RECQL4 Antibody

What applications are RECQL4 antibodies most commonly used for in research?

RECQL4 antibodies are widely employed across several experimental applications with varying recommended dilutions:

When selecting an application, consider that RECQL4 shows both nuclear and mitochondrial localization. Multiple studies have confirmed RECQL4's presence in mitochondria using various antibodies from different vendors, with coefficients of co-localization with MitoTracker Red ranging from 0.128 to higher values in perinuclear regions .

How do I choose the right RECQL4 antibody for my research?

Selection should be based on:

• Target region specificity: Different antibodies target distinct domains of RECQL4:

  • N-terminal antibodies (AA 1-652): Detect the Sld2-like domain critical for DNA replication

  • C-terminal antibodies (AA 1134-1162): Recognize the region containing the helicase motifs

  • Internal region antibodies (AA 200-337): Target conserved regions

• Experimental validation: Choose antibodies with published validation in your application of interest. For example, antibodies verified in RECQL4 knockdown/knockout models provide greater confidence in specificity .

• Host species compatibility: Consider the host species (rabbit, mouse) when designing multiplex staining experiments to avoid cross-reactivity.

• Reactivity with target species: Verify that the antibody has been validated in your species of interest (human, mouse) .

How can I validate the specificity of my RECQL4 antibody?

Proper validation is essential to ensure experimental rigor:

• Positive controls: Use cell lines known to express RECQL4 (HeLa, HepG2) .

• Knockdown/knockout validation:

  • Use siRNA-mediated knockdown (targeting sequence: CAAUACAGCUUACCGUACA) to reduce RECQL4 expression by ~90% .

  • Alternatively, employ lentiviral shRNA constructs for stable knockdown .

  • Verify knockdown efficiency by western blotting and Q-PCR before using for antibody validation.

• Molecular weight confirmation: Verify that detected bands match the expected molecular weight of RECQL4 (calculated: 133 kDa; observed: 145-150 kDa) .

• Multiple antibody confirmation: Use antibodies targeting different epitopes to confirm specificity. Studies have employed antibodies from Sigma, Santa Cruz, and custom sources with consistent results .

How can I detect RECQL4 recruitment to DNA damage sites?

RECQL4 is rapidly recruited to DNA double-strand breaks (DSBs). To study this phenomenon:

• Laser microirradiation approach:

  • Use confocal laser scanning microscopy with calibrated laser settings

  • At >10% laser intensity, both single-strand breaks (SSBs) and DSBs are produced

  • RECQL4 recruitment can be observed within 5 seconds after microirradiation

  • Co-stain with γ-H2AX to confirm DSB induction and 53BP1 as a DSB repair factor

• Live-cell imaging with GFP-RECQL4:

  • Transiently transfect cells with GFP-RECQL4 expression constructs

  • Monitor real-time recruitment to laser-induced damage

  • Compare dynamics with other RecQ helicases (GFP-WRN or GFP-BLM) as controls

• Key controls:

  • Use 8% laser intensity (generates only SSBs) versus 18% intensity (generates DSBs)

  • Verify DSB induction by immunostaining for γ-H2AX

  • Include XRCC1 (SSB repair marker) and 53BP1 (DSB repair marker) as controls

How do I detect RECQL4 interactions with other DNA repair proteins?

RECQL4 interacts with several key DNA repair proteins. To study these interactions:

• Co-immunoprecipitation protocol:

  • Prepare cell extracts with RIPA buffer containing protease inhibitors

  • Add benzonase (0.1 U/μl) with 1 μM Mg²⁺ to eliminate DNA-mediated interactions

  • Use 100-200 μg of clarified lysate with anti-RECQL4 antibody or anti-tag antibody

  • Include IgG control to assess non-specific binding

  • Wash beads with high salt (500 mM NaCl) followed by IP buffer washes

  • For PARP1 interaction studies, also perform reciprocal IPs with anti-PAR antibody

• In vitro protein interaction assays:

  • Mix purified RECQL4 (10 pmol) with purified partner protein (e.g., BLM, 20 pmol)

  • Incubate in helicase buffer for 90 min at 4°C

  • Use antibody-coated protein G agarose beads for pull-down

  • Analyze interactions by western blotting

• Enhanced detection in specific cell cycle phases:

  • The interaction between RECQL4 and BLM is enhanced during S-phase and after ionizing radiation

  • Consider synchronizing cells or treating with DNA damaging agents to enhance detection of certain interactions

What methods can detect post-translational modifications of RECQL4?

RECQL4 undergoes several post-translational modifications, particularly PARylation by PARP1:

• Detection of PARylated RECQL4:

  • Immunoprecipitate RECQL4 using specific antibodies

  • Probe with anti-PAR antibodies (e.g., Trevigen 4336-BPC-100)

  • Alternatively, pull down with anti-PAR antibodies and probe for RECQL4

  • Include PARP inhibitor treated controls to confirm specificity

• Mapping modification sites:

  • Generate truncation mutants of RECQL4 (N-terminal and C-terminal regions)

  • Express as tagged constructs in cells

  • Perform immunoprecipitation followed by western blotting with anti-PAR antibodies

  • RECQL4 is PARylated at both N- and C-terminal regions

• Functional impact assessment:

  • Use PARP inhibitors to prevent PARylation

  • Compare recruitment of RECQL4 to DNA damage sites with and without PARP inhibition

  • Assess strand annealing activity of purified RECQL4 with and without PARylation in vitro

How should I optimize Western blotting for RECQL4 detection?

RECQL4 can be challenging to detect due to its size (145-150 kDa) and variable expression levels:

• Sample preparation optimizations:

  • For nuclear RECQL4: Use nuclear extraction protocols with high salt (500 mM NaCl)

  • For mitochondrial RECQL4: Isolate mitoplasts using digitonin treatment

  • Include protease inhibitors in all buffers to prevent degradation

• Gel and transfer considerations:

  • Use 4-15% gradient SDS-PAGE gels for optimal separation

  • Consider longer transfer times (overnight at lower voltage) for high molecular weight proteins

  • Validate transfer efficiency with Ponceau staining

• Antibody dilution and incubation:

  • Start with 1:1000 dilution for most antibodies

  • Incubate primary antibody overnight at 4°C

  • Use 5% non-fat milk in TBST for blocking (45 minutes at room temperature)

• Detection systems:

  • Enhanced chemiluminescence (ECL) substrates work well for RECQL4 detection

  • Image acquisition using digital systems (e.g., Bio-Rad ChemiDoc)

What controls should I include when studying RECQL4 knockdown effects?

When studying the effects of RECQL4 depletion:

• Knockdown approaches and validation:

  • siRNA transfection: Use 100 pmol of RECQL4-targeted siRNA (sequence: CAAUACAGCUUACCGUACA) with Lipofectamine 2000

  • For more efficient knockdown, perform sequential transfections 24 hours apart

  • Always include non-targeting siRNA controls (e.g., ON-TARGET plus non-targeting siRNA #1, Dharmacon)

  • Verify knockdown by both western blotting and qPCR (expect ~90% reduction)

• Experimental controls for functional studies:

  • Include both wild-type cells and cells with other RecQ helicase knockdowns (BLM, WRN) for comparison

  • For rescue experiments, use RECQL4 constructs resistant to siRNA targeting

  • Include helicase-dead mutants (K508A) to distinguish helicase-dependent functions

  • When investigating RECQL4-BLM cooperation, examine both single and double knockdowns

• Phenotypic assays to verify knockdown effects:

  • Proliferation assays (show reduced growth in RECQL4-deficient cells)

  • Sister chromatid exchange frequency (increased in RECQL4-deficient cells)

  • γ-H2AX and 53BP1 foci persistence after DNA damage (prolonged in RECQL4-deficient cells)

How can I monitor RECQL4 biochemical activities in vitro?

RECQL4 possesses several biochemical activities that can be measured:

• Helicase activity assay:

  • Use radiolabeled fork duplex substrates (0.5 nM concentration)

  • Reaction buffer: 30 mM Tris-HCl pH 7.4, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA, 10% glycerol, 5 mM ATP

  • Incubate for 30 minutes at 37°C

  • Analyze by 10% native PAGE electrophoresis

  • Note: RECQL4 has weak helicase activity often masked by its strand annealing activity

  • Use short fork duplex DNA oligonucleotides to better visualize helicase activity

• Strand annealing assay:

  • Use complementary oligonucleotides with one strand 5'-end labeled with [γ-³²P] ATP

  • Reaction buffer: 30 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT, 5 mM MgCl₂, 100 μg/ml BSA

  • Incubate for 10 minutes at 37°C

  • Analyze by 10% native PAGE

  • RECQL4 exhibits strong strand annealing activity that can be enhanced by PARP1

• Replication protein A (RPA) displacement assay:

  • Use single-stranded DNA coated with RPA

  • Add RECQL4 protein and assess displacement

  • This activity promotes microhomology annealing during alternative NHEJ repair

How do I reconcile conflicting reports about RECQL4 helicase activity?

The helicase activity of RECQL4 has been debated in the literature:

• Historical contradiction:

  • Early reports claimed RECQL4 lacked helicase activity (Macris et al., 2006)

  • Later studies demonstrated that RECQL4 does possess weak helicase activity (Rossi et al., 2010)

• Technical solutions:

  • The strong strand annealing activity of RECQL4 can mask its helicase activity

  • Add excess single-stranded DNA to helicase reactions to visualize activity

  • Alternatively, use short fork duplex DNA substrates for direct detection without excess ssDNA

  • Optimize reaction conditions: 30 mM Tris-HCl pH 7.4, 50 mM KCl, 5 mM MgCl₂

• Experimental approach to resolve contradiction:

  • Compare RECQL4 helicase activity with other RecQ helicases (BLM, WRN) using identical substrates

  • Test influences of interacting partners (PARP1, PAR) on helicase activity

  • Use RECQL4 helicase-dead mutant (K508A) as a negative control

How can I address the dual role of RECQL4 in cancer (tumor suppressor vs. oncogene)?

RECQL4 exhibits context-dependent roles in cancer progression:

• Contradictory observations:

  • Mutations in RECQL4 cause Rothmund-Thomson Syndrome with cancer predisposition (tumor suppressor role)

  • High expression of RECQL4 correlates with poor prognosis in several cancers (oncogenic role)

• Experimental approaches to resolve this contradiction:

  • Compare RECQL4 expression levels across cancer types using tissue microarrays

  • Correlate expression with patient outcomes in specific cancer subtypes

  • In melanoma, high RECQL4 expression limits survival and serves as an independent prognostic factor

  • In TNBC, high RECQL4 expression correlates with better response to cisplatin therapy

• Mechanistic studies:

  • Investigate RECQL4 overexpression effects on RAD51 foci formation

  • Measure DSB levels using neutral comet assay in cells with RECQL4 overexpression

  • Assess the relationship between RECQL4 levels and tumor mutation burden

  • Examine correlation between RECQL4 expression and immune cell infiltration in tumors

How do I investigate the relationship between RECQL4 and immune checkpoint inhibitor response?

Recent research has identified RECQL4 as a regulator of immune responses:

How can I investigate RECQL4's dual subcellular localization (nuclear vs. mitochondrial)?

RECQL4 is unique among RecQ helicases in its significant mitochondrial localization:

• Visualization approaches:

  • Use immunofluorescence with multiple RECQL4 antibodies (Sigma, Santa Cruz) to confirm mitochondrial localization

  • Co-stain with MitoTracker Red to visualize mitochondria

  • Calculate coefficient of co-localization (reported values around 0.128)

• Fractionation methods:

  • Isolate mitochondria using differential centrifugation

  • Generate mitoplasts by digitonin treatment to remove outer membrane

  • Use markers for different compartments: VDAC (outer mitochondrial membrane), Cox IV (inner membrane), tubulin (cytosolic contamination)

  • Analyze fractions by western blotting for RECQL4

• Functional studies:

  • Measure mtDNA damage accumulation in RECQL4-deficient cells using Q-PCR

  • Perform microarray analysis to assess impact on mitochondrial bioenergetic pathways

  • Measure mitochondrial reserve capacity in RECQL4 knockdown cells

  • Investigate potential interaction between RECQL4 and mitochondrial DNA polymerase γ