KLF7 Antibody

What criteria should I use when selecting a KLF7 antibody for my research?

When selecting a KLF7 antibody, consider the following key criteria:

• Specificity: Verify the antibody specifically recognizes KLF7 without cross-reactivity to other KLF family members.

• Application validation: Ensure the antibody has been validated for your intended application (Western blot, IHC, ChIP).

• Host species compatibility: Consider the host species and avoid potential cross-reactivity with your experimental system.

• Clonality: Monoclonal antibodies offer higher specificity, while polyclonal antibodies may provide stronger signals.

Published research has successfully utilized several KLF7 antibodies, including anti-KLF7 (sc-101,034, Santa Cruz) at 1:1000 dilution for Western blotting and anti-KLF7 (Abcam, ab10802) for immunohistochemistry .

How can I validate the specificity of my KLF7 antibody?

Validate KLF7 antibody specificity through the following approaches:

• Knockdown/knockout controls: Compare antibody signals in wildtype samples versus KLF7 knockdown/knockout samples. For example, compare protein expression in cells transfected with KLF7 siRNA versus control siRNA .

• Western blot analysis: Confirm a single band at the expected molecular weight for KLF7 (approximately 39-42 kDa).

• Positive and negative controls: Use tissues or cell lines with known KLF7 expression levels. Colon cancer cell lines (SW620 and LoVo) have been documented to express different levels of KLF7 and can serve as comparative controls .

• Peptide competition: Pre-incubate the antibody with a blocking peptide to confirm signal specificity.

What are the most commonly used KLF7 antibodies in different experimental applications?

Based on published research, the following antibodies have been successfully used in KLF7 research:

• Western blotting: Anti-KLF7 (sc-101,034, Santa Cruz) at 1:1000 dilution, with normalization to GAPDH (ab8245, Abcam, 1:5000) .

• Immunohistochemistry: Anti-KLF7 (Abcam, ab10802) for detection in tissue microarrays and mouse tumor tissues .

• Functional studies: Antibodies specific to KLF7-regulated pathways, such as phosphorylated STAT3, AKT, and ERK, can be used as complementary tools to validate KLF7 function .

What is the optimal protocol for Western blot analysis using KLF7 antibodies?

For optimal Western blot detection of KLF7, follow this protocol:

• Sample preparation:

  • Lyse cells in appropriate buffer

  • Quantify protein concentration

  • Load 20-50 μg protein per lane

• Electrophoresis and transfer:

  • Separate proteins by SDS-PAGE

  • Transfer to membrane (PVDF or nitrocellulose)

• Antibody incubation:

  • Block membrane with appropriate blocking buffer

  • Incubate with anti-KLF7 (sc-101,034, Santa Cruz) at 1:1000 dilution

  • Wash thoroughly

  • Incubate with appropriate secondary antibody

• Detection and quantification:

  • Visualize using enhanced chemiluminescence

  • Capture images using a chemiluminescence imaging system

  • Quantify bands densitometrically and normalize to GAPDH

Perform at least three independent experiments for statistical validity, as practiced in published research .

How should I optimize KLF7 antibody protocols for immunohistochemistry (IHC)?

For optimal IHC results with KLF7 antibodies:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde overnight

  • Embed in paraffin

  • Section tissues at 10 μm thickness

• Staining procedure:

  • Deparaffinize and rehydrate sections

  • Perform antigen retrieval (method should be optimized)

  • Block endogenous peroxidase activity

  • Apply KLF7 antibody (Abcam, ab10802)

  • Detect using appropriate visualization system

• Analysis:

  • Have two experienced pathologists independently assess staining

  • Alternatively, analyze digital images using ImageJ software

  • Calculate percentage of KLF7-positive cells

  • Perform statistical analysis using GraphPad Prism or similar software

How can I use KLF7 antibodies to study protein-DNA interactions?

To investigate KLF7-DNA interactions:

• ChIP-seq analysis:

  • Cross-link protein-DNA complexes in cells

  • Lyse cells and shear chromatin

  • Immunoprecipitate with KLF7 antibody

  • Sequence enriched DNA fragments

  • Analyze binding peaks (e.g., KLF7 binding peak was identified upstream of the CDKN3 gene in preadipocytes)

• Validation of binding sites:

  • Create reporter constructs containing potential binding regions

  • Perform 5′-truncating mutation analysis to locate minimal promoter regions

  • Delete putative KLF7 binding sites to confirm functional relevance

  • Conduct dual-luciferase reporter assays to measure promoter activity with or without KLF7 overexpression

Western blot issues:

IHC issues:

What controls should I include when using KLF7 antibodies?

Include these essential controls in KLF7 antibody experiments:

• Western blot controls:

  • Loading control: GAPDH (ab8245, Abcam, 1:5000)

  • Positive control: Cell line with known KLF7 expression (e.g., LoVo cells)

  • Negative control: KLF7 knockdown cells (shKLF7)

• IHC controls:

  • Positive tissue control: Tissue known to express KLF7 (e.g., colon adenocarcinoma)

  • Negative tissue control: Adjacent non-tumor tissues

  • Technical control: Omission of primary antibody

• Functional validation controls:

  • KLF7 knockdown: siRNA or shRNA targeting KLF7

  • KLF7 overexpression: Transfection with KLF7 expression construct (e.g., pCMV-Myc-KLF7)

How do I address contradictory results when studying KLF7 expression?

When encountering contradictory results:

• Verify antibody specificity: Ensure your antibody specifically detects KLF7 by using knockout/knockdown controls .

• Check experimental conditions: Small variations in experimental conditions can affect results:

  • Cell density at transfection (60-70% confluence is optimal)

  • Incubation times after transfection (typically 48 hours)

  • Protein extraction methods

• Consider biological context: KLF7 functions may vary across:

  • Different cell types (cancer cells vs. preadipocytes vs. neurons)

  • Disease states vs. normal tissues

  • Developmental stages

• Validate with multiple approaches: Combine techniques (Western blot, IHC, RT-qPCR) to build a more complete picture of KLF7 expression and function .

How can I investigate the transcriptional regulatory functions of KLF7?

To study KLF7's role as a transcription factor:

• Promoter analysis:

  • Identify putative KLF7 binding sites in promoters of interest

  • Create reporter constructs with varying promoter lengths

  • Generate site-specific mutations of potential binding sites

  • Perform dual-luciferase reporter assays to measure promoter activity

  • Example: KLF7 binding to the CDKN3 promoter was validated by creating constructs with different promoter lengths (nt –1912/–7, –758/–7, –450/–7, and –160/–7) and measuring their activity

• Target gene validation:

  • Overexpress or knock down KLF7 in relevant cell models

  • Measure changes in target gene expression by RT-qPCR and Western blot

  • Example: KLF7 overexpression increased PDGFB promoter activity in SW620 cells

• Genome-wide approaches:

  • Perform ChIP-seq to identify global KLF7 binding sites

  • Conduct RNA-seq after KLF7 manipulation to identify differentially expressed genes

  • Example: RNA-seq analysis in KLF7-overexpressing SW620 cells identified differentially expressed genes involved in multiple signaling pathways

What signaling pathways does KLF7 regulate and how can they be studied?

KLF7 has been shown to regulate several important signaling pathways:

• JAK/STAT3 pathway:

  • KLF7 overexpression increases STAT3 phosphorylation

  • KLF7 knockdown decreases STAT3 phosphorylation

• PI3K/AKT pathway:

  • KLF7 promotes preadipocyte proliferation via Akt phosphorylation

  • This effect is mediated through regulation of CDKN3

  • KLF7 overexpression increases AKT phosphorylation

• MAPK/ERK pathway:

  • KLF7 overexpression increases ERK phosphorylation

  • KLF7 knockdown decreases ERK phosphorylation

• PDGFB signaling:

  • KLF7 directly regulates PDGFB expression

  • KLF7 activates PDGFB promoter in luciferase assays

  • KLF7 affects secretion of PDGF-BB protein

To study these pathways:

• Assess phosphorylation status of key signaling molecules by Western blot

• Measure secreted factors by ELISA (e.g., PDGF-BB)

• Use pathway inhibitors to determine causal relationships

• Perform RNA-seq and pathway enrichment analysis

How can I use KLF7 antibodies to study cellular processes and phenotypes?

KLF7 antibodies can be used in combination with functional assays to study:

• Cell proliferation:

  • MTT assay to measure viability and proliferation

  • Colony formation assay to assess clonogenic potential

  • Flow cytometry for cell cycle analysis (G1/S transition)

  • Example: KLF7 promotes ICP1 cell proliferation via Akt phosphorylation by regulating CDKN3

• Cell migration and invasion:

  • Wound-healing assay to measure scratch closure rate

  • Transwell migration assay to quantify cell migration

  • Example: KLF7 overexpression accelerated scratch healing in SW620 cells

• Epithelial-mesenchymal transition (EMT):

  • Western blot analysis of EMT markers (E-cadherin, Vimentin, Snail)

  • Example: KLF7 overexpression decreased E-cadherin and increased Vimentin and Snail expression

• In vivo tumor growth and metastasis:

  • Xenograft models to assess tumor growth

  • Experimental metastasis models (tail vein injection)

  • IHC staining of tumor tissues for KLF7, proliferation markers (Ki67), and EMT markers (Vimentin)

What statistical approaches are appropriate for analyzing KLF7 expression data?

Based on published methodologies, the following statistical approaches are recommended:

• For comparing two groups:

  • Two-tailed Student's t-test

  • Report results as mean ± standard deviation (SD)

  • Consider p < 0.05 as statistically significant

• For multiple group comparisons:

  • Two-way ANOVA followed by appropriate post-hoc tests

• For correlation analysis:

  • Spearman correlation coefficient for non-parametric data

• For survival analysis:

  • Kaplan-Meier survival curves to compare high vs. low KLF7 expression groups

  • Log-rank test to assess statistical significance

• Software recommendations:

  • GraphPad Prism for general statistical analysis

  • R packages (e.g., limma) for differential expression analysis

  • ImageJ for quantification of immunohistochemical staining

How can I correlate KLF7 expression with disease progression or clinical outcomes?

To correlate KLF7 expression with clinical parameters:

How should I interpret contradictory findings in KLF7 research across different biological contexts?

When interpreting contradictory findings:

• Consider tissue/cell type specificity:

  • KLF7 functions in neuronal morphogenesis

  • KLF7 promotes preadipocyte proliferation

  • KLF7 enhances cancer progression in colon adenocarcinoma

  • These distinct roles may reflect tissue-specific transcriptional programs

• Examine experimental models:

  • in vitro cell culture vs. in vivo animal models

  • Different species (human vs. mouse vs. chicken)

  • Different disease models or stages

• Analyze methodological differences:

  • Transient vs. stable KLF7 manipulation

  • Knockout vs. knockdown approaches

  • Different antibodies or detection methods

• Integrate pathway analysis:

  • KLF7 may act through different pathways in different contexts

  • In preadipocytes: Akt pathway via CDKN3 regulation

  • In colon cancer: JAK/STAT3, PI3K/AKT, MAPK/ERK pathways via PDGFB regulation