REC2 Antibody

What is REC2 antibody and what protein does it target?

REC2 antibody targets the protein encoded by the RAD51B gene (also known as RAD51L1 or R51H2). The human version of REC2/RAD51B has a canonical amino acid length of 384 residues and a molecular weight of approximately 42.2 kilodaltons, with 5 known isoforms identified to date . This protein is primarily localized in the nucleus and is widely expressed across various tissue types. Functionally, RAD51B plays critical roles in homologous recombination-mediated DNA repair and cell cycle regulation.

What are the common applications for REC2 antibodies in research?

REC2 antibodies are versatile research tools applicable across multiple experimental platforms:

When selecting application parameters, researchers should optimize dilutions based on antibody source, host species, and sample characteristics to achieve optimal signal-to-noise ratios .

How should I validate a REC2 antibody before using it in my experiments?

Proper validation of REC2 antibodies is essential for experimental reliability:

• Positive and negative controls: Use cell lines or tissues with known RAD51B expression levels, comparing with RAD51B knockdown/knockout samples

• Blocking peptide competition: Pre-incubate antibody with the immunizing peptide to confirm binding specificity

• Cross-reactivity assessment: Test across species if planning cross-species experiments

• Multiple detection methods: Confirm findings using at least two distinct techniques (e.g., WB plus IF)

• Literature comparison: Compare findings with published molecular weights and localization patterns

These validation steps help prevent experimental artifacts and ensure that observed signals genuinely represent RAD51B protein .

How should I design experiments to investigate RAD51B/REC2 interactions with other DNA repair proteins?

When investigating protein-protein interactions involving RAD51B/REC2:

• Co-immunoprecipitation (Co-IP) design: Use anti-REC2 antibodies for pull-down experiments followed by immunoblotting for potential interacting partners (particularly other RAD51 paralogs)

• Proximity ligation assay (PLA): Optimize antibody combinations when studying RAD51B interactions with:

  • RAD51C (primary partner in BCDX2 complex)

  • RAD51D

  • XRCC2

  • XRCC3

• Experimental conditions: DNA damage induction protocols significantly impact observable interactions:

  DNA Damage Agent	Concentration Range	Exposure Time	Primary Complex Formation
  Ionizing radiation	2-10 Gy	0.5-4 hours post-IR	RAD51B-RAD51C
  Hydroxyurea	0.5-2 mM	12-24 hours	Multiple complexes
  Mitomycin C	50-200 ng/mL	6-24 hours	BCDX2 complex

• Controls: Include negative controls (IgG isotype) and positive controls (established interacting partners) .

What are the key considerations when using REC2 antibodies for immunofluorescence studies of DNA repair foci?

For optimal visualization of RAD51B-containing DNA repair foci:

• Fixation protocol: Paraformaldehyde (4%) is preferred over methanol fixation for preserving nuclear architecture

• Permeabilization optimization: Test different detergents:

  • 0.2% Triton X-100 (standard)

  • 0.5% NP-40 (alternative for difficult epitopes)

• DNA damage induction timing: RAD51B foci typically form within:

  • 2-6 hours post-ionizing radiation

  • 12-24 hours post-certain chemical agents

• Co-staining markers: Include:

  • γH2AX (DSB marker)

  • RAD51 (HR marker)

  • BRCA2 (mediator protein)

• Image acquisition settings: Use z-stack imaging (0.3-0.5μm steps) for complete nuclear volume capture

• Quantification approach: Count foci per nucleus (minimum 100 nuclei) and classify cells by foci number distribution .

How can I optimize Design of Experiments (DOE) for studying RAD51B antibody specificity across different isoforms?

When applying DOE approaches to RAD51B isoform specificity:

• Critical parameter identification:

  • Primary factors: Antibody concentration, epitope location, incubation time

  • Secondary factors: Buffer composition, blocking agent, temperature

• Factorial design implementation:

  • Begin with a full factorial design incorporating 3-4 key parameters

  • Add center points (typically 3) for curvature detection

  • Example design matrix:

  Experiment	Antibody Dilution	Buffer pH	Incubation Time (h)	Temperature (°C)
  1	1:500	7.0	1	4
  2	1:2000	7.0	1	4
  3	1:500	8.0	1	4
  ...	...	...	...	...
  16	1:2000	8.0	16	25
  17 (CP)	1:1000	7.5	8	15

• Response variable selection:

  • Primary: Signal-to-noise ratio for each isoform

  • Secondary: Cross-reactivity index between isoforms

• Model development:

  • Fit linear models with interaction terms

  • Validate model with additional confirmation runs

  • Identify optimal conditions for isoform discrimination

• Design space establishment:

  • Define regions where specificity criteria are met

  • Create contour plots to visualize parameter relationships .

What approaches should I use to address contradictory data when REC2 antibody immunostaining patterns differ from known genomic expression profiles?

When faced with discrepancies between antibody-based detection and genomic data:

• Systematic validation workflow:

  • Confirm antibody specificity via western blot with recombinant protein controls

  • Test multiple antibodies targeting different epitopes of RAD51B

  • Employ genetic knockdown/knockout validation

  • Implement epitope retrieval optimization

• Technical considerations:

  • Post-translational modifications may affect epitope accessibility

  • Alternative splicing might produce tissue-specific isoforms

  • Protein stability and half-life variations across tissues

  • Subcellular compartmentalization affecting detection sensitivity

• Complementary methods integration:

  • Parallel RNA-seq and antibody-based protein detection

  • Single-cell western blot validation

  • Mass spectrometry confirmation

  • Proximity ligation assays for in situ verification

• Data reconciliation framework:

  • Decision matrix for interpreting conflicting results:

  Genomic Data	Protein Detection	Possible Interpretation	Recommended Action
  High mRNA	Low protein	Post-transcriptional regulation	Assess protein degradation pathways
  Low mRNA	High protein	Protein stability/accumulation	Measure protein half-life
  No expression	Positive signal	Antibody cross-reactivity	Conduct specificity tests
  Tissue-specific	Ubiquitous signal	Non-specific binding	Test additional antibodies

• Reporting practices:

  • Document all validation steps

  • Present conflicting data transparently

  • Discuss limitations of both detection methods .

What protocol modifications are necessary when using REC2 antibodies for chromatin immunoprecipitation (ChIP) experiments?

Optimizing ChIP protocols for RAD51B/REC2:

• Crosslinking optimization:

  • Standard formaldehyde (1%) crosslinking may be insufficient

  • Test dual crosslinking with 1.5 mM EGS (ethylene glycol bis[succinimidylsuccinate]) followed by 1% formaldehyde

  • Extend crosslinking time to 15-20 minutes (compared to standard 10 minutes)

• Chromatin fragmentation:

  • Target DNA fragments of 200-500bp

  • Compare sonication vs. enzymatic digestion outcomes

  • Recommended sonication: 30-second pulses, 30-second rest, 10-15 cycles (device-dependent)

• Antibody selection criteria:

  • Use antibodies validated specifically for ChIP applications

  • Pre-clear chromatin with protein A/G beads

  • Include IgG controls and positive controls (γH2AX)

• Washing stringency:

  • Increase salt concentration in wash buffers (up to 500mM NaCl)

  • Add mild detergent (0.1% SDS, 1% Triton X-100)

  • Perform additional washing steps (minimum 5 washes)

• DNA recovery and analysis:

  • Implement carrier strategies for low-abundance targets

  • Consider ChIP-sequencing for genome-wide binding

  • Use qPCR primers spanning known DNA repair sites

• Cell treatment conditions:

  • Induce DNA damage to enhance RAD51B recruitment

  • Synchronize cells to S/G2 phases when HR is most active .

How can I troubleshoot weak or inconsistent signals when using REC2 antibodies in western blotting?

When facing detection issues with RAD51B in western blotting:

• Sample preparation optimization:

  • Include phosphatase and protease inhibitors

  • Test nuclear extraction protocols vs. whole-cell lysates

  • Avoid repeated freeze-thaw cycles

• Protein denaturation modifications:

  • Compare reducing vs. non-reducing conditions

  • Test different denaturation temperatures (70°C vs. 95°C)

  • Adjust denaturation time (5-10 minutes)

• Transfer protocol refinement:

  • For 42.2 kDa RAD51B, use PVDF membranes

  • Implement semi-dry transfer (15V for 30-45 minutes)

  • Consider adding SDS (0.1%) to transfer buffer

• Blocking optimization:

  • Test milk vs. BSA blocking (5%)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1% Tween-20 to reduce background

• Signal enhancement strategies:

  • Extended primary antibody incubation (overnight at 4°C)

  • Higher primary antibody concentration (1:500 instead of 1:1000)

  • HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • Consider signal amplification systems

• Systematic troubleshooting table:

  Issue	Possible Cause	Solution
  No signal	Protein degradation	Fresh sample preparation, add protease inhibitors
  Multiple bands	Isoforms or degradation	Validate with recombinant protein
  High background	Insufficient blocking	Increase blocking time, add 0.05% sodium azide
  Inconsistent results	Antibody variability	Aliquot antibody, avoid freeze-thaw cycles
  Wrong molecular weight	Post-translational modifications	Test dephosphorylation treatment

• Positive control inclusion:

  • Use cell lines with confirmed RAD51B expression

  • Consider recombinant RAD51B protein as reference standard .

How should I quantify and interpret REC2/RAD51B expression levels across different cell cycle phases?

For accurate cell cycle-dependent RAD51B analysis:

• Cell synchronization protocols:

  • Double thymidine block for G1/S boundary

  • Nocodazole treatment for M phase

  • Serum starvation for G0/G1

• Dual staining approach:

  • RAD51B antibody combined with:

    • PCNA or EdU (S phase marker)

    • Phospho-Histone H3 (M phase marker)

    • Cyclin D1 (G1 phase marker)

• Quantification methodology:

  • Flow cytometry with DNA content correlation

  • Time-lapse microscopy with cell cycle reporters

  • Immunofluorescence intensity measurement with cell cycle stage classification

• Normalization strategies:

  • Normalize to total protein content

  • Use housekeeping proteins appropriate for each cell cycle phase

  • Apply cell cycle phase correction factors

• Statistical analysis framework:

  • ANOVA for multi-phase comparisons

  • Post-hoc tests with multiple comparison correction

  • Minimum sample size: 3 biological replicates with >10,000 cells per condition for flow cytometry

• Expected expression patterns:

  • RAD51B typically shows increased expression in S/G2 phases

  • Expression pattern table:

  Cell Cycle Phase	Relative RAD51B Expression	Subcellular Localization	Co-localization Partners
  G0/G1	Low (baseline)	Diffuse nuclear	Minimal foci
  S	Moderate to high	Nuclear foci	RAD51C, BRCA2
  G2	High	Distinct nuclear foci	RAD51, RAD51C, DMC1
  M	Decreasing	Excluded from chromatin	N/A

• Interpretation guidelines:

  • Consider both expression level and localization pattern

  • Evaluate proportion of cells with nuclear foci

  • Assess colocalization with DNA damage markers .

What statistical approaches should I use when comparing REC2 antibody data across multiple experimental conditions and antibody sources?

For robust statistical analysis of complex RAD51B antibody datasets:

• Experimental design considerations:

  • Minimum 3-5 biological replicates per condition

  • Include technical replicates (2-3 per biological replicate)

  • Design balanced experiments for statistical power

• Data preprocessing steps:

  • Normality testing (Shapiro-Wilk test)

  • Variance homogeneity assessment (Levene's test)

  • Outlier identification (Grubbs' test)

  • Log transformation for non-normal distributions

• Statistical test selection framework:

  • Two-condition comparisons: Student's t-test or Mann-Whitney U

  • Multi-condition comparisons: One-way ANOVA or Kruskal-Wallis

  • Factorial designs: Two-way ANOVA with interaction terms

  • Repeated measures: RM-ANOVA or mixed models

• Multiple antibody source comparison:

  • Bland-Altman analysis for method comparison

  • Concordance correlation coefficient

  • Passing-Bablok regression

• Effect size calculation:

  • Cohen's d for parametric tests

  • r coefficient for non-parametric tests

  • η² (eta squared) for ANOVA-based analyses

• Multiple comparison correction:

  • Bonferroni correction (conservative)

  • Benjamini-Hochberg procedure (FDR control)

  • Tukey's HSD for all pairwise comparisons

• Visualization approaches:

  • Box plots with individual data points

  • Forest plots for effect size comparison

  • Heat maps for correlation matrices

• Reproducibility assessment:

  • Intraclass correlation coefficient (ICC)

  • Coefficient of variation analysis

  • Antibody reproducibility metrics table:

  Metric	Acceptable Range	Calculation Method	Application
  Inter-assay CV	<15%	SD/mean × 100%	Batch-to-batch comparison
  Intra-assay CV	<10%	SD/mean × 100%	Technical replicates
  ICC	>0.75	Between/total variance	Method consistency
  LoB	Context-dependent	mean_blank + 1.645(SD_blank)	Detection limit
  LoD	Context-dependent	LoB + 1.645(SD_low concentration)	Sensitivity

• Power analysis guidelines:

  • Calculate post-hoc power for nonsignificant results

  • Determine sample sizes for future experiments

  • Consider biological significance beyond statistical significance .

How might advanced techniques like super-resolution microscopy enhance our understanding of REC2/RAD51B localization and function?

Super-resolution microscopy offers revolutionary insights into RAD51B biology:

• Technical advantages over conventional microscopy:

  • Structural illumination microscopy (SIM): 100-120nm resolution

  • Stimulated emission depletion (STED): 30-70nm resolution

  • Single-molecule localization microscopy (PALM/STORM): 10-30nm resolution

• RAD51B-specific applications:

  • Nanoscale mapping of RAD51B within DNA repair foci

  • Co-localization precision with other repair factors

  • Temporal dynamics during foci assembly/disassembly

  • Chromatin association patterns

• Experimental design considerations:

  • Fluorophore selection (photoactivatable/photoswitchable for PALM/STORM)

  • Fixation protocol optimization for epitope preservation

  • Multi-color imaging strategies for protein interaction studies

  • Live-cell compatible antibody formats (nanobodies, Fab fragments)

• Data analysis approaches:

  • Cluster analysis algorithms (DBSCAN, Ripley's K)

  • Co-localization quantification at nanoscale resolution

  • Single-particle tracking for dynamic studies

  • 3D reconstruction of nuclear architecture

• Integration with other technologies:

  • Correlative light and electron microscopy (CLEM)

  • Expansion microscopy for physical magnification

  • Multiplexed imaging with sequential antibody labeling

  • Combination with CRISPR-based tagging approaches .

What role might REC2/RAD51B antibodies play in developing potential therapeutic approaches for DNA repair deficiency syndromes?

Therapeutic applications of RAD51B research:

• Diagnostic potential:

  • RAD51B expression as biomarker in cancer tissues

  • Functional assays for homologous recombination deficiency

  • Companion diagnostics for PARP inhibitor therapy

  • Prognostic indicator in radiation/chemotherapy response

• Therapeutic targeting strategies:

  • Synthetic lethality approaches with RAD51B inhibition

  • Combined targeting of multiple RAD51 paralogs

  • Cell-penetrating antibodies for functional inhibition

  • RAD51B complex disruption to sensitize cancer cells

• Antibody-based research applications:

  • Identification of small molecule binding sites

  • Epitope mapping for functional domains

  • Conformation-specific antibodies for activation states

  • Intrabodies for selective functional inhibition

• Clinical relevance in cancer subtypes:

  • Homologous recombination deficiency evaluation

  • Potential vulnerability in specific cancer subtypes

  • Resistance mechanism identification

  • Predictive biomarker development

• Considerations for translational applications:

  • Validation across diverse patient-derived models

  • RAD51B variant classification guidelines

  • Integration with genomic and transcriptomic data

  • Standardization of assessment methods .