KLF3 Antibody

Basic Understanding and Applications

• What is KLF3 and why is it significant for molecular biology research?

KLF3 is a transcription factor belonging to the Krüppel-like factor family of C2H2-type zinc-finger proteins. The canonical human KLF3 protein contains 345 amino acid residues with a molecular weight of approximately 38.8 kDa . KLF3 is primarily localized in the nucleus and binds to the CACCC box of erythroid cell-expressed genes .

KLF3 has significant research importance due to its widespread expression across tissue types and its roles in:

• Hematopoiesis regulation

• Inflammatory response suppression

• Muscle-specific gene expression

• Cancer metastasis regulation

Research has demonstrated that KLF3 functions as a fundamental suppressor that operates as a feedback inhibitor of RELA/p65, potentially facilitating the resolution of inflammation .

• What types of KLF3 antibodies are currently available for research applications?

Researchers have access to several types of KLF3 antibodies with varying specifications:

Most commercial antibodies target specific regions of the KLF3 protein. For example, some antibodies are raised against the N-terminal region (e.g., immunogen sequence "LMFDPVPVKQEAMD-C" ), while others target regions within aa 150 to C-terminus .

• What are the key applications for KLF3 antibodies in experimental research?

KLF3 antibodies are utilized across multiple experimental techniques:

• Western Blot (WB): Most commonly used application for detecting KLF3 protein expression levels in cell and tissue lysates, typically with dilutions of 1-3μg/ml .

• Immunohistochemistry (IHC-P): Used for visualizing KLF3 expression patterns in paraffin-embedded tissue sections .

• Immunofluorescence (IF): Employed for subcellular localization studies, typically using concentrations around 10μg/ml .

• Flow Cytometry: Used for quantifying KLF3 expression in specific cell populations .

• ELISA: Applied for quantitative detection of KLF3, with dilutions typically around 1:32,000 .

• Electrophoretic Mobility Shift Assays (EMSAs): Used to detect KLF3-DNA binding activity, particularly relevant for studying transcription factor function .

Experimental Design and Methodology

• How should researchers design control experiments when working with KLF3 antibodies?

Proper experimental controls are essential for reliable KLF3 antibody-based research:

Positive Controls:

• Cell lines known to express KLF3 (e.g., HCT116 )

• Tissue samples with documented KLF3 expression

Negative Controls:

• KLF3-knockout (Klf3-/-) cell lines or tissues

• Non-immune serum or isotype-specific immunoglobulins

• Secondary antibody-only controls

Specificity Controls:

• Peptide competition assays using the immunizing peptide

• Western blot validation showing expected molecular weight (38-40 kDa)

• Comparison with alternative KLF3 antibodies targeting different epitopes

Research by Sue et al. demonstrates the importance of proper controls, using Klf3-/- samples alongside wild-type in immunoblot and EMSA experiments to verify antibody specificity .

• What methodological considerations are important when using KLF3 antibodies for investigating inflammation pathways?

When studying KLF3's role in inflammation using antibodies, researchers should consider:

Experimental Design:

• Time-course experiments are crucial, as KLF3 expression increases significantly 8-24 hours after LPS treatment .

• Include both early (4-8h) and late (24h) timepoints to capture the complete inflammatory response dynamics.

Sample Preparation:

• When working with macrophages, both unstimulated and LPS-stimulated samples should be collected, as KLF3–DNA-binding activity is most evident 24 hours after LPS treatment .

• Consider including poly(I·C) treatment groups to assess TLR3 and NF-κB pathway activation .

Data Analysis:

• When studying inflammatory cytokine production, analyze both cellular expression (qPCR) and secreted levels in supernatants .

• Key cytokines to measure include TNF, MCP-1/CCL2, IL-6, IL-1α, and IL-1β .

Methodological Workflow:

• Isolate bone marrow-derived macrophages (BMDMs) from wild-type and Klf3-/- mice

• Treat with LPS (100 ng/ml) in a time-course experiment (0, 4, 8, 24h)

• Perform immunoblotting to detect KLF3 protein levels

• Conduct EMSA to assess KLF3-DNA binding activity

• Collect supernatants for cytokine measurements

• Extract RNA for gene expression analysis

This approach effectively revealed that KLF3 deficiency leads to enhanced inflammatory responses with elevated cytokine production .

• How can researchers optimize Western blotting protocols specifically for KLF3 detection?

Optimizing Western blot protocols for KLF3 detection requires attention to several parameters:

Sample Preparation:

• Use nuclear extracts when possible, as KLF3 is primarily localized in the nucleus .

• Add protease inhibitors to prevent protein degradation.

• Include phosphatase inhibitors if studying phosphorylated forms of KLF3.

Gel Electrophoresis:

• Use 10% SDS-PAGE gels for optimal separation of the 38-40 kDa KLF3 protein .

• Include molecular weight markers that clearly demarcate the 35-45 kDa range.

Antibody Optimization:

• Optimal primary antibody dilutions range from 1:1000 (ab154531 ) to 1-3μg/ml (A83826 ).

• Incubate primary antibody overnight at 4°C for best results.

• Use species-appropriate HRP-conjugated secondary antibodies at manufacturer-recommended dilutions.

Detection and Troubleshooting:

• Both chemiluminescence and fluorescence detection methods are suitable.

• If multiple bands appear, consider:

  • Post-translational modifications of KLF3

  • Alternative splice variants (up to 2 isoforms reported )

  • Non-specific binding (address with additional blocking)

Advanced Research Applications

• How can KLF3 antibodies be utilized to investigate chromatin immunoprecipitation (ChIP) experiments?

ChIP experiments using KLF3 antibodies are valuable for identifying KLF3 binding sites across the genome:

Protocol Optimization:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature.

• Sonication: Optimize to generate DNA fragments of 200-500bp.

• Antibody Selection: Choose ChIP-validated KLF3 antibodies; rabbit polyclonal antibodies are often preferred .

• Controls: Include IgG negative control and input samples.

• DNA Analysis: Use qPCR for specific targets or ChIP-Seq for genome-wide binding analysis.

Research Applications:
KLF3 ChIP experiments have successfully identified direct binding to:

• The Rela promoter in macrophages following LPS stimulation

• The human RELA promoter in K562 cells

• Muscle-specific gene regulatory regions

Specifically, ChIP experiments revealed KLF3 binding to the CACCC box (5′-NCN CNC CCN-3′) in the mouse Rela promoter, with binding reduced when this site was mutated .

• What are the methodological approaches for studying KLF3's interaction with other transcription factors?

Investigating KLF3's interactions with other transcription factors requires multiple complementary techniques:

Co-Immunoprecipitation (Co-IP):

• Use anti-KLF3 antibodies to pull down KLF3 protein complexes from nuclear extracts.

• Probe Western blots with antibodies against potential interacting partners.

• Perform reciprocal Co-IPs to confirm interactions.

Proximity Ligation Assay (PLA):

• Use primary antibodies targeting KLF3 and the potential interacting partner.

• Apply species-specific PLA probes with oligonucleotides.

• Visualize interactions as fluorescent spots where proteins are in close proximity.

Functional Studies:

• Dual luciferase reporter assays to assess cooperative transcriptional activity.

• Sequential ChIP (Re-ChIP) to identify co-occupancy at specific genomic regions.

Research has demonstrated that KLF3 synergizes with serum response factor (SRF) in regulating muscle-specific gene expression . Methodological approaches included:

• Using antibodies against both KLF3 and SRF

• Co-immunoprecipitation experiments to detect physical interactions

• Functional assays to assess transcriptional synergy

• How can researchers investigate the role of KLF3 in cancer progression using antibodies?

KLF3 antibodies enable multiple approaches to study its role in cancer:

Expression Analysis:

• Perform IHC on clinical cancer specimens to correlate KLF3 expression with TNM stages and patient prognosis .

• Use Western blotting to quantify KLF3 levels across cancer cell lines.

Mechanistic Studies:

• Combine KLF3 antibodies with antibodies against STAT3 and EMT markers to investigate pathway interactions .

• Use subcellular fractionation followed by immunoblotting to track KLF3 nuclear localization during cancer progression.

Functional Validation:

• Generate KLF3-silenced or KLF3-overexpressing cancer cell lines

• Validate knockdown/overexpression by Western blot and IF

• Perform functional assays (migration, invasion, colony formation)

• Use in vivo xenograft models and analyze tumor tissues by IHC

Research has demonstrated that low KLF3 expression correlates with poor prognosis and advanced TNM stages in lung cancer patients . KLF3 silencing was shown to promote epithelial-mesenchymal transition (EMT) and enhance metastasis through STAT3-dependent mechanisms .

Troubleshooting and Data Interpretation

• What are the common challenges in KLF3 antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with KLF3 antibodies:

When troubleshooting weak signals in inflammation studies, consider that KLF3 expression and DNA-binding activity become most evident 24 hours after LPS treatment, so earlier timepoints may naturally show weaker signals .

• How should researchers interpret discrepancies in KLF3 expression data between different experimental methods?

When faced with discrepancies in KLF3 expression data across different methods, consider:

Method-Specific Considerations:

• Western Blot vs. IHC: Western blot quantifies total protein from cell populations, while IHC provides spatial information but may be less quantitative.

• mRNA vs. Protein: Discrepancies between qPCR and Western blot may reflect post-transcriptional regulation of KLF3.

• Antibody Epitopes: Different antibodies targeting distinct epitopes may yield varying results if:

  • Post-translational modifications mask certain epitopes

  • Protein interactions conceal specific regions

  • Conformational changes affect antibody accessibility

Biological Explanations:

• Cell-Type Heterogeneity: In mixed cell populations, bulk analysis may mask cell-specific expression patterns.

• Temporal Dynamics: KLF3 expression changes significantly over time during cellular responses, as seen in LPS-stimulated macrophages .

• Localization Changes: KLF3 may shuttle between nuclear and cytoplasmic compartments under different conditions.

Resolution Approach:

• Validate findings using multiple antibodies targeting different KLF3 epitopes

• Complement antibody-based methods with mRNA analysis

• Use cell fractionation to distinguish nuclear vs. cytoplasmic KLF3

• Consider single-cell analysis methods for heterogeneous samples

• What methodological approaches can address the challenge of detecting KLF3 in tissues with low expression levels?

Detecting low-abundance KLF3 requires specialized approaches:

Signal Amplification Techniques:

• Tyramide Signal Amplification (TSA): Enhances IHC/IF detection by enzymatically depositing additional reporter molecules.

• Antibody Conjugates: Use highly sensitive detection systems such as QDots or polymeric HRP detection.

• Proximity Ligation Assay (PLA): Can detect low levels of protein while maintaining specificity.

Sample Enrichment Strategies:

• Nuclear Extraction: Concentrate KLF3 by isolating nuclear fractions.

• Immunoprecipitation: Enrich KLF3 prior to detection by other methods.

• Cell Sorting: Isolate specific cell populations with higher KLF3 expression.

Protocol Optimization:

• Increase antibody incubation time (overnight at 4°C)

• Optimize antigen retrieval for tissue sections

• Reduce background with careful blocking and washing

• Use high-sensitivity detection substrates

Alternative Approaches:

• Employ genetic reporter systems (e.g., KLF3-GFP) in model systems

• Consider mass spectrometry-based proteomics approaches

Emerging Research Applications

• How are KLF3 antibodies being used to investigate the interplay between inflammation and metabolism?

Recent research has revealed important connections between KLF3, inflammation, and metabolism that can be studied using antibodies:

Experimental Approaches:

• Dual Immunostaining: Co-localize KLF3 with metabolic markers in tissues from inflammatory disease models.

• ChIP-Seq Analysis: Identify KLF3 binding sites in metabolic gene promoters during inflammatory conditions.

• Phospho-Specific Antibodies: Monitor post-translational modifications of KLF3 in response to metabolic signals.

Biological Significance:
KLF3-deficient mice exhibit elevated levels of circulating Ly6C+ inflammatory monocytes and macrophage-derived inflammatory cytokines . This inflammatory phenotype suggests potential metabolic consequences, as chronic inflammation often disrupts metabolic homeostasis.

Research Applications:

• Use flow cytometry with KLF3 antibodies to analyze immune cell populations in metabolic disease models

• Employ tissue-specific KLF3 knockout models and validate using immunoblotting

• Investigate KLF3's role in macrophage polarization during metabolic challenges

• What are the latest methodological advances in studying KLF3 post-translational modifications?

Investigating KLF3 post-translational modifications (PTMs) requires specialized techniques:

Detection Methods:

• Modification-Specific Antibodies: Use antibodies that specifically recognize sumoylated KLF3 .

• Gel Mobility Shift Assays: Detect molecular weight changes associated with PTMs.

• Mass Spectrometry: Identify specific modification sites and types.

Functional Analysis:

• Site-Directed Mutagenesis: Create KLF3 variants with mutated modification sites.

• PTM Enzyme Inhibitors: Assess effects on KLF3 function and stability.

• Proximity Ligation Assays: Detect interactions between KLF3 and PTM machinery.

Recent Applications:
Sumoylation of KLF3 has been reported , suggesting that this modification may regulate KLF3's transcriptional repressor activity. Researchers can use a combination of immunoprecipitation with anti-KLF3 antibodies followed by Western blotting with anti-SUMO antibodies to detect this modification.

• How can KLF3 antibodies contribute to understanding gene regulatory networks in development and disease?

KLF3 antibodies enable researchers to map complex gene regulatory networks:

Genome-Wide Approaches:

• ChIP-Seq: Map KLF3 binding sites across the genome in different cell types and conditions.

• CUT&RUN or CUT&Tag: Higher resolution alternatives to ChIP for mapping transcription factor binding.

• ChIP-SICAP: Identify protein interactors at specific chromatin sites.

Integrative Analysis:

• Combine KLF3 ChIP-Seq with RNA-Seq from KLF3-deficient models

• Correlate KLF3 binding with chromatin accessibility (ATAC-Seq)

• Integrate with histone modification data to understand chromatin context

Disease Applications:
Research has demonstrated that KLF3 acts as a transcriptional repressor of the NF-κB subunit gene Rela , indicating its importance in inflammatory gene regulation. Using ChIP assays, researchers identified direct KLF3 binding to the Rela promoter CACCC box, establishing a mechanism by which KLF3 suppresses inflammation .

In cancer research, KLF3 has been identified as a crucial regulator of metastasis by controlling STAT3 signaling pathways . This regulatory relationship can be further investigated using ChIP-Seq and functional studies with KLF3 antibodies.