NFYC5 Antibody

What is NFYC5 and how does it function in transcriptional regulation?

NFYC5 is associated with the Nuclear transcription factor Y subunit gamma (NFYC) family, which plays a crucial role in regulating gene expression by recognizing and binding to CCAAT motifs within promoters. This transcription factor consists of approximately 458 amino acids and participates in the regulation of various genes including type 1 collagen, albumin, and β-actin.

Methodologically, studies examining NFYC5's function typically employ:

• Chromatin immunoprecipitation (ChIP) assays to identify DNA binding regions

• Reporter gene assays to assess transcriptional activity

• Co-immunoprecipitation studies to identify protein-protein interactions, particularly with other transcription factors like SP1 and FOXO3/FOXO4, which are critical for STK11 gene regulation

It's important to note that NFYC5 should not be confused with NFAT5 (Nuclear factor of activated T-cells 5), which is a distinct transcription factor involved in osmoprotective and inflammatory gene regulation .

How should I validate a NFYC5 antibody before using it in my experiments?

Antibody validation is essential for ensuring experimental reproducibility. For NFYC5 antibody validation, implement the following methodological approach:

Primary Validation Methods:

• Knockout/Knockdown Testing: Compare staining/signal between wildtype samples and those with NFYC5 knocked down or knocked out

• Epitope Competition: Pre-incubate antibody with the immunizing peptide to confirm specific binding

• Multiple Antibody Verification: Use a second antibody targeting a different epitope to confirm results

Application-Specific Validation:

• Western Blot: Confirm single band at expected molecular weight (~458 amino acids should yield approximately 50-55 kDa protein)

• Immunohistochemistry: Include positive and negative control tissues

• ELISA: Generate standard curves using recombinant protein

Critical Reporting Elements:

• Document validation for each specific experimental setup as specificity in one application does not guarantee specificity in another

• Include validation data as supplementary information in publications

• Record batch number as batch-to-batch variability can affect results

Remember: "If an antibody has not been previously validated for the specific combination of application and species used, then it should be mandatory that validation be carried out and reported" .

What controls should I include when using NFYC5 antibody in immunoassays?

Proper experimental controls are critical for reliable NFYC5 antibody research:

Essential Controls for All Applications:

• Positive Control: Include samples known to express NFYC5 (based on literature or previous validation)

• Negative Control: Include samples lacking NFYC5 expression or use isotype-matched non-specific antibodies

• Expression Gradient: When possible, incorporate samples with variable expression levels of NFYC5

Application-Specific Controls:

"Every experiment should include a positive and negative control to assess antibody performance, ideally a set of samples with variable expression levels of the protein of interest" .

How do I troubleshoot specificity issues with NFYC5 antibody?

When encountering specificity issues with NFYC5 antibody, implement this systematic troubleshooting approach:

Methodological Troubleshooting Framework:

• Epitope Analysis

  • Determine the exact epitope recognized by your antibody

  • Search protein databases for sequences with similarity to the epitope

  • Consider potential cross-reactivity with related transcription factors (NFYC family, NFAT5)

• Protocol Optimization

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Modify blocking conditions (try different blocking agents: BSA, milk, serum)

  • Adjust incubation times and temperatures

  • For Western blots: Optimize membrane washing steps and detection methods

• Signal Verification Techniques

  • Perform peptide competition assay

  • Use multiple antibodies targeting different epitopes of NFYC5

  • Compare results with NFYC5 mRNA expression data

  • Consider mass spectrometry to identify proteins bound by the antibody

• Cross-Reactivity Investigation

  • Test the antibody in samples known to lack NFYC5 (knockout models)

  • Evaluate species cross-reactivity if working with non-human samples

  • Check for post-translational modifications that might affect epitope recognition

This comprehensive approach addresses both technical and biological aspects of antibody specificity issues.

What factors contribute to the nuclear localization of NFYC5 and how can this be studied?

The nuclear localization of NFYC5 as a transcription factor is crucial for its function, and studying this localization requires specific methodological approaches:

Key Factors Affecting Nuclear Localization:

• Nuclear localization signals (NLS) within the protein sequence

• Post-translational modifications (phosphorylation, SUMOylation)

• Interactions with nuclear transport proteins

• Cell cycle stage and cellular stress conditions

Methodological Approaches to Study Nuclear Localization:

• Subcellular Fractionation and Western Blotting

  • Isolate nuclear and cytoplasmic fractions using commercial kits

  • Confirm fraction purity with markers (LaminA/C for nuclear, β-tubulin for cytoplasmic)

  • Quantify NFYC5 in different cellular compartments

• Immunofluorescence Microscopy

  • Use validated NFYC5 antibodies with proper controls

  • Co-stain with DAPI for nuclear visualization

  • Apply confocal microscopy for detailed localization patterns

  • Consider examining speckle-like staining patterns similar to those observed with NFAT5

• Live Cell Imaging

  • Create fluorescent protein fusions with NFYC5

  • Monitor dynamic localization changes in response to stimuli

  • Use photobleaching techniques (FRAP) to assess mobility

• Mutational Analysis

  • Generate constructs with mutations in potential NLS sequences

  • Transfect cells and assess localization changes

  • Identify critical residues for nuclear import/export

Drawing from research on related transcription factors like NFAT5, consider that "Nuclear NFAT5 proteins were evenly dispersed following virus infection, with only a small amount of speckle-like staining left in the cytoplasm" , suggesting that cellular stress can significantly alter localization patterns.

How can I distinguish between different isoforms of NFYC5 in my research?

Distinguishing between potential NFYC5 isoforms requires precise methodological approaches:

Identification Strategies:

• Western Blot Optimization

  • Use high-resolution SDS-PAGE (8-10% gels) for optimal separation

  • Include positive controls for each known isoform

  • Consider 2D gel electrophoresis to separate isoforms with similar molecular weights

  • Similar to NFAT5 analysis, look for distinct bands with different molecular weights

• Isoform-Specific Antibodies

  • Select antibodies targeting unique epitopes in specific isoforms

  • Validate isoform specificity using recombinant proteins

  • Consider raising custom antibodies against isoform-specific regions

• RT-PCR and qPCR Analysis

  • Design primers targeting unique exon junctions

  • Optimize PCR conditions for specificity

  • Validate with sequencing of amplicons

  • Quantify relative expression of different isoforms

• Mass Spectrometry

  • Immunoprecipitate NFYC5 from cell lysates

  • Analyze by LC-MS/MS to identify peptides unique to each isoform

  • Quantify relative abundance of isoform-specific peptides

Functional Analysis of Isoforms:

• Isoform-specific knockdown/knockout studies

• Subcellular localization comparison (nuclear vs. cytoplasmic distribution)

• DNA binding and transcriptional activity assays

As observed with NFAT5, "different NFAT5 isotypes, including NFAT5a (158 kDa), NFAT5b (11 kDa), NFAT5c (166 kDa), NFAT5d1 (167.7 kDa), and NFAT5d2 (167.8 kDa), generated via alternative splicing" exhibit different subcellular localizations and responses to stimuli. Similar complexity might exist for NFYC5.

How should I properly report NFYC5 antibody use in scientific publications?

Proper reporting of NFYC5 antibody use is essential for experimental reproducibility. Follow these comprehensive guidelines:

Essential Reporting Elements:

As emphasized in the literature: "The application the antibody was used for is of central importance. This information is normally present, but it can be hard to extract if the antibody information is listed in a 'Materials' section and separated from descriptions of the techniques. Having the antibody data and application data closely linked would avoid potential confusion" .

What strategies can minimize batch-to-batch variability when using NFYC5 antibodies?

Batch-to-batch variability is a significant concern in antibody research, especially with polyclonal antibodies . Implement these methodological strategies to minimize its impact:

Proactive Strategies:

• Inventory Management

  • Purchase larger quantities of a single batch for long-term projects

  • Document batch numbers for all experiments

  • Create internal reference standards from each batch

  • Develop a batch transition protocol when changing to a new lot

• Batch Comparison Protocol

  • When receiving a new batch:

    • Run side-by-side comparisons with the previous batch

    • Test multiple applications (Western, IHC, etc.)

    • Quantify signal intensity and background

    • Document and standardize dilution adjustments needed

• Internal Controls System

  • Maintain frozen aliquots of standard samples

  • Create standard curves for each new batch

  • Use consistent positive and negative controls

  • Implement normalization procedures between batches

• Technical Alternatives

  • Consider using monoclonal antibodies when available (typically less batch variability)

  • Validate multiple antibodies from different vendors

  • Explore recombinant antibody technology

Documentation Framework:

"This type of variability is likely to be a particular issue with polyclonal antibodies, but may affect monoclonal antibodies" . Therefore, systematic approaches to manage batch variations are essential for research reproducibility.

How can NFYC5 antibodies be applied in studying protein-protein interactions in transcription complexes?

Investigating NFYC5's role in transcription complexes requires specialized methodological approaches:

Advanced Interaction Analysis Techniques:

• Co-Immunoprecipitation (Co-IP) Optimization

  • Use NFYC5 antibody as bait to pull down interaction partners

  • Optimize lysis conditions to preserve nuclear protein complexes

  • Consider dual IP (sequential IP) to isolate specific complexes

  • Validate interactions with reverse Co-IP using antibodies against suspected partners

  • Analysis protocol:

    1. Crosslink protein complexes with formaldehyde (0.1-1%)

    2. Lyse cells in buffer containing protease inhibitors

    3. Immunoprecipitate with NFYC5 antibody

    4. Analyze by Western blot or mass spectrometry

• Proximity Ligation Assay (PLA)

  • Detect protein-protein interactions in situ with subcellular resolution

  • Requires antibodies from different species for NFYC5 and potential partners

  • Quantify interaction signals across different cellular conditions

  • Particularly useful for studying interactions with SP1 and FOXO3/FOXO4 transcription factors

• Chromatin Immunoprecipitation (ChIP) Applications

  • Single ChIP: Identify NFYC5 binding sites on DNA

  • Sequential ChIP (Re-ChIP): Determine co-occupancy with other factors

  • ChIP-seq: Genome-wide mapping of binding sites

• Functional Interaction Assessment

  • Luciferase reporter assays with CCAAT motif-containing promoters

  • Mutational analysis of interaction domains

  • siRNA-mediated knockdown of interaction partners

Drawing from NFAT5 research, consider that "NFAT5 also participates in oxidative phosphorylation (OXPHOS) because hypoxia-exposed Nfat5-deficient pulmonary artery elevated the levels of OXPHOS-related transcripts" , suggesting that exploring metabolic connections could provide novel insights into NFYC5 function.

What are the considerations for using NFYC5 antibodies in cross-species studies?

Cross-species application of NFYC5 antibodies requires careful methodological consideration:

Cross-Species Validation Framework:

As noted in antibody research: "If a study uses samples from more than one species then it is also important to clearly link which antibodies were used in which species" . This principle is particularly important when working with transcription factors like NFYC5 that may have species-specific variants.

How does studying NFYC5 complement research on related transcription factors like NFAT5?

Comparative analysis of NFYC5 and related transcription factors provides valuable research insights:

Methodological Approaches for Comparative Studies:

• Parallel Expression Analysis

  • Co-stain tissues/cells for both NFYC5 and NFAT5

  • Compare subcellular localization patterns

  • Analyze co-expression or mutually exclusive expression

  • Quantify relative expression levels across different tissues

• Functional Relationship Investigation

  • Knockdown one factor and assess effects on the other

  • Analyze binding site overlap using ChIP-seq data

  • Identify co-regulated genes through RNA-seq after perturbation

  • Investigate competitive or cooperative binding to similar DNA motifs

• Stress Response Comparison

  • Given NFAT5's role in hypertonic stress response , compare NFYC5's behavior under:

    • Osmotic stress conditions

    • Inflammatory stimuli

    • Viral infection (as NFAT5 restricts virus productive infection )

    • Oxidative stress

• Structural and Mechanistic Studies

  • Compare DNA binding domains and specificities

  • Analyze post-translational modification patterns

  • Investigate potential heterodimer formation

  • Study nuclear import/export mechanisms

Research Integration Framework:

*Based on available search results

Understanding NFYC5 in relation to better-characterized factors like NFAT5 can accelerate research by leveraging established methodologies and conceptual frameworks.

What emerging technologies show promise for improving NFYC5 antibody specificity and applications?

Several cutting-edge technologies are poised to transform NFYC5 antibody research:

Emerging Methodological Approaches:

• Recombinant Antibody Development

  • Single B-cell antibody cloning for highly specific monoclonals

  • Phage display selection against specific NFYC5 epitopes

  • CRISPR-engineered antibody optimization

  • Benefits: Reduced batch variation, renewable source, defined epitope targeting

• Nanobody and Single-Domain Antibody Technology

  • Small (15 kDa) antibody fragments derived from camelid antibodies

  • Enhanced tissue penetration and epitope accessibility

  • Potential applications:

    • Super-resolution microscopy of NFYC5 in nuclear complexes

    • Intracellular expression as "intrabodies" to track native NFYC5

    • Higher density epitope labeling for advanced imaging

• Proximity-Based Protein Interaction Technologies

  • BioID/TurboID: Biotin ligase fusion for identifying proximal proteins

  • APEX2: Peroxidase-based proximity labeling

  • Split-protein complementation assays

  • Applications for mapping NFYC5 interaction networks in living cells

• Advanced Validation Methodologies

  • CRISPR-Cas9 knockout cell lines as gold-standard controls

  • Orthogonal validation with proteomic and transcriptomic data

  • Machine learning algorithms to predict cross-reactivity

  • Multiplexed epitope mapping using peptide arrays

These methodologies represent significant advancements beyond traditional antibody applications, offering unprecedented specificity, versatility, and functional insights for NFYC5 research.

How can computational approaches enhance NFYC5 antibody research?

Computational methodologies are increasingly valuable for advancing NFYC5 antibody research:

Computational Research Strategies:

• Epitope Prediction and Antibody Design

  • In silico analysis of NFYC5 protein structure

  • Identification of surface-exposed, unique epitopes

  • Prediction of epitope conservation across species

  • Computational screening of antibody-antigen binding interfaces

• Cross-Reactivity Assessment

  • Proteome-wide sequence similarity searches

  • Structural homology modeling with related transcription factors

  • Prediction of potential off-target binding

  • Epitope uniqueness scoring algorithm

• Image Analysis and Quantification

  • Machine learning-based signal quantification

  • Automated subcellular localization analysis

  • Multi-parameter phenotypic profiling

  • Correlation of NFYC5 expression with cellular features

• Multi-omics Data Integration

  • Correlation of antibody-based detection with RNA-seq data

  • Integration with ChIP-seq/CUT&RUN data for functional validation

  • Network analysis of NFYC5 within transcriptional programs

  • Systems biology modeling of NFYC5 regulatory networks

These computational approaches complement experimental methods, enhancing the specificity, reproducibility, and biological context of NFYC5 antibody research.

How do I address common technical issues when using NFYC5 antibodies in immunoassays?

This systematic troubleshooting guide addresses common technical challenges with NFYC5 antibodies:

Western Blot Issues:

Immunohistochemistry/Immunocytochemistry Issues:

ELISA Issues: