KLF5 Antibody, Biotin conjugated

What is KLF5 and why is it significant in research?

KLF5 is a zinc finger-containing transcription factor belonging to the KLF family of transcription regulators that play diverse roles in cell proliferation, differentiation, inflammation, and pluripotency. KLF5 has gained significant research attention due to its involvement in various cancer-related processes, including epithelial-mesenchymal transition (EMT), invasion, and metastasis . Recent studies have highlighted KLF5's critical role in prostate cancer, where it is expressed at high levels in a subset of castration-resistant prostate cancer (CRPC) tissues and models, including neuroendocrine prostate cancer (NEPC) . In this context, KLF5 supports oncogenic phenotypes and transcriptionally opposes androgen receptor (AR) to induce ERBB2 and basal cell identity . Additionally, KLF5 deficiency specific to renal collecting duct epithelial cells has been shown to impair cardiac stress adaptation response coordinated between the heart and kidney .

What are the key specifications of commercially available biotin-conjugated KLF5 antibodies?

Biotin-conjugated KLF5 antibodies are available with various specifications to suit different research applications. Most commercially available options are polyclonal antibodies raised in rabbits . These antibodies may target either the total KLF5 protein or specific phosphorylation sites, such as pSer275 . The reactivity spectrum varies by product, with some antibodies recognizing human KLF5 only , while others can detect KLF5 across multiple species including human, mouse, rat, cow, pig, and dog . The immunogens used for antibody production typically involve KLH-conjugated synthetic phosphopeptides (for phospho-specific antibodies) or recombinant KLF5 protein fragments (such as amino acids 69-346) . Most biotin-conjugated KLF5 antibodies have been validated for applications including Western blotting , ELISA , and potentially immunofluorescence and immunohistochemistry based on the validation of their non-biotinylated counterparts.

How should biotin-conjugated KLF5 antibodies be stored and handled to maintain optimal activity?

Proper storage of biotin-conjugated KLF5 antibodies is essential to maintain their activity and specificity. The standard recommended storage condition is -20°C for long-term preservation . To prevent degradation from repeated freeze-thaw cycles, it is advisable to aliquot the antibody upon receipt . Protection from light is critical as exposure can degrade the biotin conjugate and reduce antibody effectiveness . These antibodies are typically supplied in buffers containing stabilizers such as PBS with glycerol (50%) and preservatives like Proclin-300 (0.03%) . The antibody solution should be stored in non-reactive containers, preferably polypropylene tubes. For short-term use (1-2 weeks), working solutions can be kept at 4°C, but prolonged storage at this temperature should be avoided as it may lead to reduced activity and increased bacterial contamination risk. When handling these antibodies, avoid introducing microbial contamination, excessive agitation, or exposure to extreme pH conditions that could compromise antibody integrity.

What applications are biotin-conjugated KLF5 antibodies best suited for in research?

Biotin-conjugated KLF5 antibodies are versatile research tools suited for multiple applications. Based on the available information, these antibodies are primarily validated for Western blotting, particularly for phospho-specific detection such as pSer275 KLF5 . They are also effective in ELISA applications for detecting human KLF5 . The biotin-streptavidin system provides signal amplification advantages that make these antibodies potentially valuable for immunohistochemistry and immunofluorescence applications, though specific validation for the biotin-conjugated forms may be required. While not directly evident from the search results for biotin conjugates, related KLF5 antibodies have been validated for flow cytometry (as seen with the Alexa Fluor 488 conjugate) , suggesting biotin-conjugated versions could also be utilized in flow cytometry with appropriate streptavidin-fluorophore detection systems. For chromatin immunoprecipitation (ChIP) studies investigating KLF5's role in transcriptional regulation and super-enhancer binding, biotin-conjugated antibodies may require special optimization to prevent interference from the biotin-streptavidin interaction with DNA-protein complexes.

What are the known expression patterns of KLF5 in different cell and tissue types?

KLF5 exhibits distinct expression patterns across various cellular and tissue types that are relevant to research applications. In prostate cancer, which has been extensively studied, KLF5 shows variable expression with notably higher levels in castration-resistant prostate cancer (CRPC) tissues and models compared to hormone-sensitive tumors . Cell line studies have shown that CRPC-derived lines (such as C4-2B, 16D, 49F, and 42D) display higher baseline levels of KLF5 than androgen-sensitive parental lines like LNCaP . KLF5 expression can be dynamically regulated by androgens, with sustained induction observed over 24-48 hours in C4-2B cells treated with physiological doses (1 nM) of dihydrotestosterone (DHT) . Interestingly, cells previously exposed to AR-targeted therapy (such as enzalutamide) show enhanced and more sustained KLF5 induction in response to subsequent androgen treatment . Beyond prostate tissues, KLF5 (also known as intestinal KLF) is expressed in epithelial cells of the gastrointestinal tract . It also plays important roles in renal collecting duct epithelial cells, cardiovascular development, and has been implicated in various other epithelial tissues where it typically marks proliferating cell populations.

How do phospho-specific KLF5 antibodies (like pSer275) differ from pan-KLF5 antibodies in research applications?

Phospho-specific KLF5 antibodies (such as pSer275) and pan-KLF5 antibodies serve fundamentally different research purposes and require distinct experimental considerations. Phospho-specific antibodies recognize KLF5 only when phosphorylated at specific residues (like Ser275) , providing critical information about the post-translational modification state rather than total protein levels. This specificity makes them invaluable for studying signal transduction pathways that regulate KLF5 activity through phosphorylation. When working with phospho-specific KLF5 antibodies, researchers must implement more stringent sample preparation protocols to preserve phosphorylation status, including the use of phosphatase inhibitors during protein extraction and specialized blocking agents to reduce non-specific binding . Validation of phospho-specific antibodies requires specialized controls such as phosphatase treatment or peptide competition assays using phosphorylated versus non-phosphorylated peptides.

What experimental controls should be implemented when using biotin-conjugated KLF5 antibodies?

Rigorous experimental controls are essential when using biotin-conjugated KLF5 antibodies to ensure reliable and interpretable results. Primary negative controls should include the appropriate isotype control (typically rabbit IgG) conjugated to biotin and used at the same concentration as the KLF5 antibody . This controls for non-specific binding of the antibody structure itself. When studying phospho-specific KLF5 (such as pSer275), additional negative controls should include samples treated with phosphatase to demonstrate specificity for the phosphorylated form .

For positive controls, cell lines or tissues known to express high levels of KLF5 should be used. Based on the search results, CRPC cell lines such as C4-2B, 16D, 49F, and 42D show higher baseline levels of KLF5 and could serve as positive controls . Additionally, androgen treatment (1 nM DHT for 24-48 hours) can be used to induce KLF5 expression in appropriate cell models . When using biotin-conjugated antibodies, researchers must also implement controls for endogenous biotin, particularly in tissues or cells with high biotin content. This includes using avidin/biotin blocking kits prior to antibody application.

For quantitative applications, standard curve controls using recombinant KLF5 protein at known concentrations can help establish the linear detection range. Finally, biological validation controls such as KLF5 knockdown/knockout samples provide the most stringent confirmation of antibody specificity and should be included whenever possible, especially when investigating novel KLF5 functions or expression patterns.

How can researchers optimize protocols for detecting KLF5 in prostate cancer models?

Optimizing protocols for KLF5 detection in prostate cancer models requires careful consideration of the dynamic regulation of KLF5 in these systems, as revealed by recent research . Based on the search results, several key optimization strategies can be implemented:

First, researchers should select appropriate cell models representing different stages of prostate cancer progression. The search results indicate significant differences in KLF5 expression between androgen-dependent cell lines (like LNCaP) and castration-resistant models (such as C4-2B, 16D, 49F, and 42D) . Therefore, positive and negative controls should be carefully selected based on the specific research question.

Timing considerations are crucial when studying KLF5 in response to treatments. The search results demonstrate that sustained induction of KLF5 by androgens occurs over 24-48 hours, with different kinetics depending on the cell line . For example, C4-2B cells show sustained KLF5 induction with 1 nM DHT, while LNCaP cells require higher concentrations (10 nM R1881) for similar effects . Time-course experiments should therefore span at least 48 hours to capture the full dynamics of KLF5 regulation.

For Western blotting applications, nuclear extraction protocols should be optimized as KLF5 is a transcription factor primarily localized to the nucleus. When using phospho-specific antibodies like anti-pSer275 , phosphatase inhibitors must be included during sample preparation. For immunohistochemistry or immunofluorescence, optimization of antigen retrieval methods is essential, with citrate buffer (pH 6.0) often providing good results for nuclear transcription factors.

When using biotin-conjugated KLF5 antibodies in prostate cancer tissues, blocking endogenous biotin is particularly important as prostate tissues can contain significant levels of endogenous biotin. A specialized blocking step using avidin-biotin blocking kits should precede antibody application. Additionally, researchers should consider the influence of previous treatments on KLF5 expression—the search results demonstrate that prior exposure to enzalutamide affects the transcriptional response of KLF5 to androgens , which could impact experimental outcomes and interpretation.

What strategies can be employed to study KLF5 and AR crosstalk in prostate cancer?

The intricate relationship between KLF5 and Androgen Receptor (AR) signaling in prostate cancer represents a complex area of research requiring specialized experimental approaches. Based on the search results, several strategic approaches can effectively investigate this crosstalk:

Hormonal manipulation experiments provide critical insights into KLF5-AR interactions. Researchers should design time-course studies with physiological doses of androgens (1 nM DHT) as well as stronger synthetic androgens (10 nM R1881) to observe differential KLF5 induction patterns . The search results reveal that androgen stimulation sustains KLF5 induction over 24-48 hours in CRPC cell lines, with different kinetics than in androgen-sensitive lines . Additionally, testing the effects of anti-androgens such as enzalutamide on KLF5 expression is valuable—interestingly, pre-treatment with enzalutamide enhances subsequent KLF5 induction by androgens .

For mechanistic studies of KLF5-AR crosstalk at the genomic level, chromatin immunoprecipitation (ChIP) experiments targeting both factors can reveal shared or opposing binding sites. The search results mention a KLF5 super-enhancer region that displays AR binding in prostate cancer cells . ChIP-seq studies comparing KLF5 and AR binding patterns before and after treatments can illuminate how these factors cooperate or compete at specific genomic loci.

Functional studies should examine how KLF5 affects AR-mediated transcription and vice versa. The search results suggest that KLF5 "transcriptionally opposes AR to induce ERBB2 and basal cell identity" , indicating that gene expression studies examining AR target genes with and without KLF5 manipulation (overexpression or knockdown) would be informative. Similarly, examining how AR activation or inhibition affects KLF5 target gene expression can reveal reciprocal regulation.

Finally, phenotypic studies investigating how the KLF5-AR balance affects cancer progression are essential. The search results indicate that KLF5 overexpression is oncogenic in AR-positive prostate cancer cells . Therefore, researchers should assess how manipulating both factors affects cancer-relevant phenotypes such as proliferation, invasion, treatment resistance, and lineage plasticity, particularly the transition to AR-independent phenotypes like neuroendocrine prostate cancer.

How can researchers troubleshoot common issues with biotin-conjugated KLF5 antibodies in multiplex assays?

Multiplex assays using biotin-conjugated KLF5 antibodies present unique challenges that require systematic troubleshooting. One common issue is high background signal, which can result from endogenous biotin in samples. Researchers should implement a specific avidin-biotin blocking step prior to applying the biotin-conjugated KLF5 antibody . Additionally, using biotin-free culture media for cell-based assays and biotin-free blocking buffers (typically BSA-based rather than milk-based) can significantly reduce background. If high background persists, titrating both the biotin-conjugated KLF5 antibody and the streptavidin detection reagent is recommended.

Another common challenge is signal interference between multiple detection channels in multiplex experiments. When combining biotin-conjugated KLF5 antibodies with other detection methods, sequential rather than simultaneous detection often yields better results. For example, complete the biotin-streptavidin detection for KLF5 before proceeding with additional antibodies. If crossover signal remains problematic, implementing a biotin blocking step between detection rounds can prevent the second streptavidin conjugate from binding to the first biotin-conjugated antibody.

For flow cytometry applications, suboptimal signal separation may occur. Based on the search results showing successful use of fluorophore-conjugated KLF5 antibodies in flow cytometry , researchers should carefully select streptavidin conjugates with minimal spectral overlap with other fluorophores in the panel. Additionally, proper compensation controls are essential for accurate data interpretation.

In tissue samples, inadequate penetration of detection reagents can lead to inconsistent KLF5 staining. Optimizing tissue preparation, including fixation time, permeabilization conditions, and antigen retrieval methods, is crucial for reliable results. The nuclear localization of KLF5 may require more stringent permeabilization conditions than cytoplasmic proteins.

Finally, variable KLF5 expression levels across different models can lead to inconsistent results. The search results indicate significantly different baseline levels and induction patterns of KLF5 between androgen-sensitive and castration-resistant prostate cancer models . Researchers should adjust exposure times, antibody concentrations, and detection sensitivity based on the expected KLF5 expression level in each model system.

What are the optimized protocols for using biotin-conjugated KLF5 antibodies in chromatin immunoprecipitation (ChIP)?

Chromatin immunoprecipitation with biotin-conjugated KLF5 antibodies requires careful optimization, particularly when studying KLF5's role in transcriptional regulation. An optimized ChIP protocol should begin with effective crosslinking—for KLF5, which binds to super-enhancer regions as indicated in the search results , dual crosslinking often yields better results. This involves treating cells with 2 mM disuccinimidyl glutarate (DSG) for 45 minutes followed by 1% formaldehyde for 10 minutes at room temperature to capture both direct and indirect protein-DNA interactions.

Chromatin fragmentation should aim for fragments between 200-500 bp, which provides sufficient resolution to distinguish individual binding sites while maintaining enough DNA for efficient immunoprecipitation. Sonication conditions must be empirically determined for each cell type, with verification by agarose gel electrophoresis. For studying KLF5 binding to super-enhancer regions described in the search results , thorough fragmentation is particularly important.

The immunoprecipitation step requires special considerations when using biotin-conjugated antibodies. Pre-clearing chromatin with protein A/G beads helps reduce non-specific binding. For the immunoprecipitation itself, 2-5 μg of biotin-conjugated KLF5 antibody should be incubated with pre-cleared chromatin overnight at 4°C. Importantly, streptavidin-coated magnetic beads must be used instead of protein A/G beads to capture the biotin-conjugated antibodies. To reduce background, high-stringency washes with buffers containing increasing salt concentrations are recommended.

Essential controls include input chromatin (5-10% of starting material), a negative control using biotin-conjugated IgG from the same species, and a positive control targeting a known KLF5 binding region. The search results identify the KLF5 super-enhancer region as a potential positive control locus . For data analysis, qPCR primers should be designed to cover known or predicted KLF5 binding sites, with normalization to input and calculation of percent input or fold enrichment relative to the IgG control.

How can researchers effectively use biotin-conjugated KLF5 antibodies in flow cytometry?

Flow cytometry using biotin-conjugated KLF5 antibodies requires specific optimization strategies to achieve reliable detection of this primarily nuclear transcription factor. The search results indicate successful use of fluorophore-conjugated KLF5 antibodies (specifically Alexa Fluor 488) in flow cytometry , suggesting that biotin-conjugated versions can be similarly applied with appropriate modifications.

Cell preparation is a critical first step. Since KLF5 is predominantly nuclear, effective fixation and permeabilization are essential. A recommended protocol includes fixation with 2-4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 or commercial permeabilization buffers optimized for nuclear antigens. When studying phosphorylated KLF5 forms such as pSer275 , phosphatase inhibitors should be included throughout the sample preparation process.

For staining, an initial dilution of 1:50 for the biotin-conjugated KLF5 antibody is suggested based on similar conjugated antibody recommendations , though this should be optimized for each specific antibody and cell type. After incubation with the primary antibody (typically 30-60 minutes at room temperature or overnight at 4°C), cells should be washed thoroughly before adding an appropriate streptavidin-fluorophore conjugate. The choice of fluorophore should consider the cytometer configuration and other markers in multiplex panels.

Essential controls include unstained cells, cells stained with biotin-conjugated isotype control antibody followed by streptavidin-fluorophore, and positive control cells known to express high levels of KLF5 (such as C4-2B cells based on the search results ). For accurate gating, fluorescence minus one (FMO) controls should be included, especially in multiplex panels. When studying KLF5 in response to treatments such as androgen stimulation, time-course experiments should be considered, as the search results show dynamic changes in KLF5 expression over 24-48 hours post-treatment .

For data analysis, care should be taken to gate on single, viable cells before analyzing KLF5 expression. Given the nuclear localization and potentially heterogeneous expression of KLF5, researchers should consider analyzing both the percentage of positive cells and the mean fluorescence intensity (MFI) to fully characterize KLF5 expression patterns.

What methods can be used to validate the specificity of biotin-conjugated KLF5 antibodies?

Validating the specificity of biotin-conjugated KLF5 antibodies is crucial for generating reliable research data. A comprehensive validation strategy should incorporate multiple complementary approaches. Genetic validation techniques provide the most definitive evidence of specificity. CRISPR/Cas9-mediated knockout of KLF5 serves as the gold standard—the antibody signal should be absent or dramatically reduced in knockout cells compared to wild-type controls. Similarly, siRNA or shRNA knockdown of KLF5 should result in a corresponding reduction in signal intensity proportional to the knockdown efficiency. Conversely, overexpression of KLF5 should lead to increased antibody signal.

For phospho-specific KLF5 antibodies such as pSer275 , additional validation is necessary. Samples should be treated with phosphatase to remove the phosphorylation, which should eliminate the signal from phospho-specific antibodies while leaving total KLF5 detection unaffected. Peptide competition assays provide another layer of validation—pre-incubation of the antibody with the immunizing phosphopeptide should abolish specific signal, while pre-incubation with non-phosphorylated peptide should not affect binding.

Western blot validation should confirm that the detected protein migrates at the expected molecular weight for KLF5 (approximately 55-60 kDa). Multiple band patterns may indicate detection of isoforms, post-translational modifications, or degradation products and should be carefully characterized. Comparison with other validated KLF5 antibodies targeting different epitopes can provide further confidence in specificity.

Application-specific validation is also important. For immunohistochemistry or immunofluorescence, the staining pattern should be consistent with the expected nuclear localization of KLF5 as a transcription factor. For flow cytometry, comparing the staining pattern with that obtained using other detection methods provides additional validation. When studying KLF5 in prostate cancer models, the antibody should detect the expected differences in expression levels between androgen-sensitive and castration-resistant cell lines as described in the search results .

How do biotin-conjugated antibodies compare with other conjugation methods for studying KLF5?

Biotin conjugation represents one of several antibody labeling strategies available for KLF5 research, each with distinct advantages and limitations for particular applications. Comparative analysis with other conjugation methods, such as direct fluorophore labeling (exemplified by the Alexa Fluor 488 conjugate mentioned in the search results ), reveals important differences that can influence experimental outcomes.

Biotin-conjugated KLF5 antibodies offer significant signal amplification capabilities through the high-affinity biotin-streptavidin interaction. This amplification is particularly valuable when detecting low-abundance targets or studying specific post-translational modifications like phosphorylated KLF5 (pSer275) . The small size of the biotin molecule (244 Da) creates minimal steric hindrance, allowing the antibody to access KLF5 when it is complexed with other proteins or bound to DNA, which is particularly relevant when studying transcription factors like KLF5 in chromatin contexts.

In contrast, direct fluorophore conjugation, such as the Alexa Fluor 488 conjugate mentioned in the search results , offers a streamlined, single-step detection process without requiring secondary reagents. This simplifies workflow and reduces variability, particularly beneficial in high-throughput or clinical applications. Fluorophore-conjugated antibodies typically provide more linear signal response for quantitative applications, which is advantageous when measuring dynamic changes in KLF5 levels following treatments such as androgen stimulation as described in the search results .

For multiplex applications studying KLF5 alongside other proteins, each conjugation method presents different considerations. Biotin conjugation limits multiplexing options as all biotinylated antibodies compete for the same streptavidin detection reagent. Conversely, direct fluorophore conjugation enables more straightforward multiplexing with antibodies carrying spectrally distinct fluorophores. When studying the complex interplay between KLF5 and AR signaling described in the search results , multiplex capabilities become particularly important.

Application-specific performance also varies. For flow cytometry, the search results demonstrate successful use of fluorophore-conjugated KLF5 antibodies , while for Western blotting and ELISA, biotin-conjugated antibodies offer enhanced sensitivity through signal amplification . For challenging applications like ChIP-seq investigating KLF5's association with super-enhancer regions mentioned in the search results , the choice between conjugation methods depends on whether sensitivity or workflow simplicity is prioritized.

What are the future directions for KLF5 antibody research?

The evolving landscape of KLF5 research points toward several promising future directions for KLF5 antibody development and application. As our understanding of KLF5's role in cancer progression deepens, particularly its opposition to AR signaling in prostate cancer as highlighted in the search results , there will be increasing demand for antibodies that can detect specific functional states of KLF5. Development of additional phospho-specific antibodies beyond pSer275 will be valuable, as various phosphorylation events likely regulate different aspects of KLF5 function in context-dependent manners.

The emergence of spatial transcriptomics and proteomics creates opportunities for developing KLF5 antibodies compatible with these advanced technologies. Antibodies optimized for highly multiplexed imaging methods such as Imaging Mass Cytometry (IMC) or CO-Detection by indEXing (CODEX) would enable researchers to study KLF5 in relation to dozens of other proteins simultaneously while preserving spatial context. This would be particularly valuable for understanding KLF5's role in tumor heterogeneity and microenvironmental interactions.

The search results' findings regarding KLF5's dynamic regulation in response to cancer therapies, specifically how AR-targeted therapy enhances KLF5's transcriptional response to androgens , highlight the need for antibodies suitable for longitudinal in vivo imaging. Development of KLF5 antibodies compatible with non-invasive imaging techniques could enable monitoring of KLF5 expression during treatment and disease progression in preclinical models.

As single-cell technologies continue to advance, there will be increasing demand for KLF5 antibodies optimized for single-cell protein analysis techniques such as CyTOF and CITE-seq. Such tools would allow researchers to correlate KLF5 protein levels with transcriptomic profiles at single-cell resolution, providing unprecedented insights into cellular heterogeneity in cancer and other contexts.

Finally, the therapeutic implications of KLF5's role in cancer progression suggest potential for developing function-blocking KLF5 antibodies that could serve as therapeutic agents or research tools for inhibiting specific KLF5 activities. Given KLF5's context-dependent roles in different tissues, highly specific approaches targeting cancer-specific functions of KLF5 would be particularly valuable.

How can researchers integrate KLF5 antibody data with other molecular techniques for comprehensive analysis?

Integrating KLF5 antibody-derived data with complementary molecular techniques creates a more comprehensive understanding of KLF5 biology. Multi-omics integration approaches have become increasingly important, particularly in complex research areas such as KLF5's role in cancer progression as described in the search results . Researchers can implement several strategies to effectively integrate these diverse data types.

Correlation of KLF5 protein expression data from antibody-based techniques with KLF5 mRNA expression provides insights into post-transcriptional regulation. Discrepancies between protein and mRNA levels may indicate regulation through microRNAs, RNA-binding proteins, or protein stability mechanisms. This integration is particularly relevant given the search results' findings about dynamic regulation of KLF5 in response to androgen treatment and AR-targeted therapies .

ChIP-seq data using KLF5 antibodies can be integrated with RNA-seq to correlate KLF5 binding events with transcriptional outcomes. The search results mention KLF5 binding to super-enhancer regions ; integrating this binding data with expression profiles would reveal direct transcriptional targets versus secondary effects. Additionally, integration with AR ChIP-seq data would illuminate the molecular basis of the opposing transcriptional programs of KLF5 and AR described in the search results .

For cancer research applications, integrating KLF5 immunohistochemistry data from tissue microarrays with patient clinical information enables correlation of KLF5 expression patterns with disease progression, treatment response, and survival outcomes. This integration is particularly relevant given KLF5's oncogenic effects in AR-positive prostate cancer cells mentioned in the search results .

Technological platforms enabling simultaneous analysis of proteins and nucleic acids from the same sample, such as Digital Spatial Profiling or CITE-seq, offer powerful approaches for direct integration. These methods could be applied to study how KLF5 protein levels correlate with expression of target genes like ERBB2 mentioned in the search results at single-cell or spatial resolution.

Computational integration using machine learning approaches can identify patterns across multiple data types that may not be apparent from individual analyses. This could reveal novel insights about KLF5's role in complex phenotypes such as the lineage plasticity in prostate cancer described in the search results . By implementing these integration strategies, researchers can develop a more holistic understanding of KLF5 biology that transcends the limitations of any single methodology.