RECQL4 Antibody, FITC conjugated

What is RECQL4 and why is it an important research target?

RECQL4 is a RecQ-like helicase with pivotal roles in DNA replication, DNA repair, and homologous recombination. It is crucial for maintaining genomic integrity through its involvement in the DNA damage response pathway . Mutations in RECQL4 have been associated with various human diseases, including Rothmund–Thomson syndrome, making it a significant target for both basic research and clinical investigations . RECQL4 has more prominent single-strand DNA annealing activity than helicase activity, with specific functions in regulating major DNA repair pathways such as homologous recombination and nonhomologous end joining (NHEJ) .

What experimental applications are suitable for FITC-conjugated RECQL4 antibodies?

FITC-conjugated RECQL4 antibodies are particularly valuable for:

• Immunofluorescence microscopy to visualize RECQL4 localization within cellular compartments

• Flow cytometry analysis to quantify RECQL4 expression levels in different cell populations

• Live-cell imaging to track RECQL4 dynamics during DNA replication and repair

• Co-localization studies with other proteins involved in DNA damage response pathways

• Chromatin immunoprecipitation followed by fluorescence microscopy (ChIP-FM) to visualize RECQL4 binding to specific chromatin regions

When designing experiments, researchers should account for RECQL4's dynamic localization patterns during different cell cycle phases and in response to DNA damage events .

How should I validate the specificity of my RECQL4 antibody?

Methodological validation approach:

• Genetic controls: Test antibody in RECQL4 knockout/knockdown cells versus wild-type cells

• Peptide competition assay: Pre-incubate antibody with purified RECQL4 protein or immunizing peptide before staining

• Multiple antibody verification: Compare staining patterns with at least one other verified RECQL4 antibody targeting a different epitope

• Western blot correlation: Confirm that fluorescence intensity correlates with protein levels determined by Western blot

• siRNA validation: Demonstrate reduced signal in cells treated with RECQL4-specific siRNA

These approaches ensure that the observed fluorescence signal genuinely represents RECQL4 rather than non-specific binding, which is particularly important when studying this protein in complex with other DNA repair factors .

What are the optimal fixation and permeabilization protocols for RECQL4 immunofluorescence?

For optimal RECQL4 detection while preserving cellular structures:

For permeabilization, 0.2% Triton X-100 for 10 minutes is generally effective after PFA fixation. When visualizing chromatin-bound RECQL4, pre-extraction with 0.5% Triton X-100 before fixation can remove soluble nuclear proteins and enhance the detection of chromatin-associated RECQL4 .

How can I optimize double immunofluorescence with FITC-conjugated RECQL4 antibody and other DNA repair proteins?

When studying co-localization of RECQL4 with other DNA repair proteins:

• Sequential staining: For proteins that use the same host species antibodies, apply the RECQL4 antibody first, followed by additional fixation step with 2% PFA for 5 minutes before applying the second primary antibody

• Blocking optimization: Include 2-5% BSA and 5-10% normal serum from the host species of the secondary antibody

• Signal separation: When using multiple fluorophores, ensure proper filter sets and controls to account for spectral overlap between FITC and other fluorophores

• Post-damage timing: Consider time-dependent recruitment of RECQL4 to DNA damage sites when designing co-localization experiments with PARP1 and other early responders

The recruitment of RECQL4 to DNA double-strand breaks depends on PARP1-mediated PARylation, so experimental designs should account for this temporal relationship when studying co-localization with other repair factors .

How should I design flow cytometry experiments to measure RECQL4 expression across the cell cycle?

For cell cycle-dependent RECQL4 analysis:

• Cell synchronization: Use double thymidine block or nocodazole treatment to obtain synchronized populations

• Dual staining protocol:

  • Fix cells with 70% ethanol (dropwise while vortexing)

  • Permeabilize with 0.1% Triton X-100 for 15 minutes

  • Block with 3% BSA for 30 minutes

  • Stain with FITC-conjugated RECQL4 antibody (1:100-1:500 dilution)

  • Counterstain with propidium iodide (50 μg/ml) with RNase A (100 μg/ml)

• Gating strategy: Gate on single cells using FSC-H vs. FSC-A, then analyze RECQL4-FITC intensity in G1, S, and G2/M populations

• Controls: Include isotype control antibody-FITC conjugate to establish background fluorescence levels

This approach enables quantification of RECQL4 levels across cell cycle phases, which is particularly informative given its role in DNA replication initiation .

How can I use FITC-conjugated RECQL4 antibodies to study the protein's recruitment to DNA damage sites?

For live or fixed-cell analysis of RECQL4 recruitment to DNA damage:

• Micro-irradiation approach:

  • Grow cells on glass-bottomed dishes

  • Sensitize with Hoechst 33342 (10 μg/ml) for 10 minutes

  • Apply laser micro-irradiation using UV laser (355 nm)

  • For fixed-cell analysis, fix at various time points post-irradiation

  • Stain with FITC-conjugated RECQL4 antibody

  • Image using confocal microscopy

• Chemical damage induction:

  • Treat cells with damage-inducing agents (etoposide, bleomycin, or hydrogen peroxide)

  • Optimize concentrations to induce damage without excessive cytotoxicity

  • Monitor RECQL4 recruitment at 5, 15, 30, 60, and 120 minutes post-treatment

Research shows that PARP1 specifically promotes RECQL4 recruitment to DNA double-strand breaks through PARylation, and inhibition or depletion of PARP1 significantly diminishes RECQL4 recruitment to specific DSB sites .

How can I investigate the relationship between RECQL4 and immune checkpoint inhibitor response using immunofluorescence techniques?

To study RECQL4's impact on immune response in tumor samples:

• Multiplex immunofluorescence approach:

  • Perform sequential staining with FITC-conjugated RECQL4 antibody and markers for:

    • MHC class II molecules (key downregulation target of RECQL4)

    • Immune cell infiltrates (CD4+, CD8+ T cells, B cells, dendritic cells)

    • Immune checkpoint molecules (PD-1, PD-L1)

  • Analyze correlation between RECQL4 expression and immune cell density

  • Quantify MHC-II expression in RECQL4-high versus RECQL4-low regions

• Patient sample stratification:

  • Classify samples into RECQL4-high and RECQL4-low groups

  • Compare immunoscores and tumor purity metrics

  • Correlate with clinical response to immune checkpoint inhibitor therapy

Research indicates that high RECQL4 expression correlates with lower immune scores, higher tumor purity, and reduced infiltration of CD4+ and CD8+ T cells, B cells, and dendritic cells in tumor samples . RECQL4 has been identified as a negative regulator of response to immune checkpoint inhibitor therapy and may favor an immune-evasive phenotype by downregulating MHC class II molecules .

What are the best approaches to study RECQL4 interactions with the replication machinery using fluorescence techniques?

For analyzing RECQL4's role in replication complex assembly:

• Proximity ligation assay (PLA) with FITC detection:

  • Fix cells as for standard immunofluorescence

  • Apply primary antibodies against RECQL4 and replication factors (MCM2-7, GINS, CDC45)

  • Follow PLA protocol with FITC-labeled detection reagents

  • Counterstain with DAPI and replication markers like EdU

• Chromatin fractionation followed by immunofluorescence:

  • Extract soluble proteins with CSK buffer + 0.5% Triton X-100

  • Fix chromatin-bound proteins

  • Stain with FITC-conjugated RECQL4 antibody and antibodies against replication factors

  • Analyze co-localization at different cell cycle stages

RECQL4 interactions with replication components are crucial for the assembly of active CDC45-MCM2-7-GINS replicative helicase on chromatin to initiate DNA synthesis . Studies have shown that the RECQL4 C-terminus plays a role in antagonizing its N-terminus, thereby suppressing replication initiation .

What could cause high background when using FITC-conjugated RECQL4 antibodies, and how can I resolve it?

Common causes and solutions for high background:

For RECQL4 specifically, extraction of soluble nuclear proteins before fixation can significantly improve the signal-to-noise ratio when visualizing chromatin-bound RECQL4 .

How should I interpret changes in RECQL4 localization patterns after DNA damage?

Interpretation guidelines for damage-induced RECQL4 dynamics:

• Normal response pattern:

  • Within 1-5 minutes: Initial recruitment to damage sites (dependent on PARP1)

  • 15-30 minutes: Peak accumulation at damage sites

  • 1-4 hours: Gradual dissociation as repair progresses

  • Colocalization with γH2AX foci indicates proper recruitment to DNA breaks

• Abnormal patterns and interpretations:

  • Failure to recruit: May indicate defective PARP1 activity or RECQL4 PARylation

  • Prolonged retention: Possible defect in PARG-mediated dePARylation or repair progression

  • Diffuse nuclear accumulation without focal recruitment: Potential issue with DNA damage signaling

  • Cytoplasmic retention: Possible defect in nuclear localization signals

When analyzing RECQL4 recruitment dynamics, it's important to note that after DNA damage, PARG dePARylates RECQL4 and stimulates its end-joining activity .

How can I accurately quantify changes in RECQL4 expression levels using FITC-conjugated antibodies?

For reliable quantification of RECQL4 expression:

• Flow cytometry approach:

  • Use calibration beads with known FITC molecules per bead

  • Establish standard curve of mean fluorescence intensity

  • Calculate molecules of equivalent soluble fluorochrome (MESF)

  • Compare samples using MESF rather than arbitrary fluorescence units

• Immunofluorescence quantification:

  • Use identical exposure settings across all samples

  • Include internal control cells (e.g., non-transfected cells) in each field

  • Normalize RECQL4 signal to nuclear area or DAPI intensity

  • Use automated image analysis software to measure nuclear FITC intensity

  • Report data as relative fluorescence units compared to control

• Western blot correlation:

  • Perform parallel Western blot analysis from the same samples

  • Correlate fluorescence intensity with protein levels to validate quantification

This methodological approach is particularly important when studying RECQL4 in the context of cancer samples, where expression levels correlate with prognosis and response to therapy .

How might single-molecule fluorescence techniques advance our understanding of RECQL4 function?

Emerging single-molecule approaches applicable to RECQL4 research:

• Single-molecule FRET (smFRET):

  • Label purified RECQL4 protein with donor and acceptor fluorophores

  • Monitor conformational changes during DNA binding and strand annealing

  • Observe RECQL4's DNA annealing activity at the single-molecule level

  • Study how PARP1 interaction affects RECQL4's conformation and activity

• Single-molecule tracking in live cells:

  • Use FITC-conjugated Fab fragments against RECQL4

  • Track individual RECQL4 molecules at damaged and undamaged sites

  • Analyze diffusion coefficients to distinguish between free and DNA-bound states

  • Determine residence times at replication and repair sites

These approaches could provide unprecedented insights into RECQL4's dynamic behavior during DNA repair and replication, particularly its PARP1-dependent recruitment to DNA damage sites and displacement of replication protein A from single-stranded DNA .

What new insights might be gained from studying RECQL4 in patient-derived samples using FITC-conjugated antibodies?

Novel applications in clinical research:

• Tumor microenvironment analysis:

  • Multiplex RECQL4 with immune cell markers in patient samples

  • Correlate RECQL4 expression with T cell infiltration patterns

  • Associate expression levels with response to checkpoint inhibitors

  • Develop predictive algorithms for immunotherapy efficacy

• Liquid biopsy applications:

  • Detect RECQL4 in circulating tumor cells using flow cytometry

  • Correlate expression with disease progression and treatment response

  • Monitor changes in RECQL4 levels during therapy

Research has demonstrated that RECQL4 expression correlates with immune suppression in tumors, with high RECQL4 expression associated with lower immune scores, higher tumor purity, and reduced infiltration of immune cells . This suggests that RECQL4 could serve as both a predictive biomarker and therapeutic target for optimizing immunotherapeutic strategies across various cancer types .

How might understanding RECQL4's interaction with PARP1 and other repair factors inform the development of new therapeutic approaches?

Translational implications of RECQL4-PARP1 interactions:

• Combination therapy strategies:

  • Targeting both RECQL4 and PARP1 could synergistically impair DNA repair

  • RECQL4 inhibition might sensitize PARP-inhibitor resistant tumors

  • Monitoring RECQL4 recruitment using fluorescence techniques could assess PARP inhibitor efficacy

• Biomarker development:

  • FITC-based assays to measure RECQL4 PARylation status

  • Correlation of RECQL4 PARylation with PARP inhibitor sensitivity

  • Development of companion diagnostics for PARP inhibitor therapy

The identification of PARP1's role in promoting RECQL4 PARylation and recruitment to DNA double-strand breaks opens new avenues for therapeutic intervention . Given RECQL4's role in both classical-NHEJ- and alternative-NHEJ-mediated DSB repair, targeting this interaction could provide novel approaches for cancer treatment .