rec14 Antibody

What is REC14 and what are its alternative names in scientific literature?

REC14 is synonymous with WDR61 (WD repeat-containing protein 61), a protein component of the PAF1 complex (PAF1C). In scientific literature, it may be referred to by several names including Meiotic recombination REC14 protein homolog, Recombination protein REC14, Ski8, SKI8 homolog, WD repeat containing protein 61, and WD repeat domain 61 . When searching databases or literature, researchers should use these alternative terms to ensure comprehensive results, as nomenclature varies across research groups and model organisms.

What is the functional role of the REC14/WDR61 protein in cellular processes?

REC14/WDR61 functions as a component of the PAF1 complex (PAF1C), which plays multiple critical roles during transcription by RNA polymerase II. This complex is implicated in the regulation of development and maintenance of embryonic stem cell pluripotency. Specifically, PAF1C associates with RNA polymerase II through interaction with POLR2A CTD in both non-phosphorylated and phosphorylated forms. It is involved in transcriptional elongation, acting both independently and synergistically with TCEA1 and in cooperation with the DSIF complex and HTATSF1 . The complex is required for transcription of Hox and Wnt target genes and is involved in hematopoiesis by stimulating transcriptional activity of KMT2A/MLL1, which has implications for leukemogenesis. PAF1C also participates in histone modifications, including ubiquitination of histone H2B and methylation on histone H3 'Lys-4' (H3K4me3) .

What types of REC14/WDR61 antibodies are available for research applications?

Based on current research tools, REC14/WDR61 antibodies are available in various forms, predominantly as rabbit polyclonal antibodies. These antibodies are primarily unconjugated and designed for applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) . While polyclonal antibodies offer advantages in terms of recognizing multiple epitopes, researchers should be aware that monoclonal antibodies against REC14/WDR61 may also be available from specialized manufacturers for applications requiring higher specificity.

What are the optimal storage conditions for maintaining REC14/WDR61 antibody activity?

For optimal preservation of REC14/WDR61 antibody activity, storage at -20°C or -80°C is recommended upon receipt. It is crucial to avoid repeated freeze-thaw cycles, which can lead to denaturation and loss of antibody function . For working solutions, small aliquots should be prepared and stored separately to minimize freeze-thaw cycles. The antibody is typically supplied in a buffer containing constituents such as 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative , which helps maintain stability during storage.

What controls should be included when validating REC14/WDR61 antibodies for experimental use?

When validating REC14/WDR61 antibodies, several controls are essential: (1) Positive controls using cell lines or tissues known to express WDR61 (such as those involved in active transcription processes); (2) Negative controls using tissues or cell lines with minimal or no WDR61 expression; (3) Peptide competition assays where the antibody is pre-incubated with the immunogen peptide to confirm binding specificity; (4) Western blot validation to confirm the antibody detects a protein of the expected molecular weight (~34 kDa for human WDR61); and (5) Cross-reactivity testing in multiple species if working with non-human models, as the antibody may show reactivity with human and mouse WDR61 . Additionally, knockout or knockdown cell lines can provide definitive validation of antibody specificity.

What dilution ranges are optimal for different applications of REC14/WDR61 antibodies?

Optimal dilution ranges for REC14/WDR61 antibodies vary by application and must be empirically determined for each specific antibody lot and experimental system. Generally, for Western blotting, initial dilutions of 1:500-1:2000 may be appropriate, while for immunohistochemistry, dilutions of 1:100-1:500 are often used. For ELISA applications, higher dilutions in the range of 1:1000-1:10,000 may be suitable . It is strongly recommended to perform a dilution series during initial optimization experiments, expanding around these ranges to determine the optimal signal-to-noise ratio for your specific experimental conditions, sample types, and detection methods.

How can REC14/WDR61 antibodies be utilized in chromatin immunoprecipitation (ChIP) studies?

For chromatin immunoprecipitation studies involving REC14/WDR61, specific optimizations are necessary given its role in transcriptional regulation. Researchers should: (1) Perform crosslinking with 1% formaldehyde for 10-15 minutes to capture protein-DNA interactions; (2) Use sonication conditions optimized to generate DNA fragments of 200-500 bp; (3) Implement a pre-clearing step with protein A/G beads to reduce non-specific binding; (4) Incubate chromatin with 2-5 μg of REC14/WDR61 antibody overnight at 4°C; (5) Include appropriate controls such as IgG negative control and a positive control antibody against a known PAF1 complex component; and (6) Validate enrichment through qPCR targeting known PAF1C-associated gene regions. Since WDR61 functions within the PAF1 complex, ChIP-seq analysis would be expected to show enrichment at transcriptionally active regions, particularly at Hox and Wnt target genes .

What are the considerations for using REC14/WDR61 antibodies in co-immunoprecipitation studies of transcriptional complexes?

When designing co-immunoprecipitation (co-IP) experiments with REC14/WDR61 antibodies to study transcriptional complexes, several factors must be considered: (1) Cell lysis conditions must preserve protein-protein interactions—mild non-ionic detergents like 0.5% NP-40 or 1% Triton X-100 are recommended; (2) The antibody quantity should be optimized, typically starting with 2-5 μg per mg of protein lysate; (3) Pre-clearing of lysates with protein A/G beads is essential to reduce background; (4) Include RNase treatment if RNA-mediated interactions could complicate interpretation; (5) Confirm interactions by reciprocal co-IP using antibodies against known interaction partners such as other PAF1 complex components; and (6) Include appropriate negative controls (isotype-matched IgG) and positive controls (known interaction partners). Researchers should be aware that the presence of detergents may disrupt certain weaker interactions within the PAF1 complex, so optimization of conditions is critical for capturing the complete interactome.

How do post-translational modifications affect REC14/WDR61 antibody recognition?

Post-translational modifications (PTMs) can significantly impact REC14/WDR61 antibody recognition and experimental outcomes. If the antibody's epitope contains or is adjacent to sites of phosphorylation, ubiquitination, or other modifications, antibody binding may be enhanced or inhibited. For comprehensive studies of WDR61 function, researchers should: (1) Determine if the antibody recognizes specific PTM states by testing recognition in samples treated with phosphatase inhibitors versus phosphatase-treated samples; (2) Consider using multiple antibodies targeting different epitopes to capture all forms of the protein; (3) Apply phosphoproteomic analysis to identify relevant modification sites; and (4) For studies focusing on specific modifications, use modification-specific antibodies if available. Since WDR61/PAF1C is involved in processes regulated by phosphorylation cascades, awareness of how PTMs affect antibody recognition is crucial for accurate interpretation of results, particularly in studies examining dynamic transcriptional regulation.

What strategies can resolve weak or absent signals when using REC14/WDR61 antibodies in Western blotting?

When encountering weak or absent signals with REC14/WDR61 antibodies in Western blotting, implement these stepwise troubleshooting strategies: (1) Increase protein loading (50-100 μg total protein) as WDR61 may be expressed at relatively low levels in some cell types; (2) Optimize antibody concentration by testing a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000); (3) Extend primary antibody incubation time to overnight at 4°C; (4) Test different blocking agents (5% non-fat milk versus 5% BSA) as certain blockers may mask the epitope; (5) Verify transfer efficiency using reversible protein stains; (6) Consider membrane type (PVDF may offer better protein retention than nitrocellulose for some applications); (7) Enhance detection using high-sensitivity substrates for HRP or fluorescent secondary antibodies; and (8) Test different sample preparation methods, as certain lysis buffers may better preserve the native conformation of WDR61. If these steps fail, verify WDR61 expression in your samples through RT-qPCR before concluding antibody failure.

How can non-specific binding be minimized when using REC14/WDR61 antibodies in immunohistochemistry?

To minimize non-specific binding in immunohistochemistry with REC14/WDR61 antibodies: (1) Implement stringent antigen retrieval optimization, testing both heat-induced epitope retrieval (citrate buffer pH 6.0 and EDTA buffer pH 9.0) and enzymatic retrieval methods; (2) Extend blocking time to 1-2 hours using 5-10% normal serum from the species in which the secondary antibody was raised; (3) Add 0.1-0.3% Triton X-100 to antibody diluent to reduce membrane-associated background; (4) Include 0.1-0.3% BSA in the antibody diluent to reduce non-specific protein interactions; (5) Optimize primary antibody dilution and incubation time (consider 1:100-1:500 dilution overnight at 4°C); (6) Implement thorough washing steps (5-6 washes of 5 minutes each); (7) Perform multiple negative controls, including omission of primary antibody and use of isotype control; and (8) Consider using a biotin-streptavidin amplification system only if necessary, as it can increase background. For tissues with high autofluorescence, consider using chromogenic detection methods instead of fluorescence.

What are potential causes of batch-to-batch variability in REC14/WDR61 antibody performance?

Batch-to-batch variability in REC14/WDR61 antibody performance can significantly impact experimental reproducibility. Common causes include: (1) Variations in the immunization protocol or antigen preparation; (2) Differences in purification methods affecting antibody concentration or purity; (3) Changes in the proportion of specific to non-specific antibodies in polyclonal preparations; (4) Storage conditions during shipping or handling that may affect antibody stability; and (5) Variations in post-production quality control standards. To mitigate these issues, researchers should: (1) Test each new batch alongside the previous batch using identical samples and protocols; (2) Reserve sufficient antibody from successful batches for critical experiments; (3) Maintain detailed records of antibody performance across batches; (4) Consider validating with an alternative antibody targeting a different epitope of WDR61; and (5) When reporting results, always include the antibody manufacturer, catalog number, and lot number to enhance reproducibility across laboratories.

How can REC14/WDR61 antibodies be implemented in single-cell protein analysis techniques?

Implementing REC14/WDR61 antibodies in single-cell protein analysis requires special considerations: (1) For mass cytometry (CyTOF), conjugate the antibody with rare earth metals using commercial conjugation kits and validate signal-to-noise ratio in known positive and negative cell populations; (2) For single-cell Western blotting, optimize cell lysis conditions and antibody concentration to detect potentially low abundance WDR61; (3) In microfluidic antibody capture techniques, validate antibody performance in the microfluidic environment where surface chemistry and flow dynamics differ from traditional immunoassays; (4) For imaging mass cytometry or multiplexed ion beam imaging, optimize antibody concentration and staining protocols to ensure signal specificity in a multiplexed environment; and (5) When analyzing single-cell data, implement computational approaches to distinguish true signals from background, particularly important for nuclear proteins like WDR61 where the signal may be concentrated in a small cellular compartment. Given WDR61's role in transcriptional regulation, correlating its expression with cell cycle markers may provide valuable insights into its function at the single-cell level.

What approaches can be used to study REC14/WDR61 interactions with chromatin and transcription factors?

Advanced approaches for studying REC14/WDR61 interactions with chromatin and transcription factors include: (1) Proximity ligation assay (PLA) to visualize and quantify interactions between WDR61 and other proteins in situ with subcellular resolution; (2) ChIP-sequencing to map genome-wide binding sites of WDR61, potentially revealing new target genes beyond known Hox and Wnt pathways; (3) CUT&RUN or CUT&Tag as alternatives to traditional ChIP with potentially higher signal-to-noise ratios and lower input requirements; (4) HiChIP to simultaneously analyze chromatin interactions and protein binding, providing insights into how WDR61/PAF1C influences three-dimensional genome organization; (5) Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) to identify the complete interactome of WDR61 on chromatin; and (6) CRISPR-based approaches such as CUT&Tag-direct to study the effects of WDR61 mutations on chromatin interactions . These approaches should be complemented with functional studies, such as gene expression analysis following WDR61 depletion or mutation, to connect molecular interactions with biological outcomes.

How can recent genotype-phenotype linked antibody techniques be applied to study REC14/WDR61?

Recent advances in genotype-phenotype linked antibody techniques offer powerful approaches for studying REC14/WDR61 function: (1) Implement next-generation sequencing (NGS)-compatible functional screening methods to rapidly identify antigen-specific clones, similar to the system described for influenza antibodies ; (2) Develop a membrane-display system for WDR61-interacting proteins where antibodies are fused to fluorescent proteins like Venus and expressed on cell surfaces to enable functional screening; (3) Apply single B-cell antibody cloning approaches to generate highly specific monoclonal antibodies against different epitopes of WDR61; (4) Utilize phage display libraries to select antibodies with specific binding properties to different conformational states of WDR61; and (5) Consider nanobody development for applications requiring smaller binding molecules with potentially better access to spatially restricted nuclear complexes. These advanced antibody engineering approaches could enable more precise studies of WDR61's role in specific transcriptional contexts and potentially reveal isoform-specific or modification-specific functions that conventional antibodies might miss.

How should researchers interpret discrepancies between REC14/WDR61 antibody results and mRNA expression data?

When encountering discrepancies between REC14/WDR61 antibody results and mRNA expression data, a systematic analytical approach is necessary: (1) Verify antibody specificity through Western blot, knockdown controls, and immunoprecipitation followed by mass spectrometry; (2) Consider post-transcriptional regulation, as mRNA levels may not directly correlate with protein abundance due to miRNA regulation, RNA binding proteins, or differential translation efficiency; (3) Examine protein stability and turnover, as WDR61 may undergo regulated degradation in certain cellular contexts; (4) Investigate potential post-translational modifications that might affect antibody recognition without altering total protein levels; (5) Consider subcellular localization changes, as redistribution rather than altered expression may explain some discrepancies; and (6) Evaluate the sensitivity and dynamic range of both the antibody-based and mRNA-based detection methods. For comprehensive analysis, integrate multiple approaches such as RNA-seq, ribosome profiling, and quantitative proteomics to build a complete picture of WDR61 regulation from transcription to functional protein.

What experimental design considerations are important when studying REC14/WDR61 in different cell and tissue types?

When designing experiments to study REC14/WDR61 across different cell and tissue types, researchers must address several critical considerations: (1) Expression level variation—WDR61 levels may differ substantially between tissues, requiring adjustment of protein loading, antibody concentration, and detection sensitivity; (2) Isoform expression—determine whether different isoforms are expressed in different tissues and whether your antibody recognizes all relevant isoforms; (3) Interaction partners—the composition of the PAF1 complex may vary between cell types, affecting WDR61 function and potentially antibody accessibility; (4) Fixation and preparation protocols—optimize tissue-specific protocols as nuclear proteins often require specialized fixation; (5) Background levels—autofluorescence and endogenous peroxidase activity vary between tissues and may require additional blocking steps; and (6) Physiological state—consider how cell cycle phase, differentiation status, or pathological conditions might affect WDR61 expression or localization . Pilot studies with small sample sets representing each tissue type should be conducted before proceeding to larger-scale experiments to ensure optimal conditions for each context.