DCLRE1C Antibody

What is DCLRE1C and why is it significant in immunological research?

DCLRE1C (ARTEMIS) is a nuclease with intrinsic 5′-3′ exonuclease activity on single-stranded DNA. After phosphorylation by and in complex with DNA-dependent protein kinase catalytic subunit, ARTEMIS acquires endonuclease activity on 5′ and 3′ overhangs, and hairpins. It is essential for opening hairpins during V(D)J recombination of immunoglobulin and T-cell receptor genes in lymphocyte development .

The significance of DCLRE1C in immunological research stems from its critical role in immune system development. Null mutations cause severe combined immunodeficiency (SCID) with a block in B- and T-cell development, while hypomorphic mutations can result in a spectrum of immunodeficiency phenotypes ranging from atypical SCID to antibody deficiency . This protein provides a crucial link between DNA repair mechanisms and immune system function.

What molecular weight should I expect for DCLRE1C in Western blot analyses?

Although the calculated molecular weight of full-length DCLRE1C (692 amino acids) is approximately 78 kDa, the observed molecular weight in Western blot analysis is typically around 60 kDa . This discrepancy between calculated and observed molecular weights is not uncommon for proteins and may be attributed to:

• Protein folding affecting migration in SDS-PAGE

• Post-translational modifications

• Proteolytic processing of the full-length protein

When performing Western blot analysis, it's important to note that different isoforms may appear at different molecular weights, and truncated variants resulting from mutations might show altered migration patterns .

What are the common isoforms of DCLRE1C and which should I target with antibodies?

DCLRE1C has multiple transcript isoforms resulting from alternative splicing. The three main isoforms are:

Functional analysis demonstrated that only isoform a (the 692aa canonical sequence) induced high levels of GFP expression in recombination assays, similar to the murine Dclre1c transcript isoform a . Therefore, for most research applications, antibodies targeting isoform a would be most appropriate for studying the functional aspects of DCLRE1C.

When selecting antibodies, consider whether they target domains present in all isoforms of interest or are specific to particular isoforms.

How should I validate the specificity of a DCLRE1C antibody for my research?

Thorough validation of antibody specificity is essential for obtaining reliable results. Follow these methodological approaches:

• Positive and negative controls:

  • Use known positive samples such as HeLa cells or human kidney tissue, which have been demonstrated to express DCLRE1C

  • Include DCLRE1C knockout or knockdown samples as negative controls

  • Mouse thymus has been identified as a positive sample for DCLRE1C detection

• Western blot validation:

  • Verify a single band at the expected molecular weight (~60 kDa)

  • For polyclonal antibodies, some minor bands may be acceptable but should be consistent

• Multiple antibody approach:

  • Compare results using antibodies targeting different epitopes of DCLRE1C (N-terminal versus C-terminal)

  • Consistent results with different antibodies provide stronger validation

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide

  • Signal should be significantly reduced when the antibody is blocked with its target peptide

• Functional correlation:

  • Correlate protein detection with functional assays (V(D)J recombination activity, DNA repair capacity)

  • This is particularly important when studying mutations that may affect epitope recognition

What are optimal sample preparation methods for detecting DCLRE1C in different experimental settings?

Optimal sample preparation varies depending on the application:

For Western Blotting:

• Use recommended dilutions ranging from 1:500 to 1:2000 depending on the specific antibody

• Include protease inhibitors in lysis buffers to prevent degradation

• Consider preparing nuclear fractions as DCLRE1C is predominantly nuclear

• Load 20-50 μg of total protein per lane

For Immunohistochemistry/Immunofluorescence:

• Fixation: 4% paraformaldehyde for cells or 10% neutral buffered formalin for tissues

• Permeabilization: 0.1-0.5% Triton X-100 (10 minutes) to access nuclear antigens

• Blocking: 5% BSA or normal serum to reduce background

• Antibody incubation: Overnight at 4°C for optimal signal-to-noise ratio

• Include nuclear counterstain (DAPI) to confirm nuclear localization

For Flow Cytometry:

• Thorough permeabilization is essential as DCLRE1C is a nuclear protein

• Include appropriate isotype controls

• Use Fc receptor blocking for immune cells to reduce non-specific binding

How can I optimize detection of DCLRE1C in patient samples with hypomorphic mutations?

Detecting DCLRE1C in patient samples with hypomorphic mutations presents unique challenges as these mutations often result in reduced protein expression. Based on research findings, consider these approaches:

• Antibody selection:

  • Choose antibodies targeting protein regions preserved in the specific mutations being studied

  • For C-terminal truncations, use antibodies targeting the N-terminal region

• Enhanced detection methods:

  • Use high-sensitivity chemiluminescent substrates for Western blotting

  • Consider signal amplification systems for immunohistochemistry

  • Longer exposure times may be necessary, but control for increased background

• Sample enrichment:

  • Immunoprecipitation to concentrate the protein before detection

  • Subcellular fractionation to enrich for nuclear proteins

• Experimental controls:

  • Include samples from healthy donors processed identically

  • When available, include samples from patients with known null mutations as comparisons

Research has shown that ARTEMIS expression in fibroblasts from patients with the missense variant c.194C>T (p.T65I) was severely reduced compared to controls . Similar findings were observed in cells with compound heterozygous mutations. Western blot analyses of HEK293T cells transfected with plasmids encoding either missense or frameshift variants also yielded reduced expression of these ARTEMIS variants .

What experimental approaches can be used to study DCLRE1C function in relation to V(D)J recombination?

Several sophisticated experimental systems have been developed to study DCLRE1C function in V(D)J recombination:

• Recombination reporter systems:

  • Retroviral constructs containing inverted GFP cassettes flanked by recombination signal sequences (RSS)

  • GFP expression serves as a functional read-out of recombination activity that can be quantified by flow cytometry

  • This system has been successfully used to assess the functional impact of DCLRE1C mutations

• Cell cycle manipulation:

  • V-abl kinase transformed murine Dclre1c-/- pro-B cells can be blocked in G0/G1 cell cycle phases using imatinib (3μM)

  • This allows the Rag1/Rag2 complex to initiate recombination with higher efficiency

• Complementation studies:

  • Dclre1c-/- cells can be transduced with wild-type or mutant DCLRE1C constructs

  • Functional recovery can be measured through recombination efficiency

  • This approach allows structure-function analysis of specific mutations

• Analysis of recombination junctions:

  • Amplification and sequencing of immunoglobulin or T-cell receptor gene rearrangements

  • Examination of junctional diversity, palindromic nucleotide addition, and microhomology

  • Patients with DCLRE1C mutations show characteristic patterns, including increased palindromic nucleotides in the complementarity determining regions 3

How can I assess DCLRE1C's role in DNA repair beyond V(D)J recombination?

DCLRE1C/ARTEMIS is involved in repairing a subset of DNA double-strand breaks through the NHEJ pathway. These methodological approaches can assess this function:

• γH2AX resolution assays:

  • Measure γH2AX levels at 1h, 8h, 24h, and 36h after ionizing radiation (10Gy)

  • DNA repair efficiencies can be calculated based on mean fluorescent intensities at 36h after irradiation

  • Cells with defective DCLRE1C show impaired resolution of γH2AX foci

• Colony survival assays:

  • Assess cell survival after exposure to ionizing radiation

  • Patients with DCLRE1C mutations show decreased colony survival after irradiation

• Radiation sensitivity testing:

  • ARTEMIS-deficient cells exhibit increased sensitivity to double-strand breaks induced by ionizing radiation or chemical agents like bleomycin and neocarzinostatin

  • Dose-response curves can quantify this sensitivity

• Comet assay:

  • Single-cell gel electrophoresis to measure DNA fragmentation

  • Comparison of tail moment before and after damage induction

These approaches can be applied to cell lines, patient-derived cells, or animal models to comprehensively characterize DCLRE1C's role in maintaining genomic stability.

What approaches help resolve discrepancies between DCLRE1C protein expression and functional activity?

When protein expression does not correlate with functional activity, consider these methodological approaches:

• Isoform-specific analysis:

  • Only isoform a (692aa) shows high functional activity, while isoforms b and c have limited activity

  • Antibodies detecting all isoforms might not reflect functional capacity

• Post-translational modifications:

  • Phosphorylation by DNA-PKcs is required for DCLRE1C activation

  • Use phospho-specific antibodies (e.g., targeting pSer516) to assess activation status

• Protein-protein interactions:

  • DCLRE1C functions in complex with other proteins (DNA-PKcs, other NHEJ factors)

  • Co-immunoprecipitation can assess complex formation efficiency

• Stability and turnover:

  • Some mutations affect protein stability rather than intrinsic activity

  • Pulse-chase experiments can determine protein half-life

• Overexpression strategies:

  • Research has shown that "Overexpression of hypomorphic mutants may improve the functional defect"

  • Quantitative analysis can determine how much increased expression is needed to restore function

How does DCLRE1C expression correlate with clinical phenotypes in immunodeficiency?

Research has revealed important correlations between DCLRE1C expression patterns and clinical phenotypes:

• Expression-phenotype spectrum:

  • Null mutations causing complete absence of functional protein typically result in T-B-NK+ SCID

  • Hypomorphic mutations allowing residual protein expression can cause milder phenotypes ranging from atypical SCID to antibody deficiency

• Cellular manifestations:

  • B-cell lymphopenia is a consistent finding across phenotypes

  • T-cell counts may be normal despite functional deficits

  • Reduced naive T cells with increased terminally differentiated T cells indicate compensatory peripheral expansion

  • Restricted T-cell receptor repertoire reflects impaired V(D)J recombination

• Laboratory parameters to monitor:

  • Very low B-cell numbers and serum IgA levels are characteristic findings

  • T-cell proliferative responses to stimulation are typically impaired

  • Western blot analysis can demonstrate reduced ARTEMIS expression in patient fibroblasts

• Geographic and founder mutations:

  • The c.194C>T (p.T65I) mutation has been identified in multiple patients from the same geographic region

  • Such founder mutations may be associated with specific phenotypic presentations

What technical considerations are important when analyzing DCLRE1C in patient samples versus cell lines?

Working with patient samples presents different challenges compared to established cell lines:

• Sample limitations:

  • Limited quantity and availability of patient material

  • Variability between patients, even with identical mutations

  • Need for appropriate controls (family members, age-matched healthy donors)

• Cell type considerations:

  • Fibroblasts have been successfully used for Western blot analysis of DCLRE1C expression

  • Peripheral blood lymphocytes can be analyzed for naive T-cell counts and proliferative responses

  • Immortalized patient cell lines may show altered expression patterns compared to primary cells

• Functional assessment:

  • Colony survival after irradiation provides a functional readout in patient cells

  • T-cell receptor repertoire analysis can assess the impact on V(D)J recombination in vivo

  • V(D)J recombination reporter assays may require transfer of patient-derived mutations to experimental systems

• Standardization:

  • Process patient and control samples simultaneously

  • Establish clear cutoffs for normal versus abnormal results

  • Consider the effect of prior treatments (e.g., immunoglobulin replacement) on results

• Ethical considerations:

  • Obtain appropriate informed consent for research use of samples

  • Consider genetic counseling implications when novel mutations are identified

Why might I observe multiple bands or unexpected molecular weights when using DCLRE1C antibodies?

Multiple bands or unexpected molecular weights in Western blots can occur for several reasons:

• Alternative splicing:

  • The three major isoforms (a, b, c) have different molecular weights

  • Additional splice variants may exist in specific cell types

• Truncated proteins from mutations:

  • Frameshift or nonsense mutations produce truncated proteins

  • For example, the frameshift variant c.1669_1670insA (p.T577Nfs*21) produces a truncated protein visible as an additional band on Western blot

• Post-translational modifications:

  • Phosphorylation by DNA-PKcs changes the migration pattern

  • Other modifications may occur in response to DNA damage or cell cycle progression

• Degradation products:

  • DCLRE1C may be subject to regulated proteolysis

  • Insufficient protease inhibition during sample preparation can cause artifactual bands

• Antibody cross-reactivity:

  • Some antibodies may recognize similar epitopes in related proteins

  • Validate with knockout controls or peptide competition assays

• Technical issues:

  • Insufficient blocking leading to non-specific binding

  • Excessive antibody concentration

  • Over-development of the blot

What are common pitfalls when analyzing DCLRE1C in primary immune cells?

Working with primary immune cells presents specific challenges:

• Low expression levels:

  • DCLRE1C expression may be lower in primary cells compared to cell lines

  • Signal amplification methods may be necessary

  • Longer exposure times for Western blots or increased antibody concentration for immunostaining

• Cell heterogeneity:

  • Mixed cell populations in blood or tissue samples

  • Consider cell sorting or co-staining with lineage markers

  • Single-cell approaches may reveal population heterogeneity

• Activation state effects:

  • Expression and localization may change with cellular activation

  • Document activation status of cells (resting vs. stimulated)

  • Compare equivalent activation states between samples

• Limited cell numbers:

  • Optimize protocols for small sample sizes

  • Consider nested PCR approaches for transcript analysis

  • Use micro-Western techniques for protein detection

• Background issues in flow cytometry:

  • Autofluorescence of primary cells, especially after fixation

  • Include proper controls (unstained, isotype, FMO)

  • Use Fc receptor blocking reagents to reduce non-specific binding