KLF7 Antibody

What is KLF7 and what are its main biological functions?

KLF7 is a transcriptional factor belonging to the Krüppel-like factors (KLFs) family, characterized by zinc finger DNA-binding domains. It functions primarily as a transcriptional activator that binds to the CACCC motif of promoters and to SP1 recognition sequences .

Biologically, KLF7 plays critical roles in:

• Neuronal morphogenesis and survival of sensory neurons

• Repression of corneal epithelium differentiation

• Metabolic regulation, including modulation of insulin sensitivity in pancreatic beta cells and skeletal muscle cells

• Inhibition of adipogenesis and repression of adipokine expression (including leptin)

• Cancer progression, particularly in ovarian and colorectal cancers

• Inflammatory regulation in adipocytes through the PKCζ/NF-κB pathway

What sample types can be analyzed using KLF7 antibodies?

KLF7 antibodies are suitable for analyzing various sample types including:

• Nuclear extracts and cell lysates from cultured cells

• Tissue sections for immunohistochemistry analysis

• Animal tissues from various organs, particularly nervous system tissues, adipose tissue, and cancer specimens

• Human patient samples, especially from cancer tissues

The commercially available antibodies typically demonstrate reactivity with human and mouse samples, making them versatile for both clinical and basic research applications .

What are the standard applications for KLF7 antibodies in research?

KLF7 antibodies are commonly employed in several standard research techniques:

• Western Blotting (WB): Used for detecting and quantifying KLF7 protein expression levels in cell or tissue lysates. Typically used at dilutions of 1/500 to 1/2000 .

• Immunohistochemistry (IHC-P): Applied for visualizing KLF7 expression patterns in formalin-fixed, paraffin-embedded tissue sections .

• ELISA: Utilized at approximately 1/5000 dilution for quantitative detection of KLF7 in solution .

• Chromatin Immunoprecipitation (ChIP): Employed to analyze KLF7 binding to specific DNA targets and identify genomic binding sites .

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to achieve reliable results.

How should KLF7 antibodies be stored and handled to maintain reactivity?

Based on available product information, proper storage and handling of KLF7 antibodies includes:

• Storage upon receipt: Aliquot and store at -20°C or -80°C to prevent repeated freeze-thaw cycles

• Shipping conditions: Typically shipped at 4°C

• Working solution preparation: Dilute in appropriate buffer containing stabilizers (often PBS with 50% glycerol, 0.5% BSA and 0.02% sodium azide)

• Long-term stability: Up to 6 months when properly stored

• Form: Typically supplied as liquid antibodies in stabilizing buffers

Avoiding repeated freezing and thawing is crucial for maintaining antibody performance and specificity over time.

How can I validate KLF7 antibody specificity for my experimental system?

Validating KLF7 antibody specificity is crucial for obtaining reliable research results. A comprehensive validation approach should include:

• Genetic manipulation validation:

  • Compare antibody signal between wild-type cells and those with KLF7 silencing (using siRNA/shRNA)

  • Use KLF7 knockout models as negative controls, such as the ΔK7/ΔK7 knockout mice described in the literature

• Multiple detection methods:

  • Confirm protein expression using multiple techniques (WB, IHC, IF)

  • Correlate protein detection with mRNA expression (RT-qPCR)

• Molecular weight verification:

  • Confirm that the detected band corresponds to the expected molecular weight of KLF7

  • Look for presence/absence of known splice variants

• Cross-reactivity assessment:

  • Test antibody against related KLF family members with similar structural domains

  • Perform peptide competition assays using the immunogenic peptide

• Positive and negative control tissues:

  • Use tissues known to express high levels of KLF7 (neuronal tissues, specific cancer samples)

  • Compare with tissues known to have low or no KLF7 expression

Proper validation ensures that experimental findings truly reflect KLF7 biology rather than non-specific interactions.

What are the optimal techniques for studying KLF7's role in transcriptional regulation?

Studying KLF7's function as a transcription factor requires specialized approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use validated KLF7 antibodies to immunoprecipitate DNA-protein complexes

  • Sequence precipitated DNA (ChIP-seq) to identify genome-wide binding sites

  • Design primers for known binding sites (like the miR-139-5p promoter in CRC cells) for ChIP-qPCR

• Transcription Factor Activity Assays:

  • Employ specialized kits like the KLF7 Transcription Factor Activity Assay

  • These assays detect active KLF7 binding to consensus sequences with high sensitivity and specificity

  • Can be used for high-throughput screening of factors affecting KLF7 activity

• Reporter Gene Assays:

  • Construct luciferase reporters containing KLF7 binding sites

  • Verify direct regulation through site-directed mutagenesis of binding sites

  • Examples include using reporter constructs for p21, p27 genes , or PKCζ

• Gene Expression Analysis After KLF7 Modulation:

  • Assess downstream gene expression changes following KLF7 overexpression or silencing

  • Target genes reported include p21, p27 , PKCζ, IL-6 , and TPD52

How can I effectively use KLF7 antibodies to investigate its role in cancer progression?

KLF7 has been implicated in various cancers, particularly high-grade serous ovarian cancer (HGSOC) and colorectal cancer (CRC) . To investigate its role:

• Expression Analysis in Clinical Samples:

  • Perform IHC on tumor microarrays to correlate KLF7 expression with clinical outcomes

  • Use Western blotting to quantify expression differences between tumor and adjacent normal tissues

  • Conduct survival analysis based on KLF7 expression levels (KLF7 has been identified as an unfavorable prognostic marker in HGSOC)

• Functional Studies in Cancer Cell Lines:

  • Silence KLF7 using siRNA approaches (specific siRNA sequences are available in the literature)

  • Assess effects on:

    • Proliferation (using assays such as CCK-8)

    • Migration and invasion capabilities (using Transwell assays)

    • Sphere formation (for cancer stem cell properties)

  • Examine downstream signaling effects, particularly on EMT markers (SNAIL, ZEB2, VIM)

• In Vivo Cancer Models:

  • Generate xenograft models using cells with modulated KLF7 expression

  • Measure tumor growth, metastasis, and response to therapies

  • Perform IHC for proliferation markers like Ki-67

• Mechanistic Investigations:

  • Identify KLF7 regulatory networks specific to cancer types

  • In CRC, investigate the KLF7/miR-139-5p/TPD52 axis

  • In HGSOC, explore EMT and stemness pathways

What are the methodological considerations when using KLF7 antibodies to study metabolic disorders?

KLF7 plays significant roles in metabolic regulation, particularly in adipocyte inflammation and glucose metabolism through the PKCζ/NF-κB pathway . When investigating these aspects:

• Animal Model Selection:

  • High-fat diet (HFD)-induced obesity models show increased KLF7 expression in epididymal white adipose tissue (Epi WAT)

  • Consider using tissue-specific KLF7 knockout models (such as fat conditional knockout mice)

  • Assess metabolic parameters including glucose tolerance tests (GTT) and insulin tolerance tests (ITT)

• Tissue-Specific Analysis:

  • Evaluate different adipose tissue depots (epididymal, mesenteric, perirenal, subcutaneous) as they may show differential KLF7 expression and function

  • Consider co-analysis of adipose tissue with liver and muscle to understand systemic effects

• Inflammatory Marker Assessment:

  • Investigate downstream inflammatory markers regulated by KLF7, particularly:

    • Interleukin-6 (IL-6)

    • Phosphorylated NF-κB components (p-p65)

    • PKCζ expression and activation

    • IκB phosphorylation status

• Experimental Verification:

  • Combine protein detection (using KLF7 antibodies) with functional assays

  • Correlate KLF7 expression with glucose uptake, insulin signaling, and inflammatory cytokine production

  • Consider in vitro adipocyte models (such as 3T3-L1 cells) for mechanistic studies

What technical optimization is necessary for Western blot analysis of KLF7?

Optimizing Western blot protocols for KLF7 detection requires attention to several technical aspects:

• Sample Preparation:

  • Nuclear extraction is often necessary as KLF7 is a nuclear transcription factor

  • Use protease inhibitors to prevent degradation during isolation

  • Phosphatase inhibitors may be important if studying KLF7 post-translational modifications

• Protein Loading and Transfer:

  • Optimal protein loading: 20-40 μg of total protein per lane

  • Use GAPDH as a loading control for normalization, as demonstrated in published studies

  • Transfer conditions may need optimization for this transcription factor

• Antibody Dilution and Incubation:

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Consider overnight incubation at 4°C to improve signal

  • Secondary antibody selection should match the host species (typically rabbit for polyclonal antibodies)

• Detection System:

  • Enhanced chemiluminescence systems work well for KLF7 detection

  • Consider using digital imaging systems like ChemiDoc for quantification

  • Densitometric analysis should normalize KLF7 to loading controls

• Troubleshooting Common Issues:

  • Multiple bands may indicate splice variants, degradation, or non-specific binding

  • Weak signals may require longer exposure times or higher antibody concentrations

  • High background may necessitate more stringent washing or blocking

How should KLF7 knockdown/silencing experiments be designed and validated?

Effective KLF7 silencing experiments are crucial for functional studies and require careful design:

• siRNA Design and Selection:

  • Published siRNA sequences are available (e.g., ON-TARGETplus SMARTpool siRNA KLF7, Dharmacon)

  • Consider using pools of 4 siRNAs to enhance knockdown efficiency and reduce off-target effects

  • Always include non-targeting siRNA controls (siC)

• Transfection Optimization:

  • Optimize transfection reagent (e.g., Transfectin, Bio-Rad) concentrations and conditions

  • Cell type-specific considerations: OV-90 and PEO1 cells have been successfully transfected in ovarian cancer studies

  • Colorectal cancer cell lines have also been effectively transfected for KLF7 silencing

• Validation of Knockdown Efficiency:

  • RT-qPCR to confirm reduction in KLF7 mRNA levels (primer sequences are available in the literature)

  • Western blot with KLF7 antibodies to verify protein reduction

  • Time-course analysis to determine optimal post-transfection time points for experiments

• Functional Readouts:

  • Cell viability assays (e.g., CCK-8)

  • Migration and invasion assays (Transwell)

  • Pathway-specific markers (e.g., EMT markers, inflammatory cytokines)

  • Genetic rescue experiments to confirm specificity

What are the best approaches for studying KLF7 in tissue samples?

Analyzing KLF7 expression and function in tissue samples presents unique challenges:

• Tissue Collection and Processing:

  • For fresh tissue: rapid freezing in liquid nitrogen to preserve protein integrity

  • For FFPE samples: optimal fixation time to preserve epitopes while allowing antibody penetration

  • Consider tissue-specific processing protocols (brain tissue versus adipose tissue)

• Immunohistochemistry Optimization:

  • Antigen retrieval methods: heat-induced epitope retrieval often necessary

  • Blocking procedures: use appropriate blocking agents to reduce background

  • Antibody dilution: typically start with manufacturer recommendations and optimize

  • Detection systems: consider amplification methods for low-abundance expression

• Quantification Approaches:

  • Digital image analysis for quantitative IHC

  • Consider multiplexed staining to correlate KLF7 with functional markers

  • Scoring systems for clinical samples (percentage positive cells, staining intensity)

• Controls and Validation:

  • Positive control tissues with known KLF7 expression (neuronal tissues , certain cancer types )

  • Negative controls (antibody omission, isotype controls)

  • Correlation with other detection methods (Western blot, RNA analysis)

Comparative Analysis of Different KLF7 Detection Methods

The choice between activity assays and antibody-based detection depends on your research questions:

Use Transcription Factor Activity Assays when:

• Investigating functional activity rather than just presence/abundance

• Studying mechanisms that alter KLF7 binding capacity without changing expression levels

• Screening compounds that might inhibit KLF7-DNA interactions

• High-throughput analysis is required

Use Antibody-Based Detection Methods when:

• Determining tissue/cellular localization is important

• Quantifying total KLF7 protein levels

• Investigating post-translational modifications (with modification-specific antibodies)

• Performing co-immunoprecipitation to identify protein interaction partners

For comprehensive studies, combining both approaches provides complementary information about both KLF7 expression and functional activity.