KLF13 Antibody, FITC conjugated

What is KLF13 and what are its key biological functions?

KLF13 is a member of the Kruppel-like transcription factor family, containing three zinc finger DNA-binding domains. These transcription factors bind to CG-rich sequences and related GT and CACCC boxes within the genome. KLF13 plays multiple important roles in biological systems, including:

• Activation of transcription for various genes in erythroid lineage cells, including GATA1 and glycophorin B

• Binding to the A and A/B RANTES promoter sites, playing a crucial role in T-lymphocyte activation

• Functioning as a transcriptional regulator in cardiac development, with differential expression in atrial versus ventricular cardiomyocytes

• Dual role in inflammation by both inducing leukocyte recruitment and dampening immune responses via apoptosis regulation

• Negative regulation of Bcl-XL, suggesting involvement in apoptotic pathways

Understanding these functions is essential when designing experiments using KLF13 antibodies in research contexts.

What are the molecular characteristics of KLF13 that researchers should account for in experimental design?

KLF13 has several key molecular characteristics that researchers must consider when designing experiments:

• Calculated molecular weight: 31 kDa, though observed molecular weight typically appears as 37-45 kDa in Western blot applications

• Contains multiple potential phosphorylation sites, particularly the CDC4-phosphodegron (CPD) sites that regulate protein stability

• Gene ID (NCBI): 51621, UniProt ID: Q9Y2Y9

• Contains distinct domains for DNA binding and protein-protein interactions, particularly with GATA transcription factors

• Expression varies significantly between tissue types, with notably higher abundance in atrial versus ventricular cardiac cells

These characteristics affect antibody binding, experimental conditions, and interpretation of results in immunoassays targeting KLF13.

What advantages does FITC conjugation provide for KLF13 antibody applications?

FITC (Fluorescein isothiocyanate) conjugation of KLF13 antibodies offers several methodological advantages:

• Direct detection without secondary antibody requirements, reducing background signal and potential cross-reactivity issues

• Compatibility with standard fluorescence microscopy and flow cytometry instrumentation using 488nm excitation

• Enables multicolor staining when combined with antibodies conjugated to spectrally distinct fluorophores

• Facilitates live-cell imaging applications where membrane permeabilization may be undesirable

• Provides stable fluorescence signal for quantitative analyses of KLF13 expression patterns

When using FITC-conjugated KLF13 antibodies, researchers should account for potential photobleaching and optimize fixation protocols to maintain fluorescence intensity.

What are the validated applications for KLF13 antibodies and their recommended protocols?

KLF13 antibodies have been validated for multiple research applications, each requiring specific protocols and considerations:

Each application requires optimization for specific sample types, and the antibody should be titrated in each testing system to obtain optimal results .

How should researchers prepare samples for optimal KLF13 detection in different applications?

Sample preparation significantly impacts KLF13 detection quality and specificity:

For Western Blot applications:

• Optimal lysis buffers should contain protease inhibitors to prevent degradation

• Nuclear extraction protocols are recommended as KLF13 is predominantly nuclear

• Both reducing and non-reducing conditions have been validated

For Immunohistochemistry applications:

• Antigen retrieval is critical, with TE buffer pH 9.0 showing optimal results for human brain tissue

• Alternative retrieval can be performed with citrate buffer pH 6.0

• Fixation with 4% paraformaldehyde has been validated for cardiac tissue sections

For Immunofluorescence with FITC-conjugated antibodies:

• Minimize exposure to light throughout the protocol to prevent photobleaching

• Blocking with BSA (bovine serum albumin) reduces non-specific binding

• Nuclear counterstains should be spectrally distinct from FITC (avoid FITC overlapping fluorophores)

What are the proper storage and handling protocols for maintaining KLF13 antibody activity?

To maintain optimal activity of FITC-conjugated KLF13 antibodies:

• Store at -20°C for short-term or -80°C for long-term stability

• Avoid repeated freeze-thaw cycles; aliquoting is recommended for antibodies without glycerol

• For 20μl sizes containing 0.1% BSA, aliquoting is unnecessary for -20°C storage

• Protect from light exposure, particularly critical for FITC-conjugated antibodies to prevent photobleaching

• The antibody remains stable for one year after shipment when stored properly

• Storage buffer composition (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) maintains antibody stability

Upon receipt of FITC-conjugated KLF13 antibody, researchers should immediately transfer to appropriate storage conditions and prepare working aliquots if necessary .

How can researchers validate KLF13 antibody specificity for their experimental systems?

Validating antibody specificity is crucial for reliable experimental results. For KLF13 antibodies:

• Western blot validation should confirm the expected molecular weight (37-45 kDa observed vs. 31 kDa calculated)

• Positive controls include HT-29 cells and rat liver tissue for Western blot applications

• Knockdown/knockout validation provides the most stringent specificity confirmation, with multiple publications supporting this approach

• Cross-reactivity testing with other KLF family members is essential due to structural similarities between family members

• Peptide competition assays can verify binding specificity, as demonstrated by elimination of CACCC/KLF13 complexes with anti-KLF13 antibody in cardiomyocyte extracts

For the FITC-conjugated antibody specifically, validation should include comparison with unconjugated antibody performance to ensure conjugation has not affected specificity .

What factors affect KLF13 protein detection and how can researchers mitigate these challenges?

Several factors can impact KLF13 detection:

• Post-translational modifications: KLF13 contains multiple phosphorylation sites that may affect antibody binding. The second CPD (residues 119-123, SPAWS) is particularly important for protein stability

• Protein degradation: KLF13 is subject to both proteasomal and lysosomal degradation, with Fbw7γ-mediated ubiquitination controlling protein levels

• Temporal expression patterns: KLF13 protein expression varies significantly during development and cellular activation, despite constitutive mRNA expression

• Tissue-specific expression: Higher abundance in atrial versus ventricular cardiomyocytes requires consideration when analyzing cardiac tissues

To mitigate these challenges:

• Include proteasome inhibitors in lysis buffers when studying endogenous KLF13

• Consider the temporal dynamics of KLF13 expression, particularly in T lymphocytes where protein is undetectable in resting cells

• Account for translational regulation through 5'-UTR and miR-125a when designing experiments to manipulate KLF13 expression

What are the common technical issues when using FITC-conjugated antibodies and how can they be addressed?

FITC-conjugated antibodies present specific technical challenges:

For optimal results, researchers should always perform titration experiments to determine the ideal concentration of FITC-conjugated KLF13 antibody for their specific application and sample type .

How can KLF13 antibodies be used to investigate transcriptional complexes and protein-protein interactions?

KLF13 participates in complex transcriptional networks through protein-protein interactions. Advanced applications include:

• Co-immunoprecipitation (Co-IP) studies to investigate KLF13 interactions with GATA transcription factors, as demonstrated by physical interactions between KLF13 and GATA-4

• Chromatin immunoprecipitation (ChIP) to identify KLF13 binding sites on promoters containing CACCC elements

• Proximity ligation assays to visualize in situ protein-protein interactions between KLF13 and interaction partners

• Pull-down assays to characterize interaction domains, as shown with KLF13 and the N-terminal activation domains of GATA-4

• Investigation of transcriptional enhancesomes, as KLF13 recruits complex transcriptional machinery including MAPK Nemo-like kinase, p300/CBP, p300/CBP-associated factor, and BRG1 to the RANTES promoter

These approaches can be significantly enhanced with FITC-conjugated antibodies, which facilitate direct visualization of interaction complexes through fluorescence microscopy.

What are the cutting-edge applications of KLF13 antibodies in developmental and disease research?

Recent research highlights several advanced applications for KLF13 antibodies:

• Developmental biology: Tracking spatiotemporal KLF13 expression during cardiac development, where KLF13 displays distinct expression patterns in atrial versus ventricular tissue and plays roles in cardiac cushion formation

• T-cell immunology: Investigating KLF13's role in T-lymphocyte activation and RANTES expression, particularly important in inflammatory responses

• Degradation pathways: Studying Fbw7γ-mediated degradation of KLF13 and its impact on preventing RANTES expression, offering insights into inflammation regulation

• Translational regulation: Examining cap-dependent translation through eIF4E and miR-125a regulation of KLF13 expression in T lymphocytes

• Cardiac disease models: Analyzing BNP promoter activation through GATA-4/KLF13 cooperativity and its implications in heart failure research

FITC-conjugated antibodies enable live-cell tracking of these processes with temporal resolution that is particularly valuable for developmental studies.

How can researchers integrate KLF13 antibody data with other molecular techniques for comprehensive pathway analysis?

Integrative approaches combining KLF13 antibody data with other molecular techniques offer powerful research insights:

• Transcriptomic-proteomic correlation: Comparing KLF13 protein levels (detected by antibodies) with mRNA expression can reveal post-transcriptional regulation mechanisms, particularly relevant given the discrepancy between constitutive KLF13 mRNA expression and regulated protein expression in T lymphocytes

• Functional genomics: Combining ChIP-seq data using KLF13 antibodies with RNA-seq after KLF13 knockdown to identify direct transcriptional targets

• Protein modification analysis: Using KLF13 antibodies in conjunction with phospho-specific antibodies to track the impact of phosphorylation at CPD sites (particularly residues 119-123) on protein stability and function

• Interactome mapping: Integrating KLF13 co-IP data with mass spectrometry to comprehensively map the KLF13 interactome under different cellular conditions

• Multi-omics approaches: Correlating KLF13 binding sites with epigenetic modifications and chromatin accessibility data to understand the regulatory context of KLF13 function

These integrative approaches can provide mechanistic insights into KLF13's role in complex cellular processes beyond what can be achieved with antibody-based detection alone.