LIG4 antibodies to confirm pathway status in patient samples

  • Developing personalized therapy approaches based on DNA repair profiles

• CRISPR-Cas9 editing and cancer models:

  • Using LIG4 antibodies to study the role of NHEJ in CRISPR-induced off-target effects

  • Developing cancer models with LIG4 mutations

  • Exploring therapeutic gene editing approaches for LIG4-related disorders

• Chemosensitization strategies:

  • Monitoring LIG4 inhibition using antibody-based assays

  • Developing combination therapies that target LIG4-dependent repair

  • Identifying biomarkers of sensitivity to DNA-damaging agents

• Liquid biopsy development:

  • Exploring the potential for detecting LIG4 or LIG4-containing complexes in circulation

  • Correlating with tumor burden or treatment response

  • Developing antibody-based capture systems for circulating tumor cells

These research directions highlight the importance of LIG4 antibodies in advancing our understanding of cancer biology and developing novel therapeutic approaches.

What is the role of LIG4 in immune system development and how can antibodies help study this?

LIG4 plays essential roles in immune system development, particularly through its involvement in V(D)J recombination and class switch recombination. Antibodies against LIG4 facilitate research in this area through several methodological approaches:

• Lymphocyte development tracking:

  • Use immunohistochemistry with LIG4 antibodies to map expression patterns in thymus and bone marrow

  • Perform flow cytometry to quantify LIG4 levels across developmental stages of B and T cells

  • Correlate expression with recombination events using double staining with RAG1/2

• V(D)J recombination mechanisms:

  • Use chromatin immunoprecipitation with LIG4 antibodies to identify binding sites during recombination

  • Analyze co-recruitment of NHEJ factors using sequential ChIP approaches

  • Study differential recruitment to immunoglobulin and T-cell receptor loci

• Clinical immunodeficiency research:

  • Analyze LIG4 expression in patient samples with genetic immunodeficiencies

  • Compare monoallelic versus biallelic mutation carriers

  • Correlate protein expression with immune cell counts and function

• Autoimmunity mechanisms:

  • Study the paradoxical development of autoimmunity in LIG4-deficient patients

  • Use tissue immunohistochemistry to analyze immune cell infiltration

  • Examine auto-reactive B and T cell populations for LIG4 expression abnormalities

• Potential therapeutic approaches:

  • Monitor LIG4 expression during immune reconstitution therapies

  • Use antibodies to track protein expression in gene therapy approaches

  • Develop screening assays for compounds that might stabilize mutant LIG4 proteins

• Aging immune system research:

  • Compare LIG4 expression and activity between young and aged immune cells

  • Correlate with immunosenescence markers

  • Study accumulation of DNA damage in aged lymphocytes

• Immune response to infection:

  • Analyze LIG4 expression during lymphocyte activation and proliferation

  • Study potential roles in somatic hypermutation

  • Examine genomic stability in rapidly proliferating immune cells

These research approaches can significantly advance our understanding of LIG4's role in immune system development and function, potentially leading to new therapeutic strategies for immunodeficiency disorders.

How can single-cell analysis techniques be combined with LIG4 antibodies for advanced research applications?

Single-cell analysis combined with LIG4 antibodies opens new frontiers in understanding DNA repair mechanisms with unprecedented resolution:

• Single-cell protein quantification:

  • Use mass cytometry (CyTOF) with LIG4 antibodies conjugated to metal isotopes

  • Simultaneously measure multiple DNA repair proteins in individual cells

  • Correlate LIG4 levels with cell cycle markers and differentiation status

• Spatial transcriptomics integration:

  • Combine LIG4 immunofluorescence with in situ RNA sequencing

  • Map spatial relationships between LIG4 protein expression and transcriptional profiles

  • Identify microenvironmental factors influencing LIG4 expression

• Single-cell immune profiling:

  • Analyze LIG4 expression in immune cell subsets using flow cytometry

  • Correlate with V(D)J recombination status and receptor diversity

  • Identify rare cell populations with aberrant LIG4 expression

• Live-cell imaging at single-cell resolution:

  • Use fluorescently tagged antibody fragments to track LIG4 dynamics

  • Correlate with DNA damage markers and repair outcomes

  • Measure cell-to-cell variability in repair efficiency

• Microfluidic applications:

  • Develop antibody-based microfluidic capture systems for LIG4-expressing cells

  • Perform single-cell Western blots for LIG4 and interaction partners

  • Isolate specific cell populations for downstream genomic analysis

• Single-cell multi-omics approaches:

  • Combine LIG4 protein detection with single-cell genomics

  • Correlate protein levels with mutation signatures or chromosomal abnormalities

  • Integrate with epigenomic profiling to understand regulatory mechanisms

• Patient sample applications:

  • Analyze heterogeneity of LIG4 expression in patient-derived samples

  • Identify rare cells with altered DNA repair capacity

  • Correlate with disease progression or treatment response

This integration of single-cell technologies with LIG4 antibodies enables researchers to dissect the heterogeneity of DNA repair processes across different cell types and states, potentially revealing new insights into disease mechanisms and therapeutic opportunities.