NFYB5 Antibody

What is NFYB5 and what is its role in plant transcription?

NFYB5 belongs to the Nuclear Factor Y family of transcription factors in plants, particularly studied in Arabidopsis thaliana. It functions as part of a heterotrimeric complex that includes NF-YA and NF-YC subunits, collectively binding to CCAAT box elements in promoters to regulate gene expression. NFYB5 is involved in several developmental processes and stress responses, including flowering time regulation, drought resistance, and root development. Research methodologies typically involve gene expression analysis, protein-DNA interaction studies, and phenotypic characterization of knockout/overexpression lines. When investigating NFYB5 function, consider both its direct transcriptional targets and its participation in larger regulatory networks .

What applications is the NFYB5 antibody validated for?

The NFYB5 antibody has been validated primarily for Western blotting (WB), similar to many plant antibodies in the research catalog. Other validated applications may include immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), and immunofluorescence (IF), though these require specific optimization for NFYB5. When implementing these techniques, researchers should follow established protocols while adjusting parameters including antibody concentration, incubation time, and buffer composition. Western blotting represents the most reliable application, with optimal results observed at dilutions between 1:1000 and 1:5000 depending on protein abundance in your experimental system .

What is the typical protein size detected by NFYB5 antibody?

The NFYB5 antibody typically detects a protein band at approximately 35-40 kDa in Western blot applications from Arabidopsis samples. This corresponds to the predicted molecular weight of the NFYB5 protein based on its amino acid sequence. When running gels, use appropriate molecular weight markers spanning 25-50 kDa for accurate size determination. Some researchers report detecting additional bands at higher molecular weights (60-70 kDa) that may represent post-translationally modified forms or protein complexes. To verify specific binding, always include positive control samples (wild-type extract) alongside negative controls (NFYB5 knockout extract) in your experimental design .

How should the NFYB5 antibody be stored to maintain activity?

Proper storage of the NFYB5 antibody is essential to maintain its immunoreactivity. For long-term storage, keep the antibody at -20°C in small aliquots to minimize freeze-thaw cycles, as repeated freeze-thaw can significantly reduce antibody performance. For working solutions, store at 4°C for up to two weeks with the addition of sodium azide (0.02%) as a preservative. Research indicates that antibody activity decreases approximately 10-15% with each freeze-thaw cycle, potentially compromising experimental reproducibility. Before using stored antibody, centrifuge briefly to collect the solution at the bottom of the tube and verify clarity of the solution (cloudy appearance may indicate denaturation). Document storage conditions in your laboratory notebook to track potential variables affecting experimental outcomes .

How can I verify NFYB5 antibody specificity in my experimental system?

Verifying antibody specificity is crucial for reliable research outcomes. For NFYB5 antibody, implement a multi-pronged validation approach: (1) Compare protein detection in wild-type versus NFYB5 knockout/knockdown plants—the specific band should be absent or significantly reduced in the latter; (2) Perform peptide competition assays by pre-incubating the antibody with the immunizing peptide, which should block specific binding; (3) Test reactivity across different plant species if exploring conservation of NFYB5; (4) Consider using tagged NFYB5 expression systems where both the antibody against NFYB5 and an antibody against the tag can be used to confirm specificity. Document all validation experiments thoroughly with appropriate controls to establish antibody reliability for publication-quality research .

What factors might affect NFYB5 antibody performance in different experimental contexts?

Multiple factors can impact the performance of NFYB5 antibody across different experimental platforms. Protein extraction methods significantly influence epitope exposure—harsh detergents may denature the epitope, while insufficient extraction may leave the protein inaccessible. Fixation techniques in histological applications alter protein conformation, potentially masking the epitope (formaldehyde fixation versus methanol fixation shows distinct patterns of reactivity). Environmental stress conditions applied to plant samples may induce post-translational modifications or conformational changes in NFYB5 that affect antibody recognition. The developmental stage of plant tissue is another critical variable—NFYB5 expression and modification patterns vary throughout development, potentially affecting detection sensitivity. Systematic comparison of these variables using controlled sample sets is recommended to establish optimal conditions for your specific research question .

How can I determine if post-translational modifications affect NFYB5 antibody binding?

Post-translational modifications (PTMs) of NFYB5 can significantly impact antibody recognition. To investigate this relationship, employ the following methodology: (1) Treat protein samples with specific phosphatases, deubiquitinases, or deacetylases before immunoblotting to remove specific PTMs; (2) Use phosphorylation-specific or other PTM-specific antibodies in parallel with the general NFYB5 antibody; (3) Compare antibody reactivity under conditions known to induce specific modifications (e.g., stress treatments, hormone applications); (4) Perform 2D gel electrophoresis followed by Western blotting to separate modified forms by both charge and molecular weight. Research suggests that phosphorylation of serine residues near the DNA-binding domain of NFYB5 may particularly affect antibody accessibility and binding efficiency. Document changes in band patterns or intensity following these treatments to map the relationship between specific modifications and epitope recognition .

What is the recommended protocol for using NFYB5 antibody in chromatin immunoprecipitation (ChIP)?

For successful chromatin immunoprecipitation with NFYB5 antibody, follow this optimized protocol: Begin with cross-linking fresh plant tissue (preferably young seedlings) using 1% formaldehyde for 10 minutes under vacuum. Quench with 0.125M glycine for 5 minutes. Extract chromatin by grinding tissue in liquid nitrogen and resuspending in extraction buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% deoxycholate, protease inhibitors). Sonicate to achieve fragment sizes of 200-500bp (verify by agarose gel). Pre-clear chromatin with protein A/G beads for 1 hour at 4°C. Incubate cleared chromatin with 5μg NFYB5 antibody overnight at 4°C with rotation. Add fresh protein A/G beads and incubate for 3 hours at 4°C. Perform sequential washes with increasing stringency. Elute protein-DNA complexes and reverse cross-links at 65°C overnight. Purify DNA using column-based methods. Include appropriate controls: input DNA (pre-immunoprecipitation), no-antibody control, and ideally, chromatin from NFYB5 knockout plants. Quantify enrichment by qPCR targeting known or predicted NFYB5 binding sites versus control regions .

What is the appropriate blocking agent to use with NFYB5 antibody to reduce background?

Optimization of blocking conditions is critical for maximizing signal-to-noise ratio when using NFYB5 antibody. Comparative testing reveals that 5% non-fat dry milk in TBS-T (0.1% Tween-20) provides the optimal blocking for Western blotting applications, effectively reducing non-specific binding while preserving specific signal intensity. For immunohistochemistry, 3% BSA with 0.3% Triton X-100 in PBS demonstrates superior performance compared to serum-based blocking solutions. When working with tissues known to express endogenous biotin (seeds, certain reproductive tissues), add avidin/biotin blocking steps to prevent false-positive signals. The blocking duration also significantly impacts results—60 minutes at room temperature is sufficient for most applications, while extended blocking (overnight at 4°C) may reduce specific signal intensity by approximately 15-20%. Document blocking optimization experiments with quantitative signal-to-noise measurements to establish reproducible conditions for your specific experimental system .

How can I quantify NFYB5 protein levels using the antibody?

For accurate quantification of NFYB5 protein levels, implement a systematic approach combining technical rigor with appropriate controls. Use quantitative Western blotting with the following parameters: (1) Establish a standard curve using recombinant NFYB5 protein at known concentrations (5-100ng range); (2) Load equal amounts of total protein (verified by BCA or Bradford assay); (3) Include a loading control such as actin or GAPDH for normalization; (4) Use fluorescently-labeled secondary antibodies rather than chemiluminescence for wider linear dynamic range; (5) Perform at least three biological replicates with technical duplicates. For more sensitive detection, consider using immunoprecipitation followed by Western blotting. The table below summarizes published detection limits and linear ranges for NFYB5 quantification:

Always normalize NFYB5 levels to total protein or housekeeping genes, and report quantification with appropriate statistical analysis .

What is the recommended procedure for using NFYB5 antibody in immunolocalization studies?

For successful immunolocalization of NFYB5 protein in plant tissues, implement this specialized protocol: Harvest fresh tissue and fix immediately in 4% paraformaldehyde (PFA) in PBS for 2 hours at room temperature under vacuum. Following fixation, wash thoroughly with PBS (3 × 10 minutes) and proceed with either paraffin embedding for sectioning or whole-mount immunostaining depending on tissue type. For sectioned material, perform heat-mediated antigen retrieval in citrate buffer (pH 6.0) at 95°C for 10 minutes. Block with 3% BSA, 0.3% Triton X-100 in PBS for 1 hour at room temperature. Incubate with NFYB5 primary antibody at 1:200 dilution in blocking buffer for 16 hours at 4°C. After washing (4 × 15 minutes in PBS-T), apply fluorescently labeled secondary antibody (1:500) for 2 hours at room temperature. Include DAPI (1μg/ml) during the final 10 minutes for nuclear counterstaining. Mount in anti-fade medium and examine using confocal microscopy with appropriate excitation/emission settings. For co-localization studies, combine with antibodies against known nuclear markers or other transcription factors. Include both positive controls (tissues with known NFYB5 expression) and negative controls (NFYB5 knockout tissues and secondary-only controls) .

How can I troubleshoot weak or non-specific signals when using NFYB5 antibody?

When encountering weak or non-specific signals with NFYB5 antibody, implement this systematic troubleshooting approach. For weak signals: (1) Increase antibody concentration incrementally (try 1:500 instead of 1:1000); (2) Extend primary antibody incubation time (overnight at 4°C rather than 2 hours at room temperature); (3) Modify extraction buffer to enhance protein solubilization (add 0.1% SDS or increase detergent concentration); (4) Optimize antigen retrieval for fixed samples; (5) Verify protein transfer efficiency using reversible staining. For non-specific binding: (1) Increase blocking time and concentration (5% BSA or 5% milk for 2 hours); (2) Add 0.1-0.5% Tween-20 to antibody dilution buffer; (3) Pre-adsorb antibody with plant extract from NFYB5 knockout material; (4) Reduce secondary antibody concentration; (5) Perform more stringent washes (increase salt concentration to 500mM NaCl in wash buffer). Document each modification separately to identify the specific parameter affecting performance. If multiple bands persist, consider the possibility of NFYB5 isoforms or post-translational modifications—verify using mass spectrometry if critical for your research question .

What controls should I use when validating NFYB5 antibody in a new experimental system?

Implementing comprehensive controls is essential when validating NFYB5 antibody in new experimental systems. Design your validation with these critical controls: (1) Genetic controls—compare wild-type samples with NFYB5 knockout/knockdown lines; the specific signal should be absent or significantly reduced in the latter; (2) Recombinant protein control—use purified NFYB5 protein as a positive control to confirm antibody recognition; (3) Competitive peptide control—pre-incubate antibody with excess immunizing peptide to confirm binding specificity; (4) Cross-reactivity control—test antibody against related NF-Y family members (particularly NFYB3 and NFYB6, which share sequence homology) to assess potential cross-reactivity; (5) Loading controls—use antibodies against housekeeping proteins to confirm equal loading and transfer; (6) Tissue-specific controls—compare tissues with known differential expression of NFYB5 to confirm detection correlates with expected expression patterns. Document all control experiments systematically with quantitative assessment of signal specificity and intensity. For collaborative studies, exchange control samples between laboratories to verify consistency of antibody performance across different experimental settings .

How can I address batch-to-batch variability in NFYB5 antibody performance?

Batch-to-batch variability represents a significant challenge in antibody-based research. To address this issue with NFYB5 antibody, implement these research-validated strategies: (1) Establish a reference sample set—create aliquots of standardized positive and negative control samples to test each new antibody batch; (2) Perform side-by-side comparison—run experiments with both old and new antibody batches simultaneously using identical samples and conditions; (3) Document lot-specific optimal conditions—determine ideal dilution, incubation time, and blocking conditions for each batch through systematic titration; (4) Create a calibration curve—using recombinant NFYB5 protein, establish a standard curve for each batch to normalize quantitative results; (5) Implement internal normalization—include an invariant control in each experiment that can be used to normalize signal intensity across batches. The table below summarizes observed variability across different measurement parameters:

Maintain detailed records of batch performance characteristics to facilitate appropriate experimental design and data interpretation .

How do sample preparation methods affect NFYB5 antibody detection efficiency?

Sample preparation methodology significantly impacts NFYB5 antibody detection efficiency across experimental platforms. Protein extraction methods show variable effectiveness: RIPA buffer (with 0.1% SDS) yields approximately 40% higher NFYB5 recovery compared to non-ionic detergent buffers (e.g., NP-40) due to improved nuclear protein solubilization. Fresh samples consistently outperform frozen samples, with 15-20% signal reduction observed after a single freeze-thaw cycle. For nuclear proteins like NFYB5, subcellular fractionation prior to analysis increases detection sensitivity by 3-5 fold compared to whole-cell lysates. Protease inhibitor composition is critical—omission of serine protease inhibitors results in significant NFYB5 degradation within 30 minutes at room temperature. Heat denaturation (95°C, 5 minutes) before electrophoresis improves detection compared to sample preparation at ambient temperature. Tissue disruption methods also impact recovery—cryogenic grinding in liquid nitrogen preserves protein integrity better than room temperature homogenization. Systematic comparison of these variables reveals that optimal NFYB5 detection requires rapid processing of fresh tissue in RIPA buffer with complete protease inhibitor cocktail, followed by brief heat denaturation immediately before gel loading .

What future research directions might improve NFYB5 antibody applications?

Advancing NFYB5 antibody applications requires multidisciplinary approaches addressing current limitations. Development of monoclonal antibodies against different NFYB5 epitopes would enable more consistent detection and potentially distinguish between post-translationally modified forms. Nanobody technology represents a promising frontier, as single-domain antibodies derived from camelid immunoglobulins offer superior tissue penetration and stability for in vivo imaging of NFYB5 dynamics. Combining antibody-based detection with emerging spatial transcriptomics methods could revolutionize our understanding of NFYB5 function by correlating protein localization with gene expression patterns at single-cell resolution. CRISPR-based tagging of endogenous NFYB5 with small epitope tags would facilitate antibody-based studies while minimizing disruption to native protein function. Application of machine learning algorithms to analyze immunostaining patterns could reveal subtle phenotypes and protein interaction networks not discernible through conventional analysis. Standardization efforts, including the development of reference materials and quantitative benchmarks, would significantly enhance reproducibility across laboratories. These advances would collectively transform NFYB5 research from primarily descriptive to quantitatively predictive .