RECQL4 Antibody, Biotin conjugated

What is RECQL4 and why is it significant in genetic research?

RECQL4 (RecQ protein-like 4) is an ATP-dependent DNA helicase (EC 3.6.4.12) that belongs to the RecQ family of helicases. It is also known as DNA helicase RecQ-like type 4, RecQ4, or RTS in scientific literature. This protein plays crucial roles in maintaining genome stability through its involvement in DNA replication, repair, and recombination processes. RECQL4 is particularly significant because mutations within its encoding gene underlie the autosomal recessive cancer-predisposition disorder Rothmund-Thomson syndrome, though the precise mechanisms linking these mutations to disease pathology remain incompletely understood . Unlike other RecQ helicases, RECQL4 appears to have unique regulatory functions in hematopoiesis that are distinct from its helicase activity, making it an intriguing target for fundamental genetic research .

The significance of RECQL4 extends beyond rare genetic disorders, as it has been implicated in DNA double-strand break repair through its participation in DNA end resection - an initial and essential step of homologous recombination (HR)-dependent DNA repair . Furthermore, RECQL4 has been found to physically interact with multiple DNA damage response proteins, including the MRE11-RAD50-NBS1 (MRN) complex and CtIP, positioning it as a central player in genomic integrity maintenance mechanisms . These diverse functions make RECQL4 a critical subject for research into cancer biology, aging, and DNA repair pathways.

What are the recommended applications for RECQL4 Antibody, Biotin conjugated?

The biotin-conjugated RECQL4 antibody is primarily recommended for ELISA applications, as specified in the product information . This conjugation enhances detection sensitivity through the strong biotin-streptavidin interaction, making it particularly suitable for quantitative analyses of RECQL4 protein levels. While the biotin-conjugated variant has validated ELISA applications, researchers should note that unconjugated RECQL4 antibodies have demonstrated utility in a broader range of applications that might be adaptable to the biotin-conjugated format with appropriate optimization .

For comparative purposes, unconjugated RECQL4 antibodies have been successfully employed in multiple applications including Western Blot (recommended dilution 1:1000-1:5000), Immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate), Immunohistochemistry (1:50-1:500), and Immunofluorescence (1:50-1:500 for paraffin-embedded samples; 1:200-1:800 for cell-based assays) . Researchers interested in adapting the biotin-conjugated antibody to these applications should conduct preliminary validation experiments to determine optimal working conditions, as the biotin conjugation may alter binding kinetics and background signals compared to unconjugated formats.

What storage and handling protocols ensure optimal stability of RECQL4 Antibody, Biotin conjugated?

Proper storage and handling of the biotin-conjugated RECQL4 antibody is essential for maintaining its activity and specificity. According to manufacturer recommendations, the antibody should be stored at -20°C or -80°C upon receipt . It is critical to avoid repeated freeze-thaw cycles, as these can significantly reduce antibody activity through protein denaturation and aggregation . The antibody is typically supplied in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

For longer-term storage strategies, while the product information indicates that aliquoting may be unnecessary for storage at -20°C, it is generally good laboratory practice to divide the antibody into single-use aliquots to minimize freeze-thaw cycles for antibodies not frequently used . When handling the antibody, researchers should work quickly to minimize time at room temperature and return the stock solution to -20°C promptly after use. The working dilution should be prepared fresh before each experiment to ensure consistent performance. Additionally, researchers should be aware that the biotin conjugation may have some effect on long-term stability compared to unconjugated antibodies, though specific stability data for this particular conjugate is not provided in the search results.

What validation methods confirm RECQL4 Antibody specificity and reactivity?

Validation of RECQL4 antibody specificity is essential for ensuring reliable experimental results. While the search results don't specifically detail validation methods for the biotin-conjugated variant, standard validation approaches can be inferred from practices with unconjugated RECQL4 antibodies. These antibodies have been validated in multiple human cell lines, including HeLa and HepG2 cells for Western blot applications . The observed molecular weight of RECQL4 is typically 145-150 kDa, which is slightly higher than the calculated molecular weight of 133 kDa (1208 amino acids), likely due to post-translational modifications .

Comprehensive validation should include positive and negative controls. Positive controls might include cell lines known to express RECQL4, while knockdown/knockout approaches provide excellent negative controls. Indeed, published research has utilized RECQL4 knockdown/knockout systems to validate antibody specificity . Additionally, the specificity of the antibody can be further validated by using competing peptides or recombinant RECQL4 protein to block antibody binding in Western blot or immunostaining applications.

For the biotin-conjugated antibody, researchers should also perform control experiments to assess potential background arising from endogenous biotin or biotin-binding proteins in their experimental system. This is particularly important in tissues with high endogenous biotin content, such as kidney, liver, and brain tissues, where blocking steps may be necessary to prevent non-specific signals.

How do sample preparation methods affect RECQL4 detection using antibody-based techniques?

Sample preparation significantly impacts the success of RECQL4 detection using antibody-based techniques. For Western blot applications, efficient extraction of nuclear proteins is crucial since RECQL4 is predominantly nuclear. Standard RIPA buffer supplemented with protease inhibitors is typically sufficient, though specialized nuclear extraction protocols may improve yield for certain applications. When detecting RECQL4 in Western blots, sample denaturing conditions should be optimized as the protein's large size (145-150 kDa) may require extended transfer times or specialized transfer conditions for efficient blotting .

For immunohistochemistry applications, antigen retrieval methods can dramatically affect epitope accessibility and detection sensitivity. The search results suggest that for unconjugated RECQL4 antibodies, TE buffer at pH 9.0 is recommended for antigen retrieval, though citrate buffer at pH 6.0 provides an alternative approach . These conditions may need to be optimized for the biotin-conjugated format as well. Importantly, when using biotin-conjugated antibodies in tissues with high endogenous biotin (such as kidney tissue, where RECQL4 antibodies have been tested), researchers should employ specialized blocking methods to reduce background, such as pre-blocking with avidin/biotin or using streptavidin detection systems with specialized blocking steps.

For immunofluorescence applications, fixation methods can dramatically impact epitope preservation. Paraformaldehyde fixation (typically 4%) is commonly used, but methanol fixation might better preserve nuclear antigens in some instances. Additionally, permeabilization conditions should be optimized when detecting nuclear proteins like RECQL4, with Triton X-100 (0.1-0.5%) being commonly employed for nuclear antigen detection.

How can RECQL4 Antibody be used to investigate DNA double-strand break repair mechanisms?

RECQL4 antibodies provide valuable tools for investigating the protein's critical role in DNA double-strand break (DSB) repair mechanisms. Research has demonstrated that RECQL4 promotes DNA end resection, an initial and essential step in homologous recombination (HR)-dependent DNA double-strand break repair . Immunofluorescence techniques using RECQL4 antibodies can track the protein's recruitment to laser-induced DSBs, allowing researchers to analyze the kinetics of RECQL4 localization to damage sites. This approach revealed that RECQL4 recruitment reaches its peak approximately one minute after laser damage, providing important temporal context for its function in the DSB repair cascade .

For more sophisticated analyses, researchers can combine RECQL4 immunostaining with other DNA repair proteins to study their spatial and temporal relationships. Co-localization studies have shown that RECQL4 co-localizes with MRE11 at DSBs and that the RECQL4-MRN interaction is stimulated by ionizing radiation (IR) . Similarly, RECQL4 has been shown to co-localize with CtIP at laser-induced DSBs . Dual immunostaining approaches can quantitatively assess these interactions under various experimental conditions, such as after treatment with DNA damaging agents or in cells expressing mutant variants of RECQL4.

For biochemical analyses, RECQL4 antibodies can be employed in chromatin immunoprecipitation (ChIP) assays to directly assess RECQL4 binding to damaged DNA regions. Additionally, immunoprecipitation experiments using RECQL4 antibodies have successfully identified interaction partners in the DSB repair pathway, including MRE11, RAD50, BLM, EXO1, and DNA2 . When designing such experiments, researchers should consider using appropriate controls, such as IgG controls for immunoprecipitation and pre-immune serum controls for immunostaining, to ensure specificity of the observed interactions.

What experimental approaches best characterize RECQL4's interaction with DNA repair complexes?

Characterizing RECQL4's interactions with DNA repair complexes requires multi-faceted experimental approaches. Co-immunoprecipitation (Co-IP) has proven particularly valuable, as demonstrated by studies identifying interactions between RECQL4 and the MRN complex (MRE11-RAD50-NBS1) . For optimal results, researchers should perform Co-IP experiments both with and without DNA damaging treatments, as the RECQL4-MRN interaction has been shown to be stimulated by ionizing radiation . To distinguish between DNA-mediated interactions and direct protein-protein interactions, nucleases such as benzonase or ethidium bromide should be included in Co-IP protocols .

Recombinant protein interaction studies provide another powerful approach, eliminating cellular complexities. Research has shown that purified recombinant RECQL4 directly immunoprecipitates recombinant MRE11, RAD50, and NBS1, confirming direct complex formation between RECQL4 and MRN . Similarly, recombinant RECQL4 has been shown to interact directly with CtIP . When designing such experiments, researchers should consider using truncation mutants of RECQL4 to map interaction domains, as studies have identified the N-terminal domain of RECQL4 as responsible for interactions with both MRE11 and CtIP .

Advanced microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and single-molecule tracking, can provide dynamic information about RECQL4's interactions with repair complexes in living cells. These approaches can be particularly valuable for determining how mutations in RECQL4 affect its mobility and retention at DNA damage sites. For example, studies have shown that the MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs, providing insight into the functional relationship between these proteins .

How does RECQL4 contribute to CtIP recruitment, and what methods can detect this relationship?

RECQL4 plays a crucial role in facilitating CtIP recruitment to DNA double-strand breaks (DSBs), a process essential for initiating DNA end resection. Research has demonstrated that RECQL4 co-localizes with CtIP at laser-induced DSBs and directly interacts with CtIP via its N-terminal domain . This interaction promotes CtIP recruitment to the MRN complex at DSBs, making RECQL4 an important mediator in the DNA damage response cascade. To investigate this relationship, researchers have employed several complementary methodological approaches that can be adopted in future studies.

Chromatin fractionation assays provide a powerful method for quantitatively assessing CtIP recruitment to chromatin. Studies have shown that ionizing radiation increases chromatin-bound CtIP in control cells but not in RECQL4-depleted cells, directly linking RECQL4 to the chromatin association of CtIP . Additionally, more mobility shift of chromatin-bound CtIP was detected in control cells compared to RECQL4-depleted cells after IR, indicating that RECQL4 promotes IR-induced posttranslational modification of CtIP . When conducting such experiments, researchers should carefully optimize their fractionation protocols to cleanly separate chromatin-bound proteins from soluble nuclear fractions.

Live-cell imaging using fluorescently tagged proteins offers temporal insights into the recruitment dynamics. Research has shown that RECQL4 recruitment to DNA damage sites peaks around one minute after laser damage, while CtIP recruitment occurs over a longer timeframe . This temporal difference supports a model where RECQL4 functions upstream of CtIP in the recruitment cascade. For such studies, researchers should consider potential artifacts from protein overexpression and validate their findings with endogenous protein detection using immunofluorescence approaches when possible.

What are the methodological considerations when studying RECQL4's helicase-dependent and helicase-independent functions?

Distinguishing between RECQL4's helicase-dependent and helicase-independent functions requires careful experimental design. Studies have revealed that while RECQL4's helicase activity is essential for certain functions, others are maintained even when this activity is inactivated. In hematopoiesis, for example, a RecQ helicase inactive mutated RECQL4 can fully rescue hematopoietic colony formation and B and T cell differentiation, indicating helicase-independent functions in these processes . Conversely, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's unwinding activity in DNA repair .

When designing experiments to differentiate these functions, researchers should consider using RECQL4 mutants with specific defects in helicase activity. Site-directed mutagenesis of key residues in the Walker A or B motifs (involved in ATP binding and hydrolysis) can generate helicase-dead mutants that maintain structural integrity for protein-protein interactions. These mutants can then be used in rescue experiments following RECQL4 depletion to determine which functions depend on helicase activity and which do not.

Biochemical assays can directly assess RECQL4's helicase activity in vitro. Traditional helicase assays using radiolabeled or fluorescently labeled DNA substrates can quantify the unwinding activity of wild-type RECQL4 compared to mutant variants. When coupled with functional assays such as DNA end resection measurements (using techniques like BrdU immunostaining under native conditions or quantitative PCR adjacent to induced DSBs), these biochemical approaches can link helicase activity to specific cellular functions. Additionally, researchers should consider that RECQL4's functions may depend on specific protein complexes, so analyzing its activity in the context of interacting partners such as the MRN complex may provide more physiologically relevant insights than studying the isolated protein.

What experimental designs best assess RECQL4's role in hematopoiesis using antibody-based techniques?

Investigating RECQL4's role in hematopoiesis requires specialized experimental approaches that can be enhanced with antibody-based techniques. Mouse models have been instrumental in demonstrating that somatic deletion of Recql4 leads to rapid-onset, multilineage bone marrow failure, highlighting an essential requirement for RECQL4 in hematopoiesis maintenance . Flow cytometry combined with RECQL4 antibody staining can assess RECQL4 protein levels across different hematopoietic cell populations to determine if expression varies with differentiation state.

For detailed analysis of hematopoietic stem and progenitor cells (HSPCs), researchers can combine surface marker-based sorting with intracellular RECQL4 antibody staining. Studies have shown that loss of RECQL4 particularly affects certain progenitor populations, with granulocyte-macrophage progenitors (GMPs) and common myeloid progenitors (CMPs) maintained at the expense of megakaryocyte-erythroid progenitors (MEPs) . Additionally, pre-MegE and CFU-E populations are substantially compromised in Recql4Δ/Δ bone marrow . Immunofluorescence microscopy using RECQL4 antibodies can assess subcellular localization in different hematopoietic cell types, potentially revealing lineage-specific functions.

Cell death analyses using annexinV/7AAD staining combined with RECQL4 immunostaining have demonstrated increased proportions of dead cells in Recql4Δ/Δ bone marrow, most pronounced in multipotent progenitor (MPP) and committed progenitor fractions . This approach can help determine whether RECQL4 deficiency affects cell survival differently across hematopoietic lineages. For in vitro studies, colony-forming assays using cells with manipulated RECQL4 levels (through knockdown, knockout, or overexpression) provide functional readouts of progenitor activity. Immunoblotting with RECQL4 antibodies should be used to confirm altered protein levels in these experimental systems.

What controls are essential when using RECQL4 antibodies to study Rothmund-Thomson syndrome models?

When investigating Rothmund-Thomson syndrome (RTS) using RECQL4 antibodies, rigorous controls are essential to ensure experimental validity. Since RTS is caused by mutations in the RECQL4 gene, researchers should first verify antibody specificity in their model systems. For cell line models derived from RTS patients or engineered to carry RTS-associated mutations, Western blot analysis using RECQL4 antibodies should confirm altered expression, truncation, or destabilization of the RECQL4 protein . Comparison with wild-type cells provides the necessary baseline control.

For animal models of RTS, such as conditional Recql4 knockout mice, researchers should include heterozygous and wild-type littermates as controls . When studying RECQL4 protein levels in these models, quantitative Western blot with appropriate loading controls (such as histone H3 for nuclear proteins) ensures accurate comparison. Additionally, immunohistochemistry or immunofluorescence staining of tissues from these animals can visualize RECQL4 expression patterns across different cell types and verify knockout efficiency in target tissues.

When using patient-derived cells or tissues, matched control samples from unaffected individuals with similar demographics are crucial. If studying protein function rather than just expression, researchers should include functional controls that verify the integrity of the pathways being examined. For example, when investigating DNA repair functions, control experiments assessing the recruitment of other repair factors (such as γH2AX or 53BP1) to DNA damage sites can confirm that the general DNA damage response is intact . Finally, rescue experiments - where wild-type RECQL4 is re-expressed in deficient cells - provide powerful evidence for the specificity of observed phenotypes and should be incorporated whenever possible.