KLF5 Antibody, FITC conjugated

What is KLF5 and what is its significance in cellular processes?

KLF5 (Krueppel-like factor 5) is a transcription factor belonging to the Krueppel C2H2-type zinc-finger protein family. It functions as a key regulator that binds to GC box promoter elements to control gene expression. In humans, the canonical KLF5 protein has 457 amino acid residues with a molecular mass of approximately 50.8 kDa and is primarily localized in the nucleus . KLF5 has significant roles in various biological processes, with preferential expression in testis and placenta. Additionally, it serves as a key transcriptional regulator mediating muscle atrophy . KLF5 often cooperates with other transcription factors such as MyoD, Mef2, and Foxo1 to drive transcriptional processes during cellular differentiation and atrophy . Understanding KLF5 function is critical for research in areas including cellular differentiation, muscle physiology, and various disease processes.

What are the key characteristics of FITC-conjugated KLF5 antibodies?

FITC-conjugated KLF5 antibodies are immunological tools with fluorescein isothiocyanate directly attached to the antibody molecule, enabling fluorescent detection without secondary antibody requirements. For example, the AFG Scientific KLF5 Antibody (FITC conjugated) is a polyclonal antibody developed in rabbit using recombinant Human Krueppel-like factor 5 protein (69-346AA) as the immunogen . This antibody specifically recognizes human KLF5 and is provided in liquid form with a specialized buffer composition (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) to maintain stability and functionality . The antibody undergoes Protein G purification to achieve >95% purity, ensuring research-grade quality for experimental applications . The fluorescent conjugation makes this antibody particularly suitable for applications requiring direct visualization of KLF5 protein.

How does KLF5 function differ across various tissue types?

KLF5 demonstrates tissue-specific expression patterns and functional roles. It is preferentially expressed in testis and placenta as indicated in antibody product descriptions . In skeletal muscle, KLF5 has been identified as a critical mediator of muscle atrophy, working in concert with Foxo1 to regulate genes like Fbxo32 (which encodes Atrogin-1) . Research has demonstrated that KLF5 binding to enhancer regions within the Fbxo32 gene is augmented during glucocorticoid-induced muscle atrophy . Additionally, alternative splicing generates at least four different KLF5 isoforms, which may exhibit tissue-specific functions . When designing experiments with KLF5 antibodies, researchers should consider the specific tissue context and isoform expression patterns relevant to their research questions, as antibody reactivity and experimental outcomes may vary across different biological systems.

What are the optimal applications for FITC-conjugated KLF5 antibodies?

When designing experiments, consider that FITC has an excitation maximum at approximately 495 nm and emission maximum at 519 nm, requiring appropriate filter sets for detection. The direct conjugation eliminates the need for secondary antibodies, simplifying protocols and reducing background in multi-color experiments. For optimal results in ELISA applications, researchers should establish appropriate dilution ranges (typically starting at 1:1000 for conjugated antibodies) through titration experiments and include proper blocking steps to minimize non-specific binding. Always include appropriate positive and negative controls to validate specificity in your experimental system.

How should researchers optimize KLF5 antibody-based ChIP experiments?

Chromatin immunoprecipitation (ChIP) experiments with KLF5 antibodies require careful optimization for successful results. Based on research methodologies described in the literature, KLF5 has been successfully used in ChIP-seq and ChIP-qPCR experiments to identify binding sites in gene regulatory regions .

For optimal KLF5 ChIP protocols:

• Crosslinking optimization: Start with standard 1% formaldehyde for 10 minutes at room temperature, but optimize time (5-15 minutes) based on your cell type.

• Sonication conditions: Adjust to generate DNA fragments of 200-500 bp, verifying fragment size by gel electrophoresis.

• Antibody selection: Choose ChIP-validated antibodies; while FITC-conjugated antibodies are not typically used for ChIP, unconjugated KLF5 antibodies have been successfully employed in published studies .

• Positive control regions: Include known KLF5 binding sites such as the enhancer within intron 4 of Fbxo32 (GeneHancer identifier: GH08J123534) that contains conserved KLF5 binding motifs .

• Quantification: Use qPCR with primers flanking predicted KLF5 binding sites, including appropriate normalization controls.

Research has shown that KLF5 binding to target genes can be stimulus-dependent. For instance, treatment with dexamethasone (Dex) enhanced KLF5 binding to the Fbxo32 enhancer in muscle cells . Therefore, consider the appropriate cellular stimulation conditions relevant to your research question when designing ChIP experiments.

What are the critical considerations for co-immunoprecipitation studies involving KLF5?

Co-immunoprecipitation (Co-IP) studies with KLF5 require careful attention to several critical factors to successfully detect protein-protein interactions. Research has demonstrated that KLF5 directly interacts with transcription factors such as Foxo1 , making Co-IP a valuable technique for investigating KLF5 functional partners.

Key considerations include:

• Cell lysis conditions: Use gentle lysis buffers (e.g., 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA) with protease inhibitors to preserve protein interactions while effectively releasing nuclear proteins like KLF5.

• Antibody selection: Choose antibodies with validated IP performance. While FITC-conjugated antibodies are generally not ideal for IP due to potential steric hindrance from the fluorophore, unconjugated KLF5 antibodies have been used successfully in IP experiments .

• Binding conditions: Optimize antibody concentration and incubation time/temperature to maximize target protein capture while minimizing non-specific binding.

• Washing stringency: Balance between removing non-specific interactions and preserving real interactions; typically 3-5 washes with decreasing salt concentrations.

• Detection of binding partners: Consider using specific antibodies against predicted interaction partners (e.g., Foxo1, MyoD, Mef2) for Western blot detection after IP .

• Controls: Include IgG control, input sample, and reciprocal IP (using antibody against the suspected binding partner to pull down KLF5) to validate interactions.

When investigating transient or weak interactions, consider using crosslinking agents or stimuli that enhance the interaction. For example, dexamethasone treatment has been shown to enhance KLF5 interactions with transcriptional regulatory elements .

How can researchers analyze KLF5-mediated epigenetic modifications using FITC-conjugated antibodies?

Analyzing KLF5-mediated epigenetic modifications requires a multi-faceted approach combining KLF5 detection with assessment of histone modifications. Research has demonstrated that KLF5 influences epigenetic states, particularly histone H3 lysine 27 acetylation (H3K27ac) at enhancer regions . While FITC-conjugated KLF5 antibodies are not directly used for chromatin modification studies, they can be incorporated into comprehensive experimental designs.

A methodological approach would include:

• Sequential ChIP (re-ChIP): First immunoprecipitate with an unconjugated KLF5 antibody, then with antibodies against histone modifications (such as H3K27ac ). This reveals the co-occurrence of KLF5 binding and specific epigenetic states.

• Immunofluorescence co-localization: Use FITC-conjugated KLF5 antibody alongside antibodies against histone modifications labeled with spectrally distinct fluorophores to visualize co-localization in nuclei.

• Time-course analysis: Track the temporal relationship between KLF5 binding and epigenetic changes. Research has shown that KLF5-mediated early epigenetic modifications at enhancers can persist even as KLF5 protein levels decline .

• Functional validation: Combine with luciferase reporter assays to correlate epigenetic changes with transcriptional output, as demonstrated in studies of KLF5 and Foxo1 co-regulation of the Fbxo32 promoter .

• Gene knockout controls: Compare epigenetic profiles between wild-type and KLF5 knockout/knockdown cells to establish causality. Research showed that in KLF5 conditional knockout muscle cells, dexamethasone-responsive induction of H3K27ac was suppressed at Fbxo32 enhancers .

This approach enables researchers to establish mechanistic links between KLF5 binding, epigenetic modifications, and transcriptional outcomes in diverse biological contexts.

How can researchers integrate KLF5 antibody data with transcriptomic analyses?

Integrating KLF5 antibody-generated data with transcriptomic analyses creates a powerful approach to understand KLF5's regulatory networks. A comprehensive methodological framework includes:

• ChIP-seq and RNA-seq integration: Combine KLF5 binding profiles from ChIP-seq with gene expression changes from RNA-seq to identify direct KLF5 targets. Previous research utilized this approach to identify KLF5 binding sites in the Fbxo32 gene locus .

• Temporal analyses: Conduct time-course experiments measuring KLF5 binding (via ChIP) and consequent gene expression changes (via RNA-seq) to establish cause-effect relationships. Research has shown that early KLF5-mediated epigenetic modifications can affect gene expression even after KLF5 levels decline .

• Differential binding analysis: Compare KLF5 binding patterns between experimental conditions (e.g., with/without stimulus, wild-type vs. disease models) to identify context-specific regulatory events. For example, dexamethasone treatment enhanced KLF5 binding to the Fbxo32 enhancer .

• Motif analysis: Identify enriched sequence motifs in KLF5-bound regions to understand binding preferences and potential co-factors. Research has identified putative binding motifs for KLF5 and Foxo1 in regulatory regions of atrophy-related genes .

• Network analysis: Construct gene regulatory networks based on KLF5 binding and expression data to visualize broader regulatory impacts.

• Validation experiments: Confirm key findings using reporter assays, directed mutagenesis of binding sites, and targeted gene manipulations. Research demonstrated that KLF5 and Foxo1 together transactivated the Fbxo32 promoter to a greater degree than either factor alone .

This integrated approach enables researchers to comprehensively map KLF5's regulatory influence across the genome and understand its role in complex biological processes.

What are the current understandings of KLF5's role in disease pathogenesis based on antibody studies?

Antibody-based studies have significantly contributed to our understanding of KLF5's role in disease processes. While FITC-conjugated KLF5 antibodies specifically haven't been extensively documented in disease research, KLF5 antibodies broadly have revealed critical insights:

• Muscle Atrophy: KLF5 has been identified as a key transcriptional regulator mediating muscle atrophy. Studies using KLF5 antibodies for ChIP and Western blotting demonstrated that KLF5 cooperates with Foxo1 to induce expression of atrophy-related genes like Fbxo32 (encoding Atrogin-1) and Trim63 (encoding MuRF1) . In mouse models, muscle-specific KLF5 knockout (using Ckm-Cre;Klf5 mice) protected against glucocorticoid-induced atrophy, revealing KLF5 as a potential therapeutic target .

• Cancer Research: Although not explicitly detailed in the provided search results, KLF5 antibodies are used extensively in cancer research based on the wide availability of these research tools . KLF5's role as a transcription factor that binds to GC box promoter elements suggests its involvement in regulating proliferation-related genes.

• Developmental Disorders: KLF5's interaction with developmental transcription factors like MyoD and Mef2 during myogenic differentiation indicates its potential role in developmental processes . Antibody-based studies help track KLF5 expression patterns during development.

• Therapeutic Target Validation: Research using KLF5 antibodies has validated KLF5 as a potential therapeutic target. For instance, pharmacological intervention with Am80 has been identified as a potentially preventive treatment for muscle atrophy by modulating KLF5-mediated pathways .

For researchers investigating disease mechanisms, considering KLF5's tissue-specific roles and interaction partners is crucial for experimental design and interpretation.

What are common issues with FITC-conjugated antibodies and how can they be addressed?

FITC-conjugated antibodies, including KLF5 antibodies, present several common technical challenges that researchers should anticipate and address:

• Photobleaching: FITC is relatively prone to photobleaching compared to other fluorophores.

  • Solution: Minimize exposure to light during storage and handling. Use anti-fade mounting media containing appropriate preservatives. Consider image acquisition parameters that minimize exposure while maintaining adequate signal.

• Autofluorescence interference: Cellular components can emit autofluorescence in the same spectral range as FITC.

  • Solution: Include unstained controls to assess background. Use spectral unmixing for confocal microscopy or flow cytometry. Consider tissue-specific autofluorescence quenching reagents.

• pH sensitivity: FITC fluorescence is optimal at alkaline pH (8-9) and decreases at lower pH values.

  • Solution: Maintain appropriate pH in buffers (typically pH 7.4-8.0). Be aware that intracellular compartments with acidic pH may show reduced FITC signal.

• Storage degradation: FITC conjugates can lose activity during storage.

  • Solution: Follow manufacturer recommendations to store at -20°C or -80°C and avoid repeated freeze-thaw cycles . Aliquot antibody upon first thaw to minimize freeze-thaw cycles.

• Signal strength: Direct FITC conjugation sometimes yields lower signal than indirect detection methods.

  • Solution: Optimize antibody concentration through titration experiments. Consider signal amplification systems if needed for low-abundance targets.

• Antibody specificity: Conjugation can occasionally affect binding properties.

  • Solution: Include appropriate positive and negative controls to validate specific staining. Compare staining patterns with unconjugated antibody versions when possible.

For KLF5 FITC-conjugated antibodies specifically, optimizing nuclear permeabilization is crucial since KLF5 is a nuclear protein. Consider using specialized nuclear permeabilization buffers containing appropriate detergents to ensure antibody access to nuclear antigens.

How can researchers validate KLF5 antibody specificity in their experimental systems?

Rigorous validation of KLF5 antibody specificity is critical for generating reliable research data. A comprehensive validation approach includes:

• Knockout/knockdown controls: The gold standard for antibody validation is testing in KLF5 knockout or knockdown systems.

  • Method: Compare staining/signal between wild-type cells and cells with genetic deletion of KLF5 (such as the Ckm-Cre;Klf5 mouse model mentioned in the literature ) or siRNA-mediated knockdown.

• Overexpression controls: Complementary to knockout approaches, overexpression can confirm antibody specificity.

  • Method: Compare signal between cells with endogenous KLF5 levels and those overexpressing KLF5, expecting proportional signal increases.

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide/protein.

  • Method: If the antibody was raised against a specific recombinant KLF5 protein fragment (such as the 69-346AA region mentioned in product specifications ), pre-incubation with this peptide should abolish specific staining.

• Multiple antibody comparison: Use different antibodies recognizing distinct epitopes of KLF5.

  • Method: Concordant results with antibodies targeting different regions of KLF5 increase confidence in specificity.

• Expected expression pattern and subcellular localization: KLF5 should show nuclear localization and tissue-specific expression patterns.

  • Method: Verify nuclear localization using counterstains like DAPI and check expression levels across tissues known to express KLF5 (such as testis and placenta ).

• Western blot molecular weight verification: Confirm that the detected protein matches KLF5's expected molecular weight (approximately 50.8 kDa for the canonical form ).

  • Method: Include molecular weight markers and assess band migration pattern, considering potential post-translational modifications.

• Correlation with mRNA expression: Compare protein detection with mRNA levels across experimental conditions.

  • Method: Parallel qPCR and antibody-based detection should show concordant patterns in response to experimental manipulations.

This systematic approach ensures confidence in antibody specificity, critical for accurate interpretation of experimental results.

What are best practices for storing and handling FITC-conjugated KLF5 antibodies to maintain optimal performance?

Proper storage and handling of FITC-conjugated KLF5 antibodies is essential for maintaining their performance characteristics and extending their useful life. Based on manufacturer recommendations and standard laboratory practices:

• Temperature considerations:

  • Store antibody at -20°C or -80°C for long-term storage as recommended by the manufacturer .

  • Avoid repeated freeze-thaw cycles that can degrade both the antibody and the fluorophore .

  • For working stocks, store at 4°C protected from light for up to 2 weeks.

• Aliquoting strategy:

  • Upon first thaw, divide the antibody into single-use aliquots based on your typical experimental needs.

  • Use small, opaque tubes that minimize exposure to air and light.

  • Document the date of aliquoting and number of freeze-thaw cycles on each tube.

• Protection from light:

  • FITC is particularly susceptible to photobleaching; always protect from light exposure.

  • Use amber tubes for storage or wrap regular tubes in aluminum foil.

  • Minimize light exposure during experimental procedures by working under reduced ambient lighting.

• Buffer considerations:

  • The antibody is supplied in a specialized buffer (containing 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) optimized for stability.

  • Avoid diluting in buffers that might alter pH outside the optimal range for FITC (pH 7.4-8.5).

  • If dilution is necessary, use buffers with similar composition to the original formulation.

• Contamination prevention:

  • Use sterile technique when handling antibody solutions.

  • Include preservatives (such as 0.03% Proclin 300 as used in the commercial formulation ) in any working dilutions.

  • Avoid introducing microbial contamination that can degrade the antibody.

• Quality monitoring:

  • Periodically test antibody performance using positive controls.

  • Document lot numbers and performance characteristics.

  • Consider including fluorescence intensity standards in experiments to monitor potential degradation over time.

Following these best practices will help maintain antibody performance and extend the usable life of FITC-conjugated KLF5 antibodies in research applications.

How should researchers interpret conflicting results between different KLF5 detection methods?

When faced with conflicting results between different KLF5 detection methods, researchers should implement a systematic troubleshooting and interpretation approach:

• Method-specific limitations assessment:

  • FITC-conjugated antibodies may show lower sensitivity than amplified detection systems.

  • Western blotting detects denatured protein while immunofluorescence and ChIP detect native forms, potentially affecting epitope accessibility.

  • Flow cytometry provides quantitative single-cell analysis but may lack spatial context.

• Epitope consideration:

  • Different antibodies recognize distinct epitopes that may be differentially affected by:

    • Post-translational modifications

    • Protein-protein interactions

    • Conformational changes due to experimental conditions

  • Map the epitope regions of different antibodies (e.g., the recombinant Human KLF5 protein fragment 69-346AA used as immunogen ) relative to functional domains.

• Isoform awareness:

  • KLF5 exists in multiple isoforms (at least 4 reported ).

  • Determine whether conflicting results might reflect detection of different isoforms.

  • Use isoform-specific primers for RT-PCR validation alongside protein detection.

• Context dependency:

  • KLF5 function and expression are highly context-dependent.

  • Consider whether conflicting results reflect genuine biological variation across:

    • Cell types (e.g., preferential expression in testis and placenta )

    • Experimental conditions

    • Developmental stages

    • Stimulus responsiveness (e.g., dexamethasone-induced changes )

• Validation hierarchy:

  • Establish a hierarchy of methods based on sensitivity and specificity.

  • Validate results using orthogonal approaches (e.g., if antibody-based methods conflict, add functional assays such as reporter studies ).

  • Consider genetic approaches (knockdown/knockout) as definitive validation.

• Quantification rigor:

  • Ensure appropriate quantification methods for each technique.

  • Use statistical analyses appropriate for the data type.

  • Report conflicting results transparently, discussing potential methodological explanations.

What statistical approaches are recommended for analyzing co-localization of KLF5 with other nuclear factors?

Analyzing co-localization of KLF5 with other nuclear factors requires rigorous statistical approaches to distinguish genuine biological associations from random overlap. Recommended statistical methods include:

• Pearson's and Mander's correlation coefficients:

  • Pearson's coefficient measures the linear correlation between fluorescence intensities.

  • Mander's coefficients measure the fraction of one signal overlapping with another.

  • Application: Quantify co-localization between FITC-conjugated KLF5 antibody signal and other nuclear factors (e.g., Foxo1, which has been shown to interact with KLF5 ).

  • Threshold determination: Use automated methods such as Costes' approach to set objective thresholds.

• Object-based co-localization analysis:

  • Particularly suitable for punctate nuclear structures.

  • Identifies discrete objects in each channel and measures their spatial relationships.

  • Application: Analyze KLF5 binding to specific nuclear domains or chromatin regions.

  • Statistical testing: Use nearest neighbor distance analysis with appropriate randomization controls.

• Intensity correlation analysis (ICA):

  • Examines whether pixel intensities in two channels vary synchronously.

  • Calculates intensity correlation quotient (ICQ) values.

  • Application: Assess whether KLF5 concentration correlates spatially with other factors like MyoD or Mef2, which cooperate with KLF5 during myogenic differentiation .

• Randomization tests:

  • Generate random distributions by rotating or shifting one channel relative to the other.

  • Compare observed co-localization metrics to the distribution from randomized data.

  • Application: Determine statistical significance of observed KLF5 co-localization patterns.

• Bayesian approaches:

  • Account for uncertainty in image segmentation and classification.

  • Particularly useful for noisy or low-resolution images.

  • Application: More robust analysis when signal-to-noise ratio is suboptimal.

• Spatial statistics:

  • Ripley's K-function and related methods assess spatial clustering.

  • Application: Determine whether KLF5 and interaction partners show coordinated spatial organization beyond simple overlap.

• Temporal correlation analysis:

  • For live-cell imaging data, analyze the temporal dynamics of co-localization.

  • Application: Study dynamic interactions between KLF5 and other factors during cellular responses to stimuli like dexamethasone .

When publishing co-localization analyses, researchers should report:

• Raw images alongside processed data

• Clear description of preprocessing steps

• Objective criteria for threshold determination

• Statistical tests with appropriate randomization controls

• Effect sizes along with p-values