KLF5 Antibody, HRP conjugated

What is the molecular basis for KLF5 detection using HRP-conjugated antibodies?

KLF5 (Krüppel-like factor 5) functions as a transcription factor that binds to GC box promoter elements and activates gene transcription . For detection purposes, HRP-conjugated KLF5 antibodies utilize the catalytic activity of horseradish peroxidase directly linked to the antibody, eliminating the need for secondary antibodies.

Detection is based on KLF5's molecular characteristics:

• Canonical protein length: 457 amino acids

• Molecular weight: Typically 50.8-51 kDa, but observed at 47-55 kDa depending on cell line

• Subcellular localization: Primarily nuclear

• Alternative splicing: 4 different reported isoforms

When designing experiments, researchers should note that KLF5 interacts with proteins such as SMAD3 and beta-catenin to coordinate cell turnover and tissue homeostasis , which may affect epitope accessibility in certain applications.

What are the recommended dilutions for KLF5 antibody applications across different techniques?

The optimal dilution varies by technique and specific antibody. Based on validated protocols, the following dilutions are recommended:

For HRP-conjugated variants specifically, researchers should perform titration experiments to determine optimal concentrations, as direct HRP conjugation may affect binding kinetics compared to unconjugated antibodies.

How can researchers confirm KLF5 antibody specificity for their experimental system?

Confirming antibody specificity is crucial for reliable results. Multiple approaches are recommended:

• Knockout/knockdown validation: Compare signal between wild-type and KLF5 knockout cells. Western blot data shows complete absence of the ~55 kDa band in KLF5 knockout A549 cells compared to wild-type controls .

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide before application to determine if the signal is blocked.

• Cross-validation with different antibodies: Use multiple antibodies targeting different KLF5 epitopes. Published data confirms consistent detection of KLF5 using antibodies against different regions (aa 50-350 vs. aa 300-350) .

• Multi-technique validation: Confirm expression across techniques (e.g., WB, IHC, and IF) to ensure concordant results. Published data shows consistent KLF5 detection in HeLa cells across WB, IHC, and ICC applications .

What are the optimal conditions for Western blot detection of KLF5 using HRP-conjugated antibodies?

For optimal Western blot detection of KLF5 using HRP-conjugated antibodies, follow this validated protocol:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors

  • Load 30 μg protein per lane (validated in multiple cell lines)

• Gel electrophoresis:

  • Use 10% SDS-PAGE for optimal separation

  • Include molecular weight markers spanning 40-60 kDa range

• Transfer conditions:

  • Transfer to nitrocellulose membrane at 100V for 1 hour

  • Confirm transfer with Ponceau S staining

• Blocking:

  • Block with 3-5% non-fat milk in TBS-0.1% Tween 20 for 1 hour at room temperature

• Antibody incubation:

  • For HRP-conjugated primary: Dilute 1:1000-1:5000 and incubate overnight at 4°C

  • Wash 4 times with TBS-T (5 minutes each)

• Detection:

  • Use ECL substrate optimized for HRP

  • Expected band: 51-55 kDa (primary band)

  • Note: Multiple bands may be observed (47, 51, 53, 55 kDa) depending on cell line

• Controls:

  • Include GAPDH (36 kDa) as loading control

  • Consider including KLF5 knockout/knockdown samples as negative control

How should researchers optimize KLF5 immunohistochemistry protocols to minimize background and maximize specificity?

Optimizing IHC protocols for KLF5 requires careful attention to several parameters:

• Fixation and antigen retrieval:

  • Paraformaldehyde fixation is recommended for KLF5 preservation

  • Primary recommendation: TE buffer pH 9.0 for antigen retrieval

  • Alternative: Citrate buffer pH 6.0 with extended retrieval time

• Background reduction strategies:

  • Pre-incubate sections with 0.3% H₂O₂ to quench endogenous peroxidase

  • Include 5% normal serum from the secondary antibody host species

  • Use avidin-biotin blocking for biotin-based detection systems

• Antibody concentration:

  • Empirically determine optimal concentration (typically 1:200-1:800)

  • Incubate sections overnight at 4°C in humid chamber

• Controls and validation:

  • Include KLF5-high (colon epithelium, skin) and KLF5-low tissues

  • Nuclear localization should be prominent in epithelial tissues

  • HeLa xenograft models show strong nuclear KLF5 staining at 1:500 dilution

• Signal development:

  • For HRP-conjugated antibodies, use DAB substrate with careful timing

  • Counterstain nuclei lightly with hematoxylin to preserve KLF5 signal

Researchers should note that KLF5 is highly concentrated in gastrointestinal epithelial tissues, which serve as excellent positive controls .

What strategies can improve detection sensitivity when working with low-abundance KLF5 in certain cell types?

For low-abundance KLF5 detection, multiple sensitivity enhancement strategies are recommended:

• Signal amplification systems:

  • Tyramide signal amplification (TSA) can increase sensitivity by 10-100 fold

  • Polymer-HRP detection systems offer superior sensitivity over conventional systems

• Sample enrichment techniques:

  • Nuclear fraction isolation (since KLF5 is predominantly nuclear)

  • Immunoprecipitation before Western blotting (validated in HeLa cells)

• Consideration of KLF5 regulation:

  • KLF5 is transiently induced by androgens in prostate cancer cell lines

  • Time course experiments may capture peak expression (4-8 hours post-stimulation)

  • Pre-treatment with enzalutamide enhances androgen-induced KLF5 expression

• Cell line selection:

  • KLF5 expression is higher in basal-like breast cancer cell lines (SUM149PT, HCC1937, HCC1806) compared to luminal cell lines

  • Immortalized breast epithelial cell lines (MCF10A, 184B5) show detectable KLF5 levels

  • In prostate cancer, C4-2B cells show more sustained KLF5 induction than LNCaP cells

How can KLF5 antibodies be utilized to investigate KLF5-AR interactions in prostate cancer research?

KLF5-AR interactions represent a crucial area in prostate cancer research, as these factors regulate opposing transcriptional programs. Advanced approaches include:

• Co-immunoprecipitation (Co-IP):

  • AR antibodies have successfully pulled down KLF5 peptides in RIME experiments

  • Reciprocal Co-IP with FLAG-tagged KLF5 confirms interaction with endogenous AR

  • Use stringent washing conditions to validate direct interaction

• Chromatin Immunoprecipitation (ChIP):

  • ChIP-seq reveals 2,412 genomic binding sites co-occupied by KLF5 and AR

  • Focus on KLF5/AR common binding sites enriched for both KLF5 and AR motifs

  • The ERBB2 locus represents a model site for studying KLF5-AR opposition

• Transcriptional output analysis:

  • RNA-seq following KLF5 knockdown reveals increased AR transcriptional output

  • KLF5 activates basal cell identity genes that AR represses

  • Gene set enrichment analysis (GSEA) shows KLF5 and AR have opposing effects on oncogenic signatures

• Functional studies in CRPC progression:

  • KLF5 is upregulated in castration-resistant prostate cancer models

  • KLF5 levels increase in LNCaP sub-lines derived from xenografts progressed in castrated mice

  • Monitor changes in epithelial marker expression (CK5 up, CK8/18 down) with KLF5 upregulation

What experimental approaches can detect post-translational modifications of KLF5 using modified antibodies?

Investigating post-translational modifications (PTMs) of KLF5 requires specialized approaches:

• Phospho-specific antibody applications:

  • Use phospho-specific KLF5 antibodies to track kinase signaling

  • Combine with inhibitors of PKC, MAPK, or PI3K pathways

  • Validate with lambda phosphatase treatment controls

• Detecting KLF5 ubiquitination:

  • Treat cells with proteasome inhibitors (MG132) to accumulate ubiquitinated forms

  • Use co-IP with KLF5 antibodies followed by ubiquitin detection

  • In BLBC, histone deacetylase inhibitors promote KLF5 ubiquitination and degradation

• Acetylation analysis:

  • KLF5 acetylation regulates its activity

  • Use anti-acetyl-lysine antibodies following KLF5 immunoprecipitation

  • Compare acetylation status between different cell contexts

• Mass spectrometry approaches:

  • Immunoprecipitate KLF5 using validated antibodies

  • Analysis can identify multiple simultaneous modifications

  • Integration with rapid immunoprecipitation and mass spectrometry of endogenous proteins (RIME) techniques has successfully identified KLF5-AR interactions

How can epigenetic regulation of KLF5 be studied using ChIP-seq approaches with KLF5 antibodies?

Investigating epigenetic regulation of KLF5 using ChIP-seq requires sophisticated methodological approaches:

• Combined histone mark and KLF5 ChIP-seq:

  • The KLF5 super-enhancer shows inverse relationships between AR binding and active enhancer marks (H3K27ac, H3K4me1)

  • In KLF5-high PC-3 cells, the super-enhancer shows high density of H3K27ac and H3K4me1 marks

  • In contrast, AR-positive cells show AR binding but low H3K27ac/H3K4me1 density at the KLF5 super-enhancer

• Sequential ChIP (ChIP-reChIP):

  • Perform first ChIP with KLF5 antibody

  • Re-ChIP with antibodies against transcriptional co-regulators or chromatin modifiers

  • This approach can identify co-regulatory complexes at specific genomic loci

• Integration with epigenetic inhibitor studies:

  • Treat cells with epigenetic modifiers (HDAC inhibitors, DNA methyltransferase inhibitors)

  • Perform KLF5 ChIP-seq before and after treatment

  • Correlate with expression changes and histone modification patterns

• Comparative analysis across cell types:

  • Compare KLF5 binding in AR-null versus AR-positive cell lines

  • The KLF5 super-enhancer displays different epigenetic states in different prostate cancer cell lines

  • Correlation between KLF5 expression and super-enhancer activity provides mechanistic insights

Why might Western blots using KLF5 antibodies show multiple bands, and how should researchers interpret them?

Multiple bands in KLF5 Western blots are common and require careful interpretation:

• Expected band patterns:

  • Primary KLF5 band: 51-55 kDa

  • Additional reported bands: 47, 53 kDa

  • In H1299 cells, predicted bands at 47, 51, and 53 kDa have been observed

• Biological explanations for multiple bands:

  • Alternative splicing: Four isoforms of KLF5 have been reported

  • Post-translational modifications: Phosphorylation, ubiquitination, and acetylation can alter migration

  • Proteolytic processing: KLF5 may undergo regulated proteolysis

• Validation approaches:

  • siRNA/shRNA knockdown should reduce intensity of all specific bands

  • KLF5 knockout cells show complete absence of specific bands

  • Different antibodies targeting distinct epitopes should detect the same pattern

• Technical considerations:

  • Sample preparation: Harsh lysis conditions may cause degradation

  • Gel percentage: 10% SDS-PAGE provides optimal resolution for KLF5

  • Running conditions: Lower voltage may improve band resolution

Researchers should always include positive controls (e.g., human testis tissue, colon tissue) and negative controls (KLF5 knockout cells) to aid interpretation .

What are the key considerations for analyzing KLF5 expression in cancer tissues using immunohistochemistry?

Analyzing KLF5 expression in cancer tissues requires careful methodological considerations:

• Tissue-specific expression patterns:

  • KLF5 is highly concentrated in epithelial tissues (gastrointestinal tract, skin)

  • In normal endometrium, moderate expression in glandular epithelium, faint in stroma

  • Endometrial cancer shows strong signal in tumor cells

• Cancer-specific expression changes:

  • Prostate cancer: Low/absent in early-stage, induced in CRPC subsets

  • Breast cancer: High expression in basal-like subtypes, correlates with YB-1 expression

  • Endometrial cancer: Upregulated compared to normal endometrium

• Localization assessment:

  • Primary localization: Nuclear (transcription factor)

  • Cytoplasmic staining may be observed and should be documented

  • In D3 mouse embryonic stem cells, both nuclear and cytoplasmic staining reported

• Quantification methods:

  • H-score (0-300): Intensity (0-3) × percentage of positive cells (0-100%)

  • Digital image analysis for more objective quantification

  • Compare tumor cells to adjacent normal epithelium as internal control

• Correlation with molecular features:

  • In prostate cancer, KLF5 expression inversely correlates with AR activity

  • In breast cancer, KLF5 expression positively correlates with YB-1, negatively with DACH1

How can researchers troubleshoot inconsistent results when using KLF5 antibodies across different experimental systems?

Inconsistent results with KLF5 antibodies may arise from various factors:

• Cell type-specific KLF5 regulation:

  • KLF5 levels vary dramatically across cell types: high in basal-like breast cancer cells, low in luminal cells

  • In prostate cancer, KLF5 is transiently induced by androgens (4-8h) but returns to baseline within 24h in LNCaP cells

  • CRPC cell lines show more sustained KLF5 induction compared to androgen-sensitive lines

• Epitope accessibility issues:

  • KLF5 interacts with multiple proteins (SMAD3, beta-catenin, AR, YB-1)

  • Protein-protein interactions may mask epitopes in specific contexts

  • Different fixation methods alter epitope exposure in IHC/IF applications

• Antibody validation strategies:

  • Validate using multiple techniques (WB, IP, IHC, IF) on the same samples

  • Confirm with genetic approaches (siRNA, CRISPR knockout)

  • Use multiple antibodies targeting different epitopes

• Technical optimization:

  • For IHC: Compare TE buffer pH 9.0 versus citrate buffer pH 6.0 for antigen retrieval

  • For WB: Test different lysis buffers (RIPA vs. NP-40 vs. Triton X-100)

  • For IF: Compare methanol vs. paraformaldehyde fixation

• Experimental design considerations:

  • Include time course experiments (KLF5 expression can be dynamic)

  • Consider cell density effects (confluent cultures may alter expression)

  • Document passage number, as expression can change with continued culture

How can KLF5 antibodies be used to investigate RNA-protein interactions in transcriptional regulation?

Recent research reveals KLF5 involvement in RNA-protein interactions that can be studied using specialized approaches:

• RNA immunoprecipitation (RIP) assays:

  • YB-1 antibodies pull down KLF5 mRNA in HCC1806 and HCC1937 cells

  • Protocol adaptations can examine if KLF5 protein directly interacts with specific RNAs

  • Combined with RT-qPCR or RNA-seq to identify bound transcripts

• RNA methylation studies:

  • YB-1 functions as a reader of RNA-m5C (5-methylcytosine) in KLF5 regulation

  • KLF5 RNA contains four conserved YB-1-binding motifs in coding regions

  • Methylated RNA immunoprecipitation followed by KLF5 detection can reveal this regulation

• In vitro RNA-protein binding assays:

  • Biotin-labeled KLF5 RNA fragments can pull down interacting proteins

  • EMSA (electrophoretic mobility shift assay) with labeled RNA probes

  • Fluorescence anisotropy to measure binding kinetics

• Integration with transcriptional studies:

  • Compare DNA binding (ChIP-seq) with RNA binding (RIP-seq) profiles

  • Investigate whether RNA interactions alter KLF5 transcriptional function

  • RNA interference may regulate KLF5 target gene selectivity

What approaches can elucidate the role of KLF5 in lineage plasticity during cancer progression?

KLF5's emerging role in cancer lineage plasticity can be investigated through multiple approaches:

• Single-cell analysis techniques:

  • Single-cell RNA-seq to track KLF5 expression in heterogeneous tumor populations

  • Combine with lineage marker profiling (basal: CK5; luminal: CK8/18)

  • In prostate cancer, KLF5 upregulation coincides with increased basal marker CK5 and decreased luminal markers CK8/18

• Lineage tracing experiments:

  • KLF5 reporter systems in xenograft models

  • Track cells transitioning between epithelial states

  • The LTL-331 model shows KLF5 upregulation during transition from luminal to neuroendocrine phenotype

• 3D organoid cultures:

  • Patient-derived organoids (PDOs) maintain lineage heterogeneity

  • NEPC PDOs show differential sensitivity to ERBB2 inhibitors compared to adenocarcinoma PDOs

  • Track KLF5 expression during phenotypic transitions in organoid culture

• Therapeutic resistance studies:

  • Monitor KLF5 expression before and after AR-targeted therapy

  • Enzalutamide pre-treatment enhances androgen-induced KLF5 expression

  • LNCaP cells cultured in enzalutamide for 21 days show altered KLF5 induction dynamics

• Functional validation:

  • KLF5 overexpression promotes basal epithelial cell phenotypes

  • KLF5 and AR regulate opposing transcriptional programs of prostate epithelial cell identity

  • Gene set enrichment analysis confirms KLF5 activates basal cell identity signatures

How can multiplexed imaging techniques with KLF5 antibodies advance our understanding of tumor heterogeneity?

Advanced multiplexed imaging with KLF5 antibodies offers powerful insights into tumor heterogeneity:

• Multiplex immunofluorescence approaches:

  • Combine KLF5 with lineage markers (CK5, CK8/18), proliferation markers (Ki67), and other transcription factors (AR, YB-1)

  • Tyramide signal amplification allows multiplexing of 6-8 antibodies on a single section

  • Spectral unmixing addresses fluorophore overlap issues

• Mass cytometry imaging (IMC):

  • Label KLF5 antibodies with rare earth metals instead of fluorophores

  • Simultaneously detect 40+ proteins on the same tissue section

  • Overcomes autofluorescence limitations of conventional IF

• Spatial transcriptomics integration:

  • Correlate KLF5 protein expression with spatial RNA-seq data

  • Map transcriptional programs to specific regions within heterogeneous tumors

  • Identify microenvironmental factors influencing KLF5 expression

• Digital spatial profiling:

  • Quantitative, high-plex protein and RNA analysis at cellular/subcellular resolution

  • Combines KLF5 detection with comprehensive molecular profiling

  • Region-specific analysis of KLF5-associated gene signatures

• Data analysis considerations:

  • Single-cell segmentation algorithms for accurate quantification

  • Spatial statistics to identify significant co-localization patterns

  • Machine learning approaches to classify cellular phenotypes based on marker combinations