KLF6 Antibody

What is KLF6 and what structural features should researchers be aware of?

KLF6 is a zinc finger transcription factor belonging to the Krüppel-like family of transcription factors. It functions primarily as a transcriptional activator and tumor suppressor . The protein contains a highly conserved zinc finger DNA binding domain at its C-terminus and an activation domain at its N-terminus . The full protein has a calculated molecular weight of approximately 32 kDa but is commonly observed between 32-42 kDa in western blot analyses .

KLF6 contains three zinc finger domains which are encoded by exons 2 and 3 of the gene . These zinc fingers are critical for its function as they mediate binding to GC box motifs in DNA . When selecting antibodies for KLF6 research, it's important to consider whether they target these functional domains.

What are the recommended applications and dilutions for KLF6 antibodies?

KLF6 antibodies can be used in multiple experimental applications with varying recommended dilutions:

When optimizing these applications, researchers should start with the manufacturer's recommended dilutions and adjust based on their specific sample and detection system. Multiple studies have successfully used these applications to study KLF6 in various contexts, including its role in inflammatory responses and hepatocyte function .

Which tissues and cell types express KLF6?

KLF6 shows a tissue-specific expression pattern that researchers should consider when designing experiments:

KLF6 is highly expressed in:

• Placenta (highest expression)

• Spleen and thymus

• Prostate and testis

• Small intestine and colon

KLF6 is weakly expressed in:

• Pancreas

• Lung

• Liver

• Heart and skeletal muscle

Additionally, KLF6 is expressed in fetal brain, spleen, and thymus . At the cellular level, it shows differential expression in various cell types. For example, in the hippocampus of ICH (intracerebral hemorrhage) rats, KLF6 is more abundantly expressed in neurons than in microglia and astrocytes .

How should researchers optimize Western blot protocols for KLF6 detection?

When performing Western blot analysis to detect KLF6, consider the following methodological recommendations:

• Sample preparation: For cell lines such as HEK-293, HUVEC, or NCI-H1299, standard lysis protocols with protease inhibitors are sufficient. For tissue samples (thymus, colon, etc.), ensure thorough homogenization in appropriate buffers .

• Gel percentage: Use 8-10% SDS-PAGE gels for optimal resolution of KLF6 (32-42 kDa) .

• Transfer conditions: Transfer to nitrocellulose membranes (e.g., Hybond-C) at standard conditions for proteins of this size range .

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat milk or BSA

  • Use primary KLF6 antibody at dilutions between 1:1000-1:5000

  • Incubate overnight at 4°C for optimal results

• Detection: Enhanced chemiluminescence (e.g., Super Signal reagent) provides sensitive detection .

• Controls: Include positive controls such as HEK-293 cells, mouse thymus tissue, or mouse colon tissue which show reliable KLF6 expression .

• Verification: Validate results using KLF6 knockout samples when available to confirm antibody specificity .

What are the optimal methods for immunofluorescence staining of KLF6?

For immunofluorescence detection of KLF6, researchers should consider:

• Fixation: Paraformaldehyde (4%) fixation preserves epitope accessibility.

• Permeabilization: Use 0.2% Triton X-100 or similar detergent to allow antibody access to nuclear KLF6.

• Blocking: Block with 5-10% serum (matched to secondary antibody species) to reduce background.

• Antibody incubation:

  • Primary KLF6 antibody: Use at 1:10-1:50 dilution

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • Secondary antibody: FITC-labeled or other fluorophore-conjugated antibodies (e.g., rabbit anti-mouse IgG)

• Nuclear counterstain: DAPI is recommended to visualize nuclear localization of KLF6.

• Validation: KLF6 should show primarily nuclear localization consistent with its function as a transcription factor .

• Co-localization studies: Can be performed with markers for specific cell types (e.g., NeuN for neurons, Iba1 for microglia, GFAP for astrocytes) to determine cell-specific expression patterns .

How can researchers study KLF6's transcriptional activity in different cellular contexts?

KLF6 functions as a transcriptional activator that regulates multiple genes in different cellular contexts. The following approaches are recommended:

• Luciferase reporter assays:

  • Construct reporters containing KLF6 binding elements (GC box motifs)

  • Co-transfect with KLF6 expression vectors (e.g., pCI-neo-KLF6)

  • Compare activity with empty vector controls to measure transcriptional activation

• Chromatin immunoprecipitation (ChIP):

  • Use KLF6 antibodies to immunoprecipitate KLF6-bound chromatin

  • PCR amplify regions of interest to identify direct transcriptional targets

  • This approach confirmed KLF6 as a direct transcriptional activator of autophagy genes ATG7 and BECLIN1

• Gene expression analysis:

  • Compare expression of target genes in cells with KLF6 overexpression or knockdown

  • qRT-PCR can be used to measure expression of potential target genes

  • Example: KLF6 overexpression reduces expression of IL-10-induced anti-inflammatory genes like SOCS3, BCL3, SBNO2, and IL1RA

• Co-immunoprecipitation for transcriptional complexes:

  • KLF6 cooperates with other transcription factors like Sp1

  • Immunoprecipitate with anti-KLF6 antibodies followed by Western blotting for interacting partners

  • Sequential immunoprecipitation can be used to identify multi-protein complexes

What approaches should be used to study KLF6's role in disease models?

KLF6 has been implicated in various disease processes. The following methodological approaches are recommended for disease-specific studies:

• Inflammatory bowel disease (IBD):

  • KLF6 is upregulated in myeloid cells and intestinal tissue from IBD patients

  • Use macrophage cell lines (e.g., RAW264.7) with KLF6 overexpression or knockdown to study inflammatory responses

  • Measure expression of pro-inflammatory genes and cytokines after relevant stimulation (e.g., IFNγ)

• Liver disease and regeneration:

  • KLF6 regulates hepatocyte autophagy in acute liver injury

  • Use hepatocyte-specific Klf6 knockout (ΔKlf6) mice models

  • Assess autophagy markers (LC3-II, Atg7, Beclin1) after partial hepatectomy or injury

• Intracerebral hemorrhage (ICH):

  • KLF6 mediates oxidative stress and neuronal apoptosis

  • Use RNA interference (siRNA) to silence KLF6 expression in vivo and in vitro

  • Evaluate oxidative stress markers, neurological function, and apoptosis markers

• Prostate development and cancer:

  • KLF6 regulates prostate branching morphogenesis

  • Use prostate-specific Klf6-deficient mouse models (e.g., Klf6f/fNkx3.1Cre/+)

  • Analyze components of the Shh pathway (Shh, Ptc, Gli) which are regulated by KLF6

How can researchers validate the specificity of KLF6 antibodies?

Ensuring antibody specificity is critical for reliable KLF6 research. The following validation approaches are recommended:

• Genetic validation:

  • Compare staining in wild-type versus KLF6 knockout tissues/cells

  • Tissue-specific knockout models (e.g., myeloid-specific or prostate-specific) can be generated using Cre-loxP systems

  • Verify deletion using RT-PCR to confirm absence of exons 2 and 3

• Molecular weight verification:

  • KLF6 has a calculated molecular weight of 31865 Da but is observed at 32-42 kDa

  • Alternative splice variants may show different molecular weights

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide before application

  • This should abolish specific signals if the antibody is binding to its intended target

• Subcellular localization:

  • KLF6 should localize primarily to the nucleus consistent with its role as a transcription factor

  • Immunofluorescence should show nuclear staining patterns

• Cross-validation with multiple antibodies:

  • Compare results using antibodies targeting different epitopes within KLF6

  • Consistent results across antibodies increase confidence in specificity

What are common issues when working with KLF6 antibodies and how can they be resolved?

Researchers commonly encounter several challenges when working with KLF6 antibodies:

• High background in immunohistochemistry/immunofluorescence:

  • Increase blocking time/concentration (5-10% serum)

  • Optimize antibody dilution (start with 1:20-1:200 for IHC, 1:10-1:50 for IF)

  • For IHC, try different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Include additional washing steps with 0.1% Tween-20

• Weak or absent signal in Western blot:

  • Ensure adequate protein loading (20-50 μg total protein)

  • Try different lysis buffers to improve protein extraction

  • Increase antibody concentration or incubation time

  • Use enhanced chemiluminescence detection systems

  • Consider tissue-specific expression patterns of KLF6

• Multiple bands in Western blot:

  • KLF6 has multiple splice variants

  • Use appropriate controls (KLF6 knockout samples)

  • Verify band specificity using peptide competition assays

  • The observed molecular weight range (32-42 kDa) may represent different post-translational modifications

• Flow cytometry optimization:

  • For intracellular staining, ensure proper permeabilization with 0.1% lysophosphatidylcholine

  • Use cold PBS for washing steps

  • Analyze a minimum of 10,000 cells for reliable quantification

How should researchers approach the study of KLF6 splice variants?

KLF6 has several splice variants with distinct functions. To effectively study these variants:

• Variant-specific detection strategies:

  • Design PCR primers that can distinguish between wild-type and splice variants

  • Example primer sets:

    • WT human KLF6: forward 5′-CGGACGCACACAGGAGAAAA-3′; reverse 5′-CGGTGTGCTTTCGGAAGTG-3′

    • Total human KLF6: forward 5′-CTGCCGTCTCTGGAGGAGT-3′; reverse 5′-TCCACAGATCTTCCTGGCTGTC-3′

• Antibody selection:

  • Choose antibodies that target regions common to all variants or specific to particular variants

  • Verify whether the antibody recognizes the C-terminal region (e.g., amino acids 159-186) which may be present in multiple variants

• Functional analysis:

  • Different splice variants may have distinct roles (e.g., tumor suppressor vs. oncogenic functions)

  • Design overexpression constructs for specific variants to compare their biological effects

  • Use siRNA or CRISPR approaches targeting specific exons to selectively modulate variant expression

• Quantitative assessment:

  • Use qRT-PCR to quantify the relative abundance of different splice variants

  • Normalize to housekeeping genes such as GAPDH: forward 5′-CAATGACCCCTTCATTGACC-3′; reverse 5′-GATCTCGCTCCTGGAAGATG-3′

How is KLF6 involved in autophagy regulation and what methods are optimal for studying this function?

Recent research has identified KLF6 as a transcriptional activator of autophagy in hepatocytes . To study this function:

• Gene expression analysis:

  • Monitor expression of key autophagy regulators (ATG7, BECLIN1) in response to KLF6 manipulation

  • KLF6 directly activates transcription of these genes in a p53-dependent manner

• Chromatin immunoprecipitation (ChIP):

  • Use to confirm direct binding of KLF6 to the promoters of autophagy genes

  • Compare binding in wild-type versus p53-deficient conditions to assess the dependency relationship

• Autophagy flux assessment:

  • Monitor LC3-II levels by Western blot in KLF6-manipulated cells

  • Use autophagy inhibitors (e.g., chloroquine) to assess flux

  • Compare autophagy markers between wild-type and ΔKlf6 mice livers

• Functional recovery models:

  • Partial hepatectomy (PHx) can be used as a model to study KLF6's role in liver regeneration

  • Cell proliferation following PHx is increased in ΔKlf6 mice compared to controls

• Transcriptional reporter assays:

  • Luciferase constructs containing autophagy gene promoters can be used to measure KLF6-dependent transcriptional activation

  • Include p53 co-expression to assess cooperative effects

What are the latest methodologies for studying KLF6's role in inflammatory responses?

KLF6 plays important roles in myeloid cell plasticity and inflammatory responses . The following approaches are recommended:

• Myeloid-specific knockout models:

  • Generate myeloid-specific Klf6 null mice by crossing Klf6 conditional mutants with Lyz2cre mice

  • Verify deletion by RT-PCR analysis of exons 2 and 3 excision

• Macrophage polarization studies:

  • Use bone marrow-derived macrophages (BMDMs) from wild-type and KLF6-deficient mice

  • Stimulate with polarizing cytokines (e.g., IFNγ for M1, IL-10 for M2)

  • Assess expression of polarization markers by qRT-PCR and flow cytometry

• Transcriptional regulation assessment:

  • KLF6 modulates NF-κB and PPARγ function in macrophages

  • Use co-immunoprecipitation to study KLF6 interactions with these factors

  • Compare responses to inflammatory stimuli in wild-type versus KLF6-manipulated cells

• Disease models:

  • KLF6 is upregulated in myeloid cells and intestinal tissue from IBD patients

  • Study KLF6 expression and function in experimental colitis models

  • Assess how KLF6 levels correlate with disease severity and inflammatory parameters

• Gain- and loss-of-function approaches:

  • Transfect macrophage cell lines (e.g., RAW264.7) with KLF6-expressing plasmids (pCI-neo-KLF6)

  • Compare responses to inflammatory stimuli between KLF6-overexpressing and control cells

  • Analyze expression of pro-inflammatory and anti-inflammatory genes (e.g., SOCS3, BCL3, SBNO2, IL1RA)