LIG4 Antibody

What is the optimal application range for LIG4 antibodies in immunological studies?

LIG4 antibodies have been validated across multiple applications with varying optimal dilutions. For Western Blot applications, dilutions between 1:500-1:4000 are recommended, with Picoband® antibodies showing higher sensitivity at the lower concentration range. For immunohistochemistry (IHC), optimal dilutions typically fall between 1:20-1:200, while immunofluorescence applications require 1:50-1:500 dilutions. Flow cytometry applications have been validated but require optimization based on your specific cell type and experimental conditions. Always include positive controls known to express LIG4 (HeLa, HepG2, or testis tissue) to establish optimal working concentrations for your experimental system .

Which tissue samples serve as reliable positive controls for LIG4 antibody validation?

Several tissues and cell lines consistently demonstrate reliable LIG4 expression for use as positive controls:

For antigen retrieval in IHC applications, TE buffer (pH 9.0) is suggested, though citrate buffer (pH 6.0) can serve as an alternative method .

How does LIG4 protein expression differ across normal and pathological tissues?

LIG4 expression patterns vary significantly across tissues and disease states. In normal tissues, LIG4 shows constitutive expression in lymphoid tissues, testis, and proliferating cells that require efficient DNA repair mechanisms. In pathological contexts, altered LIG4 expression has been observed in:

• Various cancer types, particularly those with genomic instability features

• Immunodeficiency disorders associated with LIG4 mutations

• Tissues from patients with developmental abnormalities

IHC studies using validated LIG4 antibodies demonstrate nuclear localization in most positive cells, consistent with its function in DNA repair. Expression levels correlate with proliferation rates and DNA damage events, making standardized quantification important when comparing across tissue types .

How should experiments be designed to investigate LIG4 haploinsufficiency versus complete knockout phenotypes?

Designing experiments to differentiate between haploinsufficiency and complete knockout requires careful consideration of experimental models and readouts:

Experimental model selection:

• Heterozygous models (Lig4+/R or monoallelic mutations) for haploinsufficiency studies

• Homozygous models (Lig4R/R) or biallelic mutations for complete deficiency

• Cell lines with inducible knockdown systems to create dose-dependent reduction

Critical readouts to assess:

• DNA repair capacity: Measure γH2AX foci formation after radiation (10Gy standard) with kinetic analysis (repair over time) to detect subtle defects in haploinsufficient models

• Cellular radiosensitivity assays: Compare survival curves between wild-type, heterozygous, and homozygous models

• V(D)J recombination efficiency: Assess T-cell and B-cell receptor diversity through high-throughput sequencing, particularly focusing on CDR3 length and N nucleotide additions

• Lymphocyte development markers: Flow cytometric analysis of naive vs. memory T-cell populations and B-cell maturation stages

• Antibody class switching: Measure serum immunoglobulin levels across isotypes (IgM, IgG, IgA)

Recent research has demonstrated that certain monoallelic LIG4 mutations (e.g., R278Q, A842D) can cause immunological abnormalities through haploinsufficiency, whereas biallelic mutations typically lead to more severe phenotypes. When designing your experimental approach, consider including reconstitution experiments with wild-type versus mutant LIG4 to establish causality .

What are the most critical controls for evaluating anti-LIG4 antibody specificity in immunoblotting applications?

Establishing definitive antibody specificity requires implementation of multiple complementary controls:

Essential negative controls:

• LIG4 knockout cell lines (using CRISPR-Cas9 technology)

• LIG4-deficient patient-derived cells with confirmed null mutations

• Competitive blocking with immunizing peptide (particularly for polyclonal antibodies)

• Secondary antibody-only control

Critical positive controls:

• Recombinant LIG4 protein (full-length or target domain)

• Overexpression systems with tagged LIG4 constructs

• Cell types with known high endogenous expression (HeLa, HepG2)

Validation methodology:

• Confirm the expected molecular weight (100-104 kDa) across multiple cell types

• Verify reduced/absent signal in knockdown/knockout systems

• Demonstrate signal reduction in competition assays

• Compare reactivity patterns across multiple antibodies targeting different LIG4 epitopes

For mutations that may affect antibody binding, consider epitope mapping to ensure your selected antibody will detect the variant of interest. The p.R278L and p.R278H mutations are particularly important to consider as they are common in Chinese populations and may impact antibody binding depending on the immunogen used .

How can researchers accurately distinguish between DNA repair defects caused by LIG4 mutations versus other NHEJ pathway components?

Distinguishing between defects in LIG4 versus other NHEJ components requires a multi-faceted experimental approach:

Molecular and biochemical approaches:

• In vitro DNA end-joining assays: Compare ligation efficiency of cell extracts from suspected LIG4-deficient samples versus controls with deficiencies in other NHEJ components

• Protein complex analysis: Use co-immunoprecipitation to assess LIG4-XRCC4 complex formation

• ATP-dependent ligation activity: Measure ATP consumption during ligation reactions as LIG4 is ATP-dependent

Cellular phenotyping:

• Immunophenotyping profiles: LIG4 deficiency typically shows more pronounced B-cell than T-cell reduction

• V(D)J recombination pattern analysis: LIG4 deficiency leads to distinctive alterations including:

  • Shorter CDR3 regions

  • Diminished N nucleotide additions

  • Increased microhomology-mediated end-joining (MHMEJ) usage

• Telomere length analysis: Assess telomere maintenance which requires functional NHEJ

Genetic complementation studies:

• Perform rescue experiments with wild-type LIG4 versus other NHEJ components

• Use dominant-negative constructs to selectively inhibit specific NHEJ factors

Clinical correlation:
Compare immunological profiles with known LIG4 syndrome characteristics (e.g., normal or elevated IgM with low IgG2b, IgG3, and IgA) versus patterns seen in other NHEJ deficiencies .

What strategies can overcome inconsistent LIG4 detection in primary lymphocytes from immunodeficient patients?

Detection challenges in primary patient samples require strategic optimization:

Sample preparation optimization:

• Fresh isolation protocol: Process samples immediately using lymphocyte separation media with protease and phosphatase inhibitors

• Nuclear extraction focus: LIG4 is predominantly nuclear; use nuclear extraction buffers with higher salt concentration (300-400mM NaCl) for efficient extraction

• Denaturation conditions: Test multiple denaturation temperatures (70°C, 95°C, 100°C) as some LIG4 mutants may have altered protein stability

Signal amplification approaches:

• Enhanced chemiluminescence: Use high-sensitivity ECL substrates with longer exposure times

• Protein concentration: Load more protein (50-100μg) when working with primary lymphocytes

• Antibody cocktails: Combine multiple validated antibodies targeting different LIG4 epitopes

Controls and normalization:

• Internal controls: Include samples from healthy donors processed identically

• Loading controls: Use nuclear-specific loading controls (e.g., Lamin B1) rather than traditional cytoplasmic markers

Protocol modifications for low abundance samples:

• Signal accumulation: For flow cytometry, increase acquisition time and event count

• Immunoprecipitation before Western blot: Concentrate target protein before analysis

• Cell expansion: When possible, expand primary cells in IL-2/IL-7 before analysis

For patients with hypomorphic LIG4 mutations, detection of residual protein may require significantly adjusted protocols compared to complete null mutations .

How should researchers interpret discrepancies between LIG4 protein levels and functional assays in hypomorphic mutation carriers?

Discrepancies between protein detection and functional outcomes require sophisticated interpretation:

Methodological considerations:

• Antibody epitope location: Assess whether the antibody binding site overlaps with the mutation site, potentially affecting detection efficiency

• Protein stability vs. catalytic activity: LIG4 mutations can affect protein stability, complex formation with XRCC4, or catalytic function independently

• Assay sensitivity thresholds: Standard Western blot may detect protein but miss subtle activity reductions that functional assays reveal

Quantitative analysis approach:

• Create titration curves comparing protein levels (by Western blot) with functional output

• Establish threshold levels required for minimal functional activity

• Compare functional kinetics (DNA repair time course) rather than single timepoint measurements

Key mutations with known discrepancies:

• p.R278L/H mutations show detectable protein levels but significant functional impairment

• C-terminal truncating mutations may show near-normal protein levels but severely reduced function due to impaired XRCC4 interaction

Case example interpretation:
In a study of compound heterozygous p.R278L/K424RFS*20 mutations, protein was detectable at approximately 50-60% of normal levels, but DNA repair capacity was reduced to <10% of normal, indicating that protein detection alone is insufficient for predicting functional consequences .

What methodological modifications are required when using anti-LIG4 antibodies for flow cytometry versus immunohistochemistry?

Each application requires specific optimization strategies:

Flow cytometry protocol adaptations:

• Fixation and permeabilization: Use paraformaldehyde (2-4%) fixation followed by Triton X-100 or saponin permeabilization for nuclear antigen access

• Buffer composition: Include 5-10% serum and 0.1% Triton X-100 in staining buffer to reduce background

• Signal amplification: Consider secondary antibody with higher fluorophore:antibody ratio

• Antibody incubation: Extend to overnight at 4°C to improve signal-to-noise ratio

• Controls: Include fluorescence-minus-one (FMO) and isotype controls specific to LIG4 antibody

Immunohistochemistry optimizations:

• Antigen retrieval: TE buffer (pH 9.0) is preferred, but comparative testing with citrate buffer (pH 6.0) is recommended

• Section thickness: 4-5μm sections provide optimal balance of morphology and antibody penetration

• Blocking: Extended blocking (1-2 hours) with 5-10% normal serum matching secondary antibody host

• Amplification systems: Biotin-free detection systems reduce background in lymphoid tissues

• Counterstaining: Hematoxylin counterstaining should be light to avoid masking nuclear LIG4 signal

Antibody selection considerations:

• For flow cytometry: Clone 66705-1-Ig (mouse monoclonal) shows superior performance

• For IHC: Clone 12695-1-AP (rabbit polyclonal) generally provides better results

Regardless of application, titration experiments are essential, as optimal concentrations vary significantly between applications and sample types .

What are the methodological considerations when using LIG4 antibodies to evaluate repair capacity in hematopoietic stem cells versus mature lymphocytes?

Analyzing LIG4 function across different hematopoietic developmental stages requires specialized protocols:

Hematopoietic stem cell (HSC) analysis:

• Cell isolation considerations: Use gentle isolation methods that preserve DNA repair complex integrity

• Protein extraction protocol: Low-detergent nuclear extraction to maintain protein-protein interactions

• Background reduction: HSCs show higher autofluorescence requiring additional controls

• Correlation with stemness markers: Co-stain with CD34, CD38, CD90 to identify true HSCs

Protocol modifications for HSCs vs. mature lymphocytes:

• Fixation parameters: HSCs require gentler fixation (2% PFA vs. 4% for lymphocytes)

• Buffer composition: Include additional antioxidants for HSC processing

• Cell cycle considerations: Normalize LIG4 expression to cell cycle phase using DNA content analysis

Functional readouts comparison:

Interpretation challenges:

• HSCs show heterogeneous repair capacity based on quiescence state

• Different lymphocyte subsets (T vs. B cells) show distinct repair dynamics

• Developmental stage affects baseline expression and repair activity

When analyzing LIG4-deficient patients, comparing HSCs with mature lymphocytes can provide insights into selective pressures during lymphocyte development and potential compensatory mechanisms in long-lived cells .

How can researchers reconcile contradictory findings regarding LIG4 expression levels and immunoglobulin production in patients with hypomorphic mutations?

Resolving contradictions between LIG4 expression and antibody production requires multifaceted analysis:

Experimental approaches to resolve contradictions:

• Comprehensive isotype profiling: Measure all Ig isotypes (not just total IgG) with age-matched controls

• Functional B cell analysis: Assess specific antibody responses to protein (T-dependent) and polysaccharide (T-independent) antigens

• B cell subset characterization: Analyze transitional, naïve, memory, and plasma cell populations

• In vitro CSR analysis: Measure class switching capacity in isolated B cells

Mechanistic explanations for observed contradictions:

• Differential requirements: Class switching to different isotypes has varying LIG4 threshold requirements

• Compensatory pathways: Alternative end-joining mechanisms may partially rescue antibody production

• Mutation-specific effects: Different mutations affect V(D)J recombination versus class switching differentially

• Homeostatic expansion: Lymphopenic environment drives compensatory B cell activation

Integration of contradictory findings:
Research indicates that patients with hypomorphic LIG4 mutations (e.g., p.R278L) can display:

• Normal or elevated IgM (reflecting preserved initial V(D)J recombination)

• Variable IgG1 levels (often preserved)

• Consistently low IgG2b, IgG3, and IgA (reflecting class switching defects)

• In some cases, elevated total IgG with autoimmune features

What experimental designs can effectively measure altered DNA repair pathway choice in cells with hypomorphic LIG4 mutations?

Assessing repair pathway alterations requires specialized experimental approaches:

DNA repair pathway interrogation methods:

• Fluorescent reporter systems: Transfect cells with plasmids containing pathway-specific repair substrates:

  • NHEJ reporters (EJ5-GFP): Measure classical NHEJ efficiency

  • Alt-NHEJ reporters (EJ2-GFP): Quantify microhomology-mediated repair

  • HR reporters (DR-GFP): Assess homologous recombination usage

• Junction sequence analysis: PCR-amplify and sequence repair junctions to identify repair signatures:

  • NHEJ: Minimal nucleotide loss, N-additions

  • MMEJ: Microhomology at junctions (2-20bp)

  • SSA: Extended homology (>25bp)

• Repair protein recruitment kinetics: Use live-cell imaging to track:

  • Classical NHEJ: 53BP1, Ku70/80, DNA-PKcs

  • Alternative NHEJ: PARP1, XRCC1, DNA ligase III

  • HR: RAD51, BRCA1, RPA

Experimental design for LIG4 hypomorphic mutations:

• Create controlled DNA damage using:

  • Site-specific endonucleases (I-SceI, CRISPR/Cas9)

  • Ionizing radiation (2-10Gy)

  • Radiomimetic drugs (bleomycin, neocarzinostatin)

• Compare repair outcomes between:

  • Wild-type cells

  • LIG4 knockout cells

  • Cells expressing hypomorphic LIG4 mutations

  • Complemented cells (rescue with wild-type LIG4)

Data interpretation framework:

This approach has revealed that cells with hypomorphic LIG4 mutations (e.g., R278H) show a 3-5 fold increase in microhomology-mediated end joining compared to wild-type cells, and this correlates with the severity of immune abnormalities in patients .

How can researchers design experiments to evaluate the impact of LIG4 mutations on immune repertoire diversity beyond simple V(D)J recombination defects?

Comprehensive immune repertoire analysis requires integrated methodologies:

Next-generation sequencing approaches:

• CDR3 spectratyping: Analyze CDR3 length distribution and N-addition patterns

• Repertoire sequencing depth: Deep sequencing (>10⁶ reads) to capture rare clonotypes

• Paired chain analysis: Single-cell sequencing of paired TCRα/β or IgH/L to assess combinatorial diversity

• Longitudinal sampling: Track repertoire evolution over time or after immune challenges

Advanced analytical parameters:

• Clonality metrics: Calculate Shannon entropy, Gini index, and clonality scores

• Convergent recombination: Identify multiple recombination events leading to identical or similar receptors

• Public vs. private clonotypes: Compare shared sequences between individuals

• Junctional diversity analysis: Quantify N-additions and P-nucleotides at V(D)J junctions

Experimental design to isolate LIG4-specific effects:

• Cell subset-specific analysis: Compare naïve vs. memory compartments

• Antigen-specific responses: Track repertoire changes after vaccination

• Thymic output assessment: Analyze recent thymic emigrants using TRECs

• Selective pressure analysis: Compare theoretical vs. observed repertoire to identify selection signatures

Integrative analysis framework:

Studies of LIG4-deficient patients have shown that beyond simple V(D)J recombination defects, selective expansion of certain clonotypes occurs, potentially as a compensatory mechanism. This results in a repertoire that appears diverse by standard diversity metrics but shows functional limitations in antigen response capabilities .

What is the optimal protocol for using anti-LIG4 antibodies to investigate the interplay between DNA repair defects and cellular metabolism in primary immune cells?

Investigating repair-metabolism connections requires integrated methodologies:

Sample preparation for coupled analysis:

• Simultaneous preservation: Use fixation methods that preserve both DNA repair complexes and metabolic enzymes

• Cell fractionation strategy: Sequential extraction of cytoplasmic metabolic components and nuclear repair factors

• Metabolic state control: Standardize nutrient conditions and collection timing

Multiparameter analysis approaches:

• Flow cytometry panels combining:

  • LIG4 and repair proteins (γH2AX, 53BP1)

  • Metabolic sensors (GLUT1, 2-NBDG uptake)

  • Mitochondrial markers (MitoTracker, TMRE)

  • ROS indicators (CellROX, MitoSOX)

• Imaging flow cytometry protocol:

  • Primary fixation: 2% PFA for 10 minutes at RT

  • Permeabilization: 0.3% Triton X-100 for 15 minutes

  • LIG4 staining: Clone 66705-1-Ig (1:100) overnight at 4°C

  • Metabolic dyes: Add before fixation according to manufacturer instructions

Functional correlation assays:

• Metabolic manipulation experiments:

  • Glucose restriction/supplementation

  • Mitochondrial inhibitors (oligomycin, rotenone)

  • Fatty acid oxidation modulators

• DNA repair capacity measurement under metabolic stress:

  • Comet assay following metabolic manipulation

  • γH2AX resolution kinetics

  • LIG4-XRCC4 complex formation efficiency

Data integration framework:

Research has revealed that LIG4-deficient primary B cells show altered metabolic profiles, including increased glycolysis and mitochondrial membrane potential, potentially reflecting compensatory mechanisms. These metabolic alterations correlate with DNA repair capacity and can be detected using properly optimized anti-LIG4 antibody protocols integrated with metabolic measurements .

How can LIG4 antibodies be employed to investigate the relationship between DNA repair deficiency and cellular senescence in immune cells?

Investigating repair-senescence connections requires specialized protocols:

Multiparameter analysis protocol:

• Sample preparation:

  • Fresh isolation of lymphocytes using density gradient

  • Gentle fixation (2% PFA, 10 min, RT) to preserve senescence markers

  • Sequential staining: senescence markers → LIG4 → DNA damage markers

• Markers panel design:

  • Senescence: p16INK4a, p21, SA-β-gal activity

  • DNA damage: γH2AX, 53BP1

  • LIG4 and NHEJ components

  • Telomere-associated proteins: TRF1, TRF2

Experimental approaches:

• Replicative senescence model:

  • Serial passaging of primary lymphocytes

  • Correlation of LIG4 expression/function with senescence onset

  • Comparison between wild-type and LIG4-deficient cells

• Stress-induced senescence:

  • Sublethal irradiation (1-5Gy)

  • Oxidative stress (H₂O₂ treatment)

  • Measurement of senescence marker induction

• Intervention studies:

  • Senolytic agents (ABT-263, dasatinib/quercetin)

  • DNA repair modulators

  • Metabolic interventions (NAD+ supplementation)

Analytical framework:

Research has shown that lymphocytes from LIG4-deficient patients exhibit premature senescence features, including increased SA-β-gal activity, elevated p16INK4a expression, and shortened telomeres. These changes correlate with impaired proliferative capacity and altered cytokine production, potentially contributing to immune dysregulation beyond simple developmental defects .

What are the methodological considerations for using LIG4 antibodies to investigate DNA repair in tissue-resident immune cells from LIG4-deficient patients?

Analyzing tissue-resident immune cells requires specialized techniques:

Tissue sample processing optimization:

• Preservation method selection:

  • Fresh frozen tissue maintains optimal epitope integrity

  • For FFPE samples, extended antigen retrieval (30-40 min) is necessary

  • Consider PAXgene fixation for combined protein/RNA analysis

• Cell extraction protocol:

  • Enzymatic digestion: Collagenase D (1mg/ml) + DNase I (0.1mg/ml)

  • Mechanical disruption: GentleMACS with protein crosslinking protection

  • Immediate processing or cryopreservation with DMSO/FBS

Antibody panel design for tissue-resident cells:

• Tissue-resident markers:

  • T cells: CD69, CD103, CD49a

  • B cells: CD69, CD27, CD138

  • Macrophages: CD68, CD163

• DNA repair assessment:

  • LIG4 (Clone 66705-1-Ig, 1:100)

  • γH2AX (Clone JBW301)

  • 53BP1

  • Ki-67 (proliferation status)

Technical modifications for tissue analysis:

• Signal amplification:

  • Tyramide signal amplification for IHC

  • Extended antibody incubation (overnight at 4°C)

  • Sequential staining approach for multiplexing

• Background reduction:

  • Extended blocking (10% serum, 2 hours)

  • FcR blocking reagents

  • Tissue-specific autofluorescence quenchers

Analysis framework: