KLF2 Antibody

What is KLF2 and why is it important in biological research?

KLF2 (Krüppel-like factor 2, also known as LKLF) is a transcription factor that regulates diverse biological processes, including immune cell differentiation, vascular endothelial cell function, and inflammatory responses. It plays crucial roles in suppressing NF-κB-mediated gene expression and maintaining an anti-thrombotic endothelial surface. KLF2 has been identified as a key regulator in the development of regulatory T cells (Tregs), making it a significant target for immunological research . The protein has a molecular weight of approximately 37-42 kDa depending on post-translational modifications . Understanding KLF2's functions is important for research in immunology, cardiovascular biology, and potential therapeutic applications.

What types of KLF2 antibodies are available for research applications?

Several types of KLF2 antibodies are available for research, including:

• Monoclonal antibodies (e.g., Clone #665333 from R&D Systems, E2Z1H from Cell Signaling Technology)

• Polyclonal antibodies (e.g., rabbit polyclonal antibodies from Merck Millipore)

• Species-specific antibodies (primarily targeting human KLF2, with some cross-reactivity to mouse and rat orthologs)

These antibodies have been validated for various applications including Western blotting, immunocytochemistry, flow cytometry, and chromatin immunoprecipitation, with each antibody having specific validated applications .

What are the key applications for KLF2 antibodies in scientific research?

KLF2 antibodies serve multiple important applications in scientific research:

• Western blotting: For detecting KLF2 protein expression levels in cell lysates (typically appearing at 37-42 kDa)

• Immunocytochemistry/Immunofluorescence: For visualizing cellular localization of KLF2, which is predominantly nuclear in cells like embryonic stem cells

• Flow cytometry: For quantitative analysis of KLF2 expression in specific cell populations, particularly in immune cells and stem cells

• Chromatin immunoprecipitation (ChIP): For studying KLF2 binding to DNA targets and its regulatory functions

Each application requires specific optimization depending on the antibody and experimental system used.

How should I optimize Western blotting protocols for detecting KLF2?

Optimizing Western blotting for KLF2 detection requires several considerations:

• Sample preparation: Use a lysis buffer containing protease inhibitors to prevent KLF2 degradation

• Protein loading: Load 20-40 μg of total protein per lane for optimal detection

• Antibody dilution: Most KLF2 antibodies perform optimally at 1:1000 dilution for Western blotting

• Expected molecular weight: Look for bands at approximately 37-42 kDa

• Controls: Include positive controls (cells known to express KLF2, such as endothelial cells) and negative controls

For enhanced specificity, consider:

• Blocking with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Optimizing primary antibody incubation (overnight at 4°C is often recommended)

• Using secondary antibodies with minimal cross-reactivity to other species

What protocols are recommended for flow cytometry detection of KLF2?

For optimal flow cytometry detection of KLF2, consider the following protocol based on validated methods:

• Cell preparation:

  • Harvest 1-5 × 10^6 cells and wash with PBS

  • Fix cells with paraformaldehyde or Flow Cytometry Fixation Buffer

  • Permeabilize with saponin or Flow Cytometry Permeabilization/Wash Buffer I

• Staining procedure:

  • Incubate with primary KLF2 antibody (10 μg/mL has been validated)

  • Use appropriate isotype control antibody in parallel samples

  • Apply fluorophore-conjugated secondary antibody (Allophycocyanin or Phycoerythrin-conjugated Anti-Mouse IgG have been validated)

• Analysis considerations:

  • Include single-stained controls for compensation

  • Use appropriate gating strategies to identify positive populations

  • Compare with isotype control to determine specific staining

This protocol has been validated for detecting KLF2 in both embryonic stem cells and induced pluripotent stem cells (iPSCs) .

How do I perform immunocytochemistry to visualize KLF2 localization?

For successful immunocytochemistry to visualize KLF2 localization:

• Cell preparation:

  • Culture cells on coverslips until appropriate confluence

  • Fix cells using immersion fixation (4% paraformaldehyde for 15-20 minutes)

• Staining protocol:

  • Permeabilize cells (0.2% Triton X-100 for 10 minutes)

  • Block with 5-10% normal serum in PBS for 30-60 minutes

  • Incubate with KLF2 primary antibody (10 μg/mL for 3 hours at room temperature has been validated)

  • Apply fluorophore-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Mouse IgG)

  • Counterstain nuclei with DAPI

  • Mount slides with anti-fade mounting medium

• Expected results:

  • KLF2 should show predominantly nuclear localization

  • Compare with appropriate negative controls (isotype control antibody)

This approach has been validated in BG01V human embryonic stem cells, showing specific localization to nuclei .

How can I investigate KLF2's role in transcriptional regulation?

Investigating KLF2's transcriptional regulatory functions requires several specialized approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use anti-KLF2 antibodies to immunoprecipitate KLF2-bound chromatin

  • Analyze binding to specific promoters of interest

  • Studies have used this approach to investigate KLF2's relationship with NFκB and HIF-1α promoters

• Gene reporter assays:

  • Construct reporter plasmids containing promoters of interest (e.g., HIF-1α promoter-luciferase)

  • Co-transfect with KLF2 expression vectors

  • Measure reporter activity with and without KLF2 overexpression

  • Previous research has shown KLF2 attenuates p65-induced HIF-1α promoter activity

• RNA-seq following KLF2 modulation:

  • Overexpress KLF2 using viral vectors (e.g., adenovirus)

  • Alternatively, knock down KLF2 using siRNA approaches

  • Perform transcriptome analysis to identify KLF2-regulated genes

  • Validate key targets with qPCR and protein analysis

These methodologies can reveal how KLF2 influences gene expression networks in various cellular contexts.

What approaches can be used to study KLF2's role in regulatory T cell development?

To investigate KLF2's function in regulatory T cell (Treg) development:

• Generation of antigen-induced Tregs (iTregs) with KLF2 modulation:

  • Isolate CD4+CD25- T cells

  • Treat with TGF-β and IL-2 to induce conversion to CD4+CD25+FoxP3+ Tregs

  • Modulate KLF2 levels using pharmaceutical agents (e.g., statins) or genetic approaches

  • Evaluate conversion efficiency by flow cytometry measuring CD25 and FoxP3

• Functional assays for Treg activity:

  • Perform suppression assays using Tregs generated with modulated KLF2 levels

  • Measure proliferation of responder T cells using methods like CFSE dilution

  • Data suggests KLF2 overexpression during iTreg induction does not impair suppressive activity

• In vivo models:

  • Use adoptive transfer of KLF2-modified Tregs

  • Evaluate impact on autoimmune disease models

  • Assess peripheral Treg generation in vivo

This research approach has demonstrated that KLF2 is necessary for the generation of antigen-induced Tregs and peripheral Tregs, making it a viable target for immunotherapy .

How can I assess the impact of KLF2 on endothelial thrombotic function?

To evaluate KLF2's role in endothelial thrombotic function:

• Gene expression analysis:

  • Measure expression of thrombotic regulators (thrombomodulin, eNOS, PAI-1, tissue factor) after KLF2 overexpression or knockdown

  • Use qPCR for mRNA and Western blotting for protein levels

  • Previous studies have shown KLF2 strongly induces thrombomodulin and eNOS while reducing PAI-1 and tissue factor expression

• Functional assays:

  • Thrombomodulin activity assay measuring activated protein C (APC) production

  • KLF2 overexpression has been shown to increase thrombomodulin activity by approximately 7.7-fold

  • In vitro clotting assays to measure blood clotting time

  • Flow rate measurements under basal and inflammatory conditions

• siRNA-mediated knockdown experiments:

  • Use siRNA to reduce KLF2 expression in endothelial cells

  • Measure changes in antithrombotic and prothrombotic factor expression

  • Assess functional impact on clotting parameters

  • Studies have demonstrated that KLF2 knockdown reduces blood clotting time and flow rates

These approaches can comprehensively assess how KLF2 regulates the thrombotic properties of the endothelium.

Why might I observe variable results in KLF2 antibody staining between different cell types?

Variability in KLF2 antibody staining across cell types can result from several factors:

• Differential expression levels:

  • KLF2 expression varies significantly between cell types (high in some endothelial cells and lymphocytes, lower in other cell types)

  • Expression can be influenced by cell activation state, culture conditions, and inflammatory stimuli

• Post-translational modifications:

  • KLF2 undergoes various modifications that may mask or expose epitopes

  • These modifications may differ between cell types and activation states

  • Consider using antibodies targeting different epitopes or regions of KLF2

• Protocol optimization needs:

  • Different cell types may require adjusted fixation conditions

  • Permeabilization requirements may vary (saponin works well for flow cytometry of stem cells)

  • Antibody concentration may need cell-type specific optimization

• Nuclear localization challenges:

  • As KLF2 is predominantly nuclear, ensure adequate nuclear permeabilization

  • Different cell types have varying nuclear membrane characteristics

To address these issues, always include positive control cells with known KLF2 expression and optimize protocols specifically for each cell type being studied.

What are the best approaches for validating KLF2 antibody specificity?

To ensure KLF2 antibody specificity:

• Genetic validation:

  • Compare staining between wild-type cells and KLF2 knockout/knockdown cells

  • siRNA-mediated knockdown has been used successfully to validate KLF2 antibodies

  • Expected result: Reduced or absent signal in KLF2-deficient samples

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of KLF2

  • Compare staining patterns between different KLF2 antibodies

  • Consistent results with multiple antibodies increases confidence in specificity

• Blocking peptide experiments:

  • Pre-incubate antibody with excess KLF2 blocking peptide

  • Run parallel experiments with blocked and unblocked antibody

  • Expected result: Blocked antibody should show minimal staining

• Overexpression systems:

  • Compare staining in cells overexpressing KLF2 versus control cells

  • This approach has validated several commercial KLF2 antibodies

These validation approaches should be combined for comprehensive confirmation of antibody specificity.

How can I address inconsistent Western blot results when detecting KLF2?

When encountering inconsistent Western blot results for KLF2:

• Sample preparation optimization:

  • Ensure complete lysis with appropriate buffers (RIPA buffer with protease inhibitors works well)

  • Consider nuclear extraction protocols as KLF2 is primarily nuclear

  • Keep samples cold throughout preparation to prevent degradation

• Gel electrophoresis considerations:

  • Use 10-12% polyacrylamide gels for optimal resolution of 37-42 kDa KLF2

  • Ensure complete transfer to membrane (confirm with Ponceau S staining)

  • Consider wet transfer methods for more efficient transfer of nuclear proteins

• Antibody optimization:

  • Test multiple antibody dilutions (starting with 1:1000 as recommended)

  • Optimize incubation time and temperature (overnight at 4°C often yields best results)

  • Consider using recombinant antibodies for improved lot-to-lot consistency

• Signal enhancement strategies:

  • Use enhanced chemiluminescence (ECL) substrates appropriate for your protein abundance

  • Consider signal enhancers for low-abundance proteins

  • Optimize exposure times to avoid oversaturation or weak signals

These approaches have been successful in laboratories working with KLF2 detection in various cell types.

How can KLF2 antibodies be utilized to investigate the role of KLF2 in inflammatory diseases?

KLF2 antibodies can be valuable tools for studying inflammatory disease mechanisms:

• Tissue immunohistochemistry:

  • Compare KLF2 expression in healthy versus diseased tissues

  • Correlate KLF2 levels with inflammatory markers

  • Dual staining with cell-type specific markers to identify KLF2-expressing cells

• Flow cytometry for immune cell populations:

  • Analyze KLF2 expression in specific immune cell subsets from patients

  • Correlate with disease activity markers

  • Study how treatments affect KLF2 expression in relevant cell populations

• Mechanistic studies:

  • Use ChIP to investigate how KLF2 regulates inflammatory gene expression

  • Studies have shown KLF2 attenuates p65-induced HIF-1α promoter activity

  • Investigate KLF2 binding partners in inflammatory contexts

These approaches can help elucidate how KLF2 regulates inflammatory responses and potentially identify novel therapeutic targets.

What methodologies are recommended for studying KLF2 in stem cell differentiation?

To investigate KLF2's role in stem cell biology:

• Temporal expression analysis:

  • Track KLF2 expression during differentiation processes

  • Use flow cytometry or immunofluorescence at defined timepoints

  • KLF2 antibodies have been validated in both embryonic stem cells and iPSCs

• Gain and loss of function studies:

  • Overexpress KLF2 during differentiation protocols

  • Use siRNA or CRISPR approaches to knock down/out KLF2

  • Assess impact on differentiation markers and cell fate decisions

• Single-cell analysis:

  • Combine KLF2 antibody staining with other pluripotency/differentiation markers

  • Perform single-cell RNA-seq with KLF2 protein validation

  • Map KLF2 expression changes during lineage commitment

These methodologies can reveal how KLF2 influences stem cell maintenance and differentiation pathways.