KLF13 Antibody, HRP conjugated

What is KLF13 and what are its main biological functions?

KLF13 (Krüppel-like factor 13) is a member of the zinc-finger family of transcription factors that binds to GC-rich DNA elements. It functions primarily as a transcriptional regulator with dual activities - it can repress transcription by binding to the BTE site in competition with activator SP1 and by interacting with corepressor Sin3A and HDAC1, while also activating RANTES expression in T-cells . KLF13 is expressed predominantly in the heart, where it binds evolutionarily conserved sequences and appears to be crucial for cardiac development . Recent studies have also implicated KLF13 in neurodevelopmental processes and psychiatric disorders , as well as in SLE (systemic lupus erythematosus) pathogenesis through chromatin modification mechanisms .

What applications can KLF13 antibodies be used for in research?

KLF13 antibodies, including HRP-conjugated variants, can be employed in multiple experimental techniques:

KLF13 antibodies have been validated in multiple species, including human, mouse, and rat samples , making them valuable tools for comparative studies across model organisms.

How should KLF13 antibodies be stored and handled to maintain optimal activity?

For maximum stability and performance, KLF13 antibodies require specific storage conditions. The HRP-conjugated polyclonal antibody should be shipped at 4°C. Upon receipt, it should be stored at -20°C for short-term storage or -80°C for long-term storage . Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity.

What are the optimal conditions for using KLF13 antibody in immunohistochemistry?

For successful immunohistochemical detection of KLF13, researchers should consider the following protocol parameters:

How can I validate the specificity of KLF13 antibody in my experimental system?

Validation of KLF13 antibody specificity is crucial for reliable research outcomes. Several complementary approaches are recommended:

• Western blot analysis: Verify that the antibody detects a protein of the expected molecular weight (31 kDa calculated, though typically observed at 37-45 kDa due to post-translational modifications) . Known positive samples include HT-29 cells and rat liver tissue .

• Knockout/knockdown controls: Use KLF13 knockout or knockdown samples as negative controls. Literature shows at least three publications have utilized this approach for antibody validation .

• Recombinant protein testing: As demonstrated in developmental studies, antibody specificity can be confirmed by testing against recombinant KLF13 and other KLF family members to ensure selective recognition .

• Cross-reactivity assessment: Test the antibody against related KLF family members, particularly those with high sequence homology, to confirm specificity. The N-terminal domain (first 135 amino acids) is the most divergent across KLF family members (<30% homology) and is often used to generate specific antibodies .

How can KLF13 antibodies be used to investigate transcriptional regulation mechanisms?

KLF13 antibodies provide powerful tools for investigating transcriptional mechanisms through several advanced techniques:

• Chromatin Immunoprecipitation (ChIP): KLF13 antibodies can be used to identify genomic binding sites. Studies have employed streptavidin precipitation (ChSP) techniques with biotinylated KLF13 to map genome-wide binding patterns . For standard ChIP protocols, ensure chromatin is appropriately fragmented and use recommended antibody concentrations (typically 2-5 μg per immunoprecipitation).

• Electrophoretic Mobility Shift Assay (EMSA): KLF13 antibodies can be used in supershift assays to confirm the identity of KLF13 in protein-DNA complexes. Previous research demonstrated that addition of anti-KLF13 antibody abrogated DNA-protein complexes in cardiomyocyte extracts , confirming the presence of KLF13 in these regulatory complexes.

• Co-immunoprecipitation: To study protein-protein interactions involving KLF13, antibodies can pull down KLF13 and associated protein complexes. This approach has revealed interactions between KLF13 and transcriptional partners like GATA-4 .

• Reporter gene assays: While not directly using the antibody, these assays complement antibody-based approaches by validating functional effects of KLF13 on target genes identified through ChIP experiments, as demonstrated with genes like Klf16, Casp3, and E2f2 .

What are the considerations for using KLF13 antibody in developmental and disease-focused research?

When applying KLF13 antibodies to developmental biology or disease research, several methodological considerations are important:

• Spatiotemporal expression analysis: For developmental studies, KLF13 immunoreactivity has been successfully tracked across embryonic stages (E9.5-E14.5) and in specific tissues. Higher antibody concentrations (1:200) have been effective for tissue sections, while cultured cells may require more dilute solutions (1:5000) .

• Co-localization studies: Combining KLF13 antibody with markers for specific cell types enhances the understanding of its role in heterogeneous tissues. For instance, co-staining with desmin (marker for cardiomyocytes) helped identify KLF13 in both desmin-positive cardiomyocytes and desmin-negative cardioblasts during development .

• Disease-specific applications: In SLE research, KLF13 antibodies have helped identify its role in chromatin modification and disease pathogenesis . For neurodevelopmental studies, these antibodies have been used to investigate KLF13's protective effects against glutamate-induced excitotoxic damage .

• Protein structure alteration detection: In studies of genetic variants, KLF13 antibodies may help detect structural changes associated with mutations, complementing bioinformatic predictions of protein disorder changes .

What are common issues with KLF13 antibody experiments and how can they be resolved?

Researchers may encounter several challenges when working with KLF13 antibodies:

How can I differentiate between true KLF13 signals and non-specific binding?

Ensuring signal specificity is crucial for accurate interpretation of results:

• Knockout/Knockdown Validation: The gold standard for antibody validation is comparison with genetic models where KLF13 is absent or significantly reduced. Several publications have utilized this approach with KLF13 antibodies .

• Peptide Competition Assay: Pre-incubating the KLF13 antibody with the immunizing peptide should abolish specific binding. This method has been demonstrated in gel shift assays where cold probe competition eliminated specific KLF13 binding .

• Known Expression Pattern Correlation: Compare your findings with established KLF13 expression patterns. For instance, in murine development, KLF13 shows strong immunoreactivity in cardiac tissues (particularly atria), epidermis, brain mesenchyme, dorsal ganglia, skeletal muscles, and endothelial cells of vascular vessels in the liver by E14.5 .

• Multiple Antibody Validation: When possible, verify findings using a second KLF13 antibody targeting a different epitope.

How do post-translational modifications affect KLF13 detection with antibodies?

KLF13 undergoes several post-translational modifications that may impact antibody recognition:

• Apparent Molecular Weight Variation: While the calculated molecular weight of KLF13 is 31 kDa, it typically appears at 37-45 kDa in Western blots , likely due to post-translational modifications.

• Phosphorylation Sites: KLF13 contains multiple potential phosphorylation sites that may alter antibody binding efficiency depending on the cellular context and signaling conditions.

• Protein Disorder Regions: Bioinformatic analyses suggest that genetic variants can alter KLF13's intrinsic disorder profile, potentially affecting epitope accessibility. PONDR analysis reveals increased disorder in specific residues (three and four) in variant forms compared to wild-type protein .

• Metal-Binding Alterations: Amino acid changes in KLF13 can potentially alter metal-binding activity , which could affect zinc finger domain structure and consequently antibody recognition of conformational epitopes.

Researchers should be aware that experimental conditions that affect post-translational modification states may influence antibody detection sensitivity and specificity.

How can KLF13 antibody be used to investigate protein-protein interactions in different cellular contexts?

KLF13 antibodies provide valuable tools for exploring the protein interaction network:

• Co-Immunoprecipitation (Co-IP): KLF13 antibodies have successfully demonstrated physical interactions between KLF13 and other transcription factors such as GATA-4 . For co-IP experiments, use 1-5 μg of antibody with 200-500 μg of protein lysate.

• Proximity Ligation Assay (PLA): This technique allows visualization of protein-protein interactions in situ. While not explicitly mentioned in the provided search results, this method could be adapted using KLF13 antibodies to investigate interactions with known partners like GATA-4, Sin3A, or HDAC1 .

• Pull-Down Assays: Complementary to Co-IP, these assays have confirmed KLF13 interactions with transcriptional partners and can be designed to investigate novel interaction partners.

• Fluorescence Resonance Energy Transfer (FRET): By labeling KLF13 antibody and antibodies against potential binding partners with appropriate fluorophores, FRET can detect close physical associations in fixed or living cells.

When using these techniques, it's important to consider tissue-specific contexts, as KLF13 interactions may vary between cell types. For example, KLF13 shows differential expression between atrial and ventricular cardiomyocytes, which may impact its protein interaction network .

How do polyclonal and monoclonal KLF13 antibodies compare in different applications?

When selecting between polyclonal and monoclonal KLF13 antibodies, consider these application-specific differences:

For the HRP-conjugated polyclonal KLF13 antibody specifically, it has been generated against recombinant human KLF13 protein (amino acids 26-147) and purified using protein G . This region contains sequences that are relatively conserved across mammalian species, explaining its cross-reactivity.

What are the methodological differences when using KLF13 antibodies in various model systems?

Optimizing KLF13 antibody usage across different experimental models requires system-specific considerations:

• Cell Culture Models:

  • For immunofluorescence in cell cultures, higher dilutions (1:5000) have been reported effective

  • HT-29 cells have been validated as positive controls for Western blot analysis

  • For HT22 neuronal cell lines, KLF13 antibodies have helped identify its role in protection against glutamate-induced excitotoxicity

• Tissue Sections:

  • Lower dilutions (1:200) are typically required for tissue immunohistochemistry compared to cell cultures

  • Antigen retrieval methods vary by tissue type, with TE buffer (pH 9.0) generally recommended, though citrate buffer (pH 6.0) may be used as an alternative

  • Human brain tissue has been validated for IHC applications

• Developmental Models:

  • In murine embryos, KLF13 antibodies have successfully tracked expression from E9.5 through postnatal stages

  • When studying developmental processes, co-staining with lineage markers (e.g., desmin) can provide valuable context for KLF13 expression

• Disease Models:

  • In SLE research, KLF13 antibodies help investigate its role in immune cell dysfunction

  • For studies of KLF13 genetic variants in psychiatric disorders, antibodies may help validate bioinformatic predictions of protein structural changes

Each model system may require specific optimization of fixation methods, antigen retrieval techniques, antibody concentration, and detection systems.