KLF13 Antibody, Biotin conjugated

What is KLF13 and what are its primary functions in cellular biology?

KLF13 is a zinc finger DNA-binding transcription factor belonging to the Kruppel-like factor family. It functions as a major mediator of glucocorticoid receptor (GR) signaling in cardiomyocytes and plays crucial roles in cardiac development and function. KLF13 binds to CG-rich sequences and related GT and CACCC boxes in DNA, regulating the transcription of various genes in the erythroid lineage, including GATA1 and glycophorin B . Additionally, KLF13 has been implicated in immune regulation through its binding to RANTES promoter regions, and recent research has identified its role in systemic lupus erythematosus (SLE) pathogenesis . At the cellular level, KLF13 provides protection against DNA damage and cell death, particularly in cardiomyocytes exposed to stressors such as hypoxia or chemotherapeutic agents like doxorubicin .

What criteria should researchers consider when selecting a KLF13 antibody for specific experimental designs?

Researchers should evaluate several critical parameters when selecting a KLF13 antibody:

• Epitope specificity: Confirm the immunogen sequence aligns with your research species and the specific KLF13 domain of interest. For example, the CAB17158 antibody targets a synthetic peptide corresponding to a sequence within amino acids 100-200 of human KLF13 .

• Cross-reactivity validation: Verify species reactivity through published validation data. Available KLF13 antibodies demonstrate confirmed reactivity with human, mouse, and rat samples .

• Application compatibility: Select antibodies validated for your specific experimental technique. The 18352-1-AP antibody, for instance, is validated for WB (1:1000-1:4000 dilution), IHC (1:20-1:200 dilution), IF, and ELISA applications .

• Molecular weight detection: Ensure the antibody detects the expected molecular weight of KLF13 (calculated at 31 kDa but typically observed at 37-45 kDa on Western blots) .

• Clone type consideration: Polyclonal antibodies offer broader epitope recognition but potentially more lot-to-lot variability compared to monoclonals.

• Biotin conjugation needs: Consider whether the signal amplification benefits of biotin conjugation outweigh potential endogenous biotin interference in your specific sample type.

How can researchers optimize chromatin immunoprecipitation (ChIP) protocols using KLF13 antibodies to identify novel glucocorticoid-responsive elements?

Optimizing ChIP protocols with KLF13 antibodies requires careful consideration of several parameters based on established research methodologies:

• Cross-linking optimization: For KLF13 ChIP, researchers should employ dual cross-linking with both formaldehyde (1% for 10 minutes) and protein-protein cross-linkers like DSG (disuccinimidyl glutarate) to capture indirect DNA associations through protein complexes, as KLF13 often functions in transcriptional complexes with partners like TBX5 .

• Antibody selection and validation: Verify ChIP-grade quality through validated assays. Research has successfully employed ChIP assays to demonstrate glucocorticoid receptor recruitment to KLF13 intronic glucocorticoid responsive elements (GREs) following 90-minute glucocorticoid treatment .

• Sonication parameters: Optimize chromatin fragmentation to 200-500bp fragments, as KLF13 binding sites are frequently located in both promoter regions (up to 5kb upstream of transcription start sites) and within intronic regions .

• Control selection: Include both negative controls (IgG) and positive controls targeting known KLF13-bound regions, such as the RANTES promoter or the functional GRE in the KLF13 intron-1 region that shows 4.37±0.47-fold enrichment after glucocorticoid treatment .

• Sequential ChIP consideration: For identifying novel glucocorticoid-responsive elements, sequential ChIP (first with GR antibody followed by KLF13 antibody) may identify genomic regions where both factors co-localize.

• Data analysis approach: Employ differential binding analysis comparing vehicle-treated versus glucocorticoid-treated samples to identify condition-specific binding events.

What are the critical experimental variables when using KLF13 antibodies to investigate cardiac protection mechanisms against doxorubicin-induced toxicity?

When investigating cardiac protection mechanisms involving KLF13 against doxorubicin-induced toxicity, researchers should control these critical variables:

• Cell model selection: Primary cardiomyocytes provide physiologically relevant responses, while the HL-1 immortalized cardiomyocyte line offers experimental consistency. Both models have been validated for KLF13-mediated cardioprotection studies .

• Treatment regimen optimization:

  • Doxorubicin concentration: 1μM has been established as effective for inducing measurable cardiotoxicity

  • Pre-treatment with dexamethasone (0.1-1μM) for 48 hours before doxorubicin exposure

  • Doxorubicin exposure duration: 24-48 hours is optimal for observing KLF13-dependent protective effects

• KLF13 manipulation approaches:

  • Knockdown studies: siRNA targeting KLF13 should achieve >80% reduction in protein levels

  • Overexpression studies: verify successful transfection through Western blot before doxorubicin challenge

• Multi-parameter toxicity assessment:

  Assessment Method	Measurement Parameter	Expected Outcome with KLF13 Protection
  Flow cytometry with PI	Plasma membrane integrity	Significant reduction in PI-positive cells
  Cell cycle analysis	Sub-G1 DNA content	Decreased sub-G1 population
  Western blot	Cleaved caspase-3 levels	Reduced cleaved caspase-3 expression

• Mechanistic validation: Include experiments demonstrating that KLF13 overexpression before doxorubicin treatment prevents DNA damage and maintains cellular viability, as previously observed where "No significant differences in cell survival were found between Dox-treated KLF13 overexpressing cells and vehicle-treated cells" .

How can KLF13 antibodies be utilized in multi-color immunofluorescence to investigate co-localization with interaction partners like TBX5?

Multi-color immunofluorescence using KLF13 antibodies to investigate co-localization with interaction partners requires:

• Antibody compatibility planning:

  • Select primary antibodies from different host species (e.g., rabbit anti-KLF13 and mouse anti-TBX5)

  • For biotin-conjugated KLF13 antibodies, use streptavidin-coupled fluorophores with minimal spectral overlap with other fluorophores

• Sample preparation optimization:

  • Fixation: 4% paraformaldehyde for 15 minutes preserves nuclear architecture

  • Permeabilization: 0.25% Triton X-100 for 10 minutes ensures nuclear protein accessibility

  • Antigen retrieval: TE buffer pH 9.0 has proven effective for KLF13 detection in tissues

• Sequential staining protocol:

  • For biotin-conjugated antibodies: block endogenous biotin before antibody incubation

  • Apply primary antibodies sequentially if using same-species antibodies

  • Include appropriate blocking steps between incubations

• Image acquisition parameters:

  • Confocal microscopy with sequential scanning to prevent bleed-through

  • Z-stack acquisition for three-dimensional co-localization analysis

  • Include single-stained controls for spectral unmixing

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficient and Manders' overlap coefficient

  • Conduct proximity ligation assay (PLA) as confirmatory approach for protein-protein interactions

  • Report co-localization in specific cellular compartments (e.g., nuclear vs. cytoplasmic)

What strategies can address non-specific background when using biotin-conjugated KLF13 antibodies in tissues with high endogenous biotin?

When working with biotin-conjugated KLF13 antibodies in tissues with high endogenous biotin (such as liver, kidney, brain, or spleen), researchers should implement these strategies:

• Endogenous biotin blocking:

  • Apply avidin/biotin blocking kit before antibody incubation

  • Use sequential application of free avidin followed by free biotin

  • Consider non-biotin detection systems for extremely biotin-rich tissues

• Sample preparation modifications:

  • Avoid biotin-containing fixatives

  • Implement shorter fixation times to reduce biotin trapping

  • Use acetone fixation as an alternative when appropriate

• Signal amplification alternatives:

  • Consider tyramide signal amplification (TSA) instead of biotin-streptavidin

  • Evaluate polymer-based detection systems

  • Use directly labeled primary antibodies for critical experiments

• Validation controls:

  • Include isotype controls at the same concentration as primary antibody

  • Perform antigen pre-absorption controls

  • Validate results with an unconjugated KLF13 antibody using conventional detection

• Tissue-specific optimization:

  • Adjust blocking reagents (5% BSA with 0.3% Triton X-100 typically effective)

  • Optimize antibody concentration (generally 1:20-1:200 for IHC applications with KLF13 antibodies)

  • Consider counterstaining strategies to improve signal-to-noise ratio

How should researchers interpret and troubleshoot variable molecular weight detection of KLF13 across different sample types?

KLF13 has a calculated molecular weight of 31 kDa but is typically observed at 37-45 kDa on Western blots . To properly interpret and troubleshoot variable molecular weight detection:

• Post-translational modification analysis:

  • KLF13 undergoes phosphorylation, SUMOylation, and potentially ubiquitination

  • Different cell/tissue types may exhibit different post-translational modification patterns

  • Consider using phosphatase treatment of lysates to determine if variable weights are phosphorylation-dependent

• Sample preparation considerations:

  • Protein extraction method: RIPA buffer with protease and phosphatase inhibitors is recommended

  • Sample heating: Limit to 70°C for 5 minutes to prevent aggregate formation

  • Loading buffer: Use reducing conditions with fresh DTT or β-mercaptoethanol

• Gel system optimization:

  • Use gradient gels (4-12%) for better resolution of different KLF13 isoforms

  • Consider longer running times for improved separation of closely migrating bands

  • Include molecular weight markers that span the 25-50 kDa range

• Antibody specificity verification:

  • Compare results with multiple antibodies targeting different KLF13 epitopes

  • Perform knockdown or knockout validation to confirm band specificity

  • Consider pre-absorption controls with the immunizing peptide

• Isoform identification strategies:

  • Cross-reference with transcript variants known for KLF13

  • Consider tissue-specific expression patterns of different isoforms

  • Consult the GenBank Accession Number (BC012741) for reference sequence comparisons

What protocol modifications are necessary for using KLF13 antibodies in challenging research models like lupus nephritis studies?

For adapting KLF13 antibody protocols to challenging research models like lupus nephritis studies:

• Sample processing adaptations:

  • For kidney tissues: Fix in 4% paraformaldehyde, followed by graded ethanol dehydration and paraffin embedding

  • Section thickness: 4-5μm optimal for IHC applications

  • Antigen retrieval: Extended retrieval using TE buffer pH 9.0 is recommended for KLF13 detection in complex tissue samples

• Staining protocol modifications:

  • Increase blocking time (2 hours at room temperature) with 5% normal serum matching secondary antibody host

  • Extend primary antibody incubation to overnight at 4°C with optimized dilution (1:20-1:100)

  • Include tissue-specific positive controls (e.g., human brain tissue shows reliable KLF13 positivity)

• Multiple marker co-detection strategies:

  • Consider sequential immunostaining for detecting KLF13 alongside lupus nephritis markers

  • Implement spectral imaging and unmixing to distinguish closely overlapping signals

  • Use nuclear counterstains (e.g., DAPI) to facilitate cellular localization of KLF13

• Specific controls for autoimmune tissue:

  • Include non-immune IgG controls at matched concentrations

  • Validate with samples from both disease and healthy individuals

  • Consider peptide competition assays to confirm antibody specificity

• Data interpretation considerations:

  • Correlate KLF13 expression with RANTES levels, as "Klf13 upregulation is associated with increased production of RANTES, a key chemokine in lupus nephritis"

  • Assess both T cell and kidney tissue compartments as separate analyses

  • Implement quantitative image analysis with threshold controls for consistency

How can KLF13 antibodies be used to investigate the relationship between glucocorticoid receptor signaling and cardioprotection in doxorubicin-treated models?

To investigate the relationship between glucocorticoid receptor signaling and KLF13-mediated cardioprotection:

• Experimental design framework:

  Experimental Group	Treatment Regimen	Analysis Timepoints	Expected Outcome
  Control	Vehicle only	24h, 48h, 72h	Baseline KLF13 expression
  GR activation	Dexamethasone (0.1-1μM)	4h, 24h, 48h	Increased KLF13 expression
  GR inhibition	RU486 (mifepristone) pretreatment before dexamethasone	24h, 48h	Blocked KLF13 induction
  Doxorubicin damage	Doxorubicin (1μM)	24h, 48h	Cell death and DNA damage
  GR-mediated protection	Dexamethasone pretreatment followed by doxorubicin	24h, 48h	Reduced cell death and DNA damage
  KLF13 knockdown + GR activation + Doxorubicin	siRNA KLF13 + Dexamethasone + Doxorubicin	24h, 48h	Abrogated protective effect

• Molecular mechanism analysis:

  • ChIP assays to demonstrate GR binding to the KLF13 gene GRE in intron-1

  • RT-qPCR to quantify KLF13 mRNA induction (confirming direct transcriptional regulation)

  • Western blot with KLF13 antibodies to verify protein upregulation following GR activation

• Cardioprotection assessment:

  • Flow cytometry for propidium iodide exclusion to measure membrane integrity

  • Cell cycle analysis to quantify sub-G1 population indicative of apoptosis

  • TUNEL assay for DNA fragmentation quantification

• Translational relevance evaluation:

  • Ex vivo heart tissue viability assays following doxorubicin exposure with/without GR pre-activation

  • Immunohistochemistry with KLF13 antibodies to measure expression in tissue sections

  • Correlation of KLF13 levels with markers of cardiomyocyte damage

• Validation through gain-of-function studies:

  • Overexpress KLF13 independent of GR activation and demonstrate protection against doxorubicin

  • Document that "No significant differences in cell survival were found between Dox-treated KLF13 overexpressing cells and vehicle-treated cells"

What methodologies are recommended for investigating KLF13 variants in congenital heart disease (CHD) using antibody-based approaches?

For investigating KLF13 variants in CHD using antibody-based approaches:

• Patient sample collection and processing:

  • Obtain cardiac tissue samples from CHD patients during corrective surgery

  • Process with controlled fixation protocols (10% neutral buffered formalin for 24 hours)

  • Create tissue microarrays for high-throughput analysis across multiple patients

• Genotype-phenotype correlation workflow:

  • Sequence KLF13 variants in CHD patients (as performed in studies with "309 CHD patients and population-matched healthy controls")

  • Categorize variants based on predicted functional impact

  • Perform immunostaining with KLF13 antibodies to assess expression pattern differences

• Functional analysis of KLF13 variants:

  • Transfect wild-type and variant KLF13 constructs in cardiomyocyte models

  • Assess protein expression by Western blot

  • Evaluate subcellular localization using immunofluorescence

  • Measure transcriptional activity on downstream targets like BNP

• Protein interaction studies:

  • Investigate physical interactions with cardiac transcription factors like TBX5

  • "Wild-type or variant KLF13 constructs were cotransfected with TBX5 using the BNP-luciferase reporter" to evaluate functional interactions

  • Perform co-immunoprecipitation assays with KLF13 antibodies to assess protein-protein interactions

• Translation to animal models:

  • Generate transgenic models expressing identified human KLF13 variants

  • Perform comprehensive cardiac phenotyping

  • Use immunohistochemistry with KLF13 antibodies to map expression patterns during heart development

How can researchers integrate KLF13 antibody-based techniques with RNA-seq data to comprehensively analyze KLF13's role in SLE pathogenesis?

Integrating KLF13 antibody-based techniques with RNA-seq for understanding SLE pathogenesis requires:

• Multi-omics experimental design:

  • Obtain matched samples for both protein (antibody-based) and RNA (RNA-seq) analysis

  • Include disease (SLE) and control groups with adequate sample sizes

  • Consider cell-type specific analyses (T cells, B cells, myeloid cells) given KLF13's role in immune regulation

• ChIP-seq and RNA-seq integration workflow:

  • Perform ChIP-seq using KLF13 antibodies to identify genome-wide binding sites

  • Conduct RNA-seq on the same samples to assess transcriptional changes

  • Implement computational integration to identify direct KLF13 transcriptional targets

  • Focus on "genes involved in mediating immune activation, including key proinflammatory cytokines/chemokines in T cells"

• Protein-RNA correlation analysis:

  • Quantify KLF13 protein levels using validated antibodies (Western blot, flow cytometry)

  • Correlate with KLF13 mRNA expression from RNA-seq

  • Identify post-transcriptional regulatory mechanisms affecting KLF13 expression

• Pathway enrichment methodology:

  • Map KLF13-regulated genes to immune signaling pathways

  • Identify "dysregulation in cytokine signaling pathways in myeloid cells in response to toll receptor ligands"

  • Validate key pathway components at protein level using antibodies

• In vivo validation approach:

  • Use lupus-prone mouse models (NZM2410-derived with Sle1/Sle3 loci)

  • Implement tissue-specific immunohistochemistry with KLF13 antibodies

  • Correlate RANTES production with KLF13 expression in both T cells and kidney tissues, as "Klf13 upregulation is associated with increased production of RANTES, a key chemokine in lupus nephritis"

How might biotin-conjugated KLF13 antibodies be utilized in single-cell protein profiling of cardiovascular disease models?

Biotin-conjugated KLF13 antibodies offer unique advantages for single-cell protein profiling in cardiovascular disease:

• Single-cell proteomics applications:

  • Integration with mass cytometry (CyTOF) by using biotin-conjugated KLF13 antibodies detected with metal-tagged streptavidin

  • Incorporation into microfluidic antibody capture platforms for single-cell protein secretion analysis

  • Adaptation for spatial proteomics using multiplexed ion beam imaging (MIBI)

• Cardiovascular disease model analysis:

  • Profile KLF13 expression in cardiomyocytes exposed to hypoxia (modeled with CoCl₂·6H₂O treatment)

  • Investigate cellular heterogeneity in doxorubicin-induced cardiotoxicity models

  • Evaluate KLF13 expression in patient-derived cardiac organoids with CHD-associated variants

• Multi-parameter cellular analysis:

  • Combine with markers of cell stress (γH2AX), apoptosis (cleaved caspase-3), and cardiac damage (troponin)

  • Correlate with glucocorticoid receptor nuclear translocation at single-cell level

  • Develop high-dimensional data analysis pipelines using dimensionality reduction techniques

• Technical optimization requirements:

  • Titrate biotin-conjugated antibodies to achieve optimal signal-to-noise ratio

  • Implement stringent controls for endogenous biotin blocking

  • Validate specificity through genetic knockdown controls

• Translational research applications:

  • Analyze KLF13 expression in endomyocardial biopsies from heart failure patients

  • Correlate with treatment response to cardioprotective therapies

  • Investigate KLF13 as a potential biomarker for cardiotoxicity risk assessment

What methodological approaches can assess the impact of KLF13 post-translational modifications on protein function using modified antibodies?

To investigate KLF13 post-translational modifications and their functional impacts:

• Modification-specific antibody development:

  • Generate antibodies specific to phosphorylated, SUMOylated, or ubiquitinated KLF13

  • Validate specificity through in vitro modification assays

  • Implement peptide competition assays with modified versus unmodified peptides

• Temporal dynamics analysis:

  • Track modification patterns following cellular stimulation (e.g., glucocorticoid treatment)

  • Develop pulse-chase experimental designs with modification-specific antibodies

  • Correlate modification status with transcriptional activity

• Site-directed mutagenesis validation:

  • Create KLF13 mutants with modified post-translational modification sites

  • Assess functional consequences on DNA binding, protein interactions, and transcriptional activity

  • Compare immunoreactivity patterns between wild-type and mutant proteins

• Mass spectrometry integration:

  • Use KLF13 antibodies for immunoprecipitation followed by mass spectrometry

  • Map modification sites comprehensively

  • Quantify stoichiometry of different modification states

• Functional assay correlation:

  Modification Type	Detection Method	Functional Impact Assessment
  Phosphorylation	Phospho-specific antibodies	DNA binding affinity, reporter gene activation
  SUMOylation	SUMO-KLF13 antibodies	Subcellular localization, protein stability
  Ubiquitination	Ubiquitin-KLF13 antibodies	Protein half-life, proteasomal degradation kinetics

How can researchers implement cutting-edge spatial transcriptomics approaches with KLF13 antibody staining to understand tissue-specific expression patterns?

Integrating spatial transcriptomics with KLF13 antibody staining requires:

• Combined workflow development:

  • Sequential approach: Perform immunofluorescence with KLF13 antibodies followed by spatial transcriptomics

  • Image registration algorithms to align protein and RNA data from serial sections

  • Consider commercial platforms (10x Visium, Nanostring GeoMx) compatible with antibody pre-staining

• Cardiac tissue optimization:

  • Fresh-frozen cardiac tissue processing to preserve both RNA integrity and protein epitopes

  • Validated fixation protocols minimizing RNA degradation while maintaining KLF13 antibody reactivity

  • Region-of-interest selection guided by KLF13 protein expression patterns

• Multi-modal data integration:

  • Spatial correlation between KLF13 protein levels and KLF13 mRNA expression

  • Identification of regions with post-transcriptional regulation (protein/mRNA discordance)

  • Mapping of KLF13-regulated gene networks in different cardiac microenvironments

• Disease-specific applications:

  • Map KLF13 expression in border zones of myocardial infarction

  • Characterize spatial heterogeneity in doxorubicin-treated cardiac tissue

  • Profile cardiac developmental abnormalities in CHD models with KLF13 variants

• Advanced computational analysis:

  • Develop spatial statistics approaches for protein-RNA correlation

  • Implement neighborhood analysis to identify cellular interactions dependent on KLF13 expression

  • Create integrative visualization tools for multi-modal spatial data presentation