Visfatin Antibody

What is visfatin and why is it significant in research?

Visfatin, also known as Pre-B cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (NAMPT), is a multifaceted molecule that plays crucial roles in inflammation, apoptosis, and metabolic regulation. It has gained significant research interest due to its involvement in various pathophysiological processes, including insulin resistance, inflammation, and cellular energy metabolism. Visfatin has been shown to exert regulatory effects on inflammatory processes in multiple cell types and tissues, making it an important target for studying conditions like nonalcoholic fatty liver disease (NAFLD), metabolic disorders, and inflammatory diseases . Research has revealed that visfatin can induce proinflammatory cytokine production and inhibit insulin signaling via critical pathways such as STAT3 and NF-κB, positioning it as a potential mediator in the development of insulin resistance and chronic inflammation .

What are the common research applications for visfatin antibodies?

Visfatin antibodies are employed across various experimental techniques in molecular and cellular biology research. Based on current literature, the primary applications include:

These techniques have been validated across multiple species including human, mouse, and rat samples, making visfatin antibodies versatile tools for comparative and translational research .

How do I select the appropriate visfatin antibody for my specific research model?

When selecting a visfatin antibody, consider these key factors based on your research objectives:

• Species reactivity: Ensure the antibody recognizes visfatin from your species of interest. Available antibodies demonstrate reactivity with human, mouse, and rat visfatin .

• Application compatibility: Verify the antibody has been validated for your specific application. For example, some antibodies perform optimally in Western blot but may not work as well for immunohistochemistry .

• Epitope recognition: Consider whether you need an antibody that recognizes a specific region of visfatin, especially if studying specific fragments or post-translational modifications.

• Validation evidence: Review the antibody's validation data, such as Western blot images showing clear bands at the expected molecular weight (~52 kDa for visfatin) .

• Clonality: Monoclonal antibodies (like clone #882104) offer high specificity for a single epitope, while polyclonal antibodies can provide enhanced sensitivity by recognizing multiple epitopes .

For verification, check published literature where the specific antibody has been successfully used in similar experimental conditions to your research model.

What are the optimal conditions for detecting visfatin using Western blot?

Based on published protocols, the following optimized conditions are recommended for visfatin detection by Western blot:

• Sample preparation:

  • Use appropriate lysis buffers (e.g., RIPA buffer with protease inhibitors)

  • Load 20-40 μg of total protein per lane

• Electrophoresis conditions:

  • 5-20% SDS-PAGE gel

  • Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

• Transfer and blocking:

  • Transfer to PVDF membrane

  • Block with 5% non-fat dry milk or BSA in TBST

• Antibody incubation:

  • Primary antibody concentration: 0.5-1.0 μg/mL

  • Incubate overnight at 4°C

  • Use HRP-conjugated secondary antibodies (e.g., Anti-Mouse IgG)

• Detection parameters:

  • Expected molecular weight: approximately 52 kDa

  • Use Immunoblot Buffer Group 1 for optimal results

  • Perform under reducing conditions

These conditions have been validated across multiple cell lines, including 293T human embryonic kidney cells, RAW 264.7 mouse monocyte/macrophage cells, Neuro-2A mouse neuroblastoma cells, and various human tissue samples .

How can I optimize immunocytochemistry protocols for visfatin detection?

For optimal immunocytochemistry results when detecting visfatin, follow this methodological approach:

• Cell preparation:

  • Culture cells on sterile coverslips in appropriate media

  • Allow cells to reach 70-80% confluence for optimal visualization

• Fixation and permeabilization:

  • Fix cells in 4% paraformaldehyde/PBS for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 if detecting intracellular visfatin

• Blocking:

  • Block with 1% bovine serum albumin in PBS for 1 hour at room temperature

• Antibody incubation:

  • Apply primary visfatin antibody at recommended dilution (typically 1-10 μg/mL)

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Wash thoroughly with PBS (3-5 times, 5 minutes each)

  • Apply appropriate Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies

• Nuclear counterstaining and mounting:

  • Mount using fluorescent mounting medium containing DAPI for nuclear visualization

  • Analyze using confocal microscopy (e.g., LSM900 confocal microscope)

This approach has been successfully employed to visualize visfatin expression and localization in various cell types, providing insights into its subcellular distribution patterns.

What controls should be included when conducting visfatin-focused experiments?

Robust experimental design for visfatin research requires appropriate controls to ensure validity and reproducibility:

• Positive controls:

  • Cell lines known to express visfatin (e.g., 293T cells, RAW 264.7 cells, A431 cells)

  • Recombinant visfatin protein (e.g., E. coli-derived recombinant human PBEF/Visfatin, Pro27-His491)

• Negative controls:

  • Primary antibody omission control

  • Isotype control antibody

  • Cell lines with PBEF/NAMPT knockdown or knockout

• Treatment validation controls:

  • When studying visfatin's effects, include baseline/untreated controls

  • For inflammation studies, include positive controls like LPS treatment

  • For signaling pathway investigations, include specific pathway inhibitors as validation controls (e.g., AG490 for JAK2 inhibition, Bay 7082 for NF-κB inhibition)

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Housekeeping genes for qPCR (GAPDH, β-actin, 18S rRNA)

  • Vehicle controls for treatments

• Quantification controls:

  • Standard curves for ELISA measurements

  • Concentration gradients for dose-response experiments

These controls help distinguish specific visfatin-mediated effects from experimental artifacts and provide necessary reference points for data interpretation.

How can visfatin antibodies be utilized to investigate inflammation pathways?

Visfatin has been identified as a key mediator in inflammatory processes across various tissue and cell types. Researchers can leverage visfatin antibodies to explore these mechanisms through several sophisticated approaches:

• Inflammatory cytokine profiling:

  • Use visfatin antibodies in combination with measurements of inflammatory markers (IL-6, TNF-α, IL-1β) to establish correlation or causation

  • Research has shown that visfatin treatment can significantly alter the expression of inflammatory cytokines in RAW264.7 cells

• Signaling pathway dissection:

  • Combine visfatin antibody detection with phospho-specific antibodies against key inflammatory signaling molecules

  • Studies have demonstrated that visfatin activates the JAK2/STAT3 and IKK/NF-κB signaling pathways

  • Use pathway inhibitors (AG490 for JAK2, Bay 7082 for NF-κB) to confirm specificity of visfatin-mediated effects

• Time-course experiments:

  • Monitor visfatin expression and inflammatory marker dynamics over multiple timepoints (6h, 12h, 18h, 24h) to establish temporal relationships

  • Research has shown time-dependent effects of visfatin on LIF, TNFRSF1B, and IL-1α expression

• Transcriptomic analysis:

  • Use RNA-seq to identify differentially expressed genes following visfatin treatment

  • Studies have identified 1225 differentially expressed genes in RAW264.7 cells exposed to visfatin, with 692 up-regulated and 527 down-regulated genes

This multi-faceted approach allows researchers to comprehensively map visfatin's role in inflammation, from receptor engagement to downstream gene expression changes.

What methods can be used to study visfatin's role in insulin resistance?

Investigating visfatin's involvement in insulin resistance requires sophisticated experimental designs incorporating several methodological approaches:

• Insulin signaling pathway analysis:

  • Use Western blotting with antibodies against key insulin signaling proteins (IR, IRS-1, AKT, GSK-3α/β) and their phosphorylated forms

  • Research has shown visfatin reduces the phosphorylation of IR, IRS-1 (Tyr612), AKT, and GSK-3α/β while increasing IRS-1 S307 phosphorylation, which is associated with insulin resistance

• Glucose metabolism assays:

  • Measure glucose uptake using radiolabeled glucose

  • Assess glycogen synthesis and glucose oxidation rates in response to visfatin treatment

  • Monitor gluconeogenic enzyme expression and activity

• Signaling inhibition experiments:

  • Apply specific inhibitors of the JAK2/STAT3 and IKK/NF-κB pathways (AG490 and Bay 7082, respectively) to determine their roles in visfatin-induced insulin resistance

  • Research has demonstrated that these inhibitors can rescue insulin signaling impaired by visfatin exposure

• Cell model systems:

  • HepG2 cells have been validated as an appropriate model for studying visfatin's effects on insulin resistance

  • Use primary hepatocytes or adipocytes for translational relevance

  • Consider tissue-specific effects in experimental design

• Gene expression analysis:

  • Measure mRNA expression of insulin signaling components and glucose metabolism genes

  • Assess changes in inflammatory cytokines that may mediate insulin resistance

A comprehensive experimental approach would involve measuring both direct effects on insulin signaling pathways and indirect effects mediated through inflammatory processes, as visfatin appears to impact insulin sensitivity through both mechanisms.

How can visfatin antibodies be used in studying the RANKL-induced osteoclastogenesis pathway?

Visfatin has emerged as a regulator of osteoclast differentiation and function through its interactions with the RANKL pathway. Researchers can employ visfatin antibodies to investigate this process through several methodological approaches:

• Visfatin secretion during osteoclastogenesis:

  • Use enzyme-linked immunosorbent assay (ELISA) to quantify visfatin secretion during RANKL-induced osteoclast differentiation

  • Culture bone marrow-derived macrophages (BMDMs) with M-CSF and RANKL and measure visfatin levels in culture media at different time points (e.g., day 1 and day 3)

• Visfatin expression pattern analysis:

  • Employ immunocytochemistry with visfatin antibodies to visualize cellular localization during different stages of osteoclast differentiation

  • Use confocal microscopy and co-staining with osteoclast markers to determine temporal and spatial expression patterns

• Functional studies:

  • Combine visfatin neutralizing antibodies with osteoclastogenesis assays to determine the requirement for visfatin in this process

  • Add exogenous visfatin at different time points during RANKL treatment to identify critical windows for visfatin's effects

• Downstream signaling pathway analysis:

  • Use Western blotting and phospho-specific antibodies to examine how visfatin modulates RANKL-induced signaling pathways

  • Focus on NF-κB, MAPK, and NFATc1 pathways that are critical for osteoclastogenesis

• Transcriptional regulation:

  • Perform ChIP assays to identify visfatin-dependent transcriptional changes during osteoclast differentiation

  • Analyze expression of osteoclast-specific genes in the presence/absence of visfatin

This multi-faceted approach allows researchers to comprehensively map visfatin's role in osteoclastogenesis, from its secretion patterns to its effects on downstream signaling and gene expression changes that drive osteoclast differentiation and function.

What are common challenges in visfatin antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with visfatin antibodies. Here are evidence-based solutions for addressing these issues:

• Weak or absent signal in Western blot:

  • Increase antibody concentration (try 0.5-1.0 μg/mL as validated in published protocols)

  • Optimize primary antibody incubation time (overnight at 4°C typically yields better results)

  • Use reducing conditions as specified in validated protocols

  • Ensure proper transfer by using validated buffer systems (e.g., Immunoblot Buffer Group 1)

  • Increase protein loading to 30-40 μg per lane

• High background in immunocytochemistry:

  • Improve blocking conditions (extend blocking time to 2 hours)

  • Dilute primary antibody further

  • Increase washing steps between antibody incubations

  • Use highly cross-adsorbed secondary antibodies to reduce non-specific binding

  • Pre-absorb primary antibodies with cell or tissue lysates

• Inconsistent ELISA results:

  • Ensure sample preparation consistency

  • Generate new standard curves with each experiment

  • Validate antibody pairs for capture and detection

  • Monitor temperature conditions during all incubation steps

  • Use technical replicates (minimum of triplicate measurements)

• Cross-reactivity concerns:

  • Validate antibody specificity using positive controls (e.g., recombinant visfatin)

  • Include knockout/knockdown controls where possible

  • Use monoclonal antibodies like clone #882104 for higher specificity

• Sensitivity limitations:

  • Employ signal amplification systems when needed

  • Use fresh reagents and avoid repeated freeze-thaw cycles of antibodies

  • Consider more sensitive detection methods (chemiluminescence for Western blot, fluorescent secondary antibodies for imaging)

These troubleshooting approaches are based on validated protocols used successfully in published research and can significantly improve experimental outcomes when working with visfatin antibodies.

How can I optimize visfatin antibody-based detection in challenging tissues or samples?

Detecting visfatin in difficult samples requires specialized optimization strategies:

• For fixed tissue samples:

  • Optimize antigen retrieval methods (heat-induced epitope retrieval at pH 6.0 or pH 9.0)

  • Test different fixation protocols (4% paraformaldehyde typically preserves epitopes better than formalin for some antibodies)

  • Use tyramide signal amplification (TSA) to enhance sensitivity

  • Consider thicker sections (8-10 μm) for improved signal detection

  • Extend primary antibody incubation time (overnight at 4°C or up to 48 hours for challenging samples)

• For high-fat tissues (adipose, liver with steatosis):

  • Incorporate additional delipidation steps in sample preparation

  • Use detergent-enhanced buffer systems for better penetration

  • Extend washing steps to remove lipid interference

  • Consider using fluorescent detection methods which may be less susceptible to lipid interference

• For samples with high autofluorescence:

  • Pre-treat with sodium borohydride to reduce autofluorescence

  • Use far-red fluorophores that minimize overlap with autofluorescence spectra

  • Employ specific autofluorescence quenching agents

  • Consider non-fluorescent detection methods like chromogenic immunohistochemistry

• For limited sample quantities:

  • Scale down protocols using microfluidic Western blotting

  • Employ ultra-sensitive ELISA techniques with lower sample requirements

  • Consider using multiplex detection platforms to maximize data from minimal sample

  • Use carrier proteins for very dilute samples

• For degraded samples:

  • Target more stable epitopes that resist degradation

  • Use antibody cocktails targeting multiple epitopes

  • Implement more stringent protease inhibition during sample preparation

  • Consider protocols optimized for formalin-fixed paraffin-embedded (FFPE) samples

These optimization strategies can significantly improve detection sensitivity and specificity in challenging tissue types, enabling successful visfatin detection even in complex biological matrices.

How should researchers interpret seemingly contradictory results regarding visfatin's roles in inflammation?

Research has revealed dual roles for visfatin in inflammatory processes, which may appear contradictory. Here's how to interpret such findings:

Researchers should explicitly address these contextual factors in experimental design and interpretation, including appropriate timepoints, concentration ranges, cell types, and pathway analyses to fully characterize visfatin's inflammatory roles.

What statistical approaches are recommended for analyzing visfatin expression data across different experimental conditions?

When analyzing visfatin expression data, researchers should employ rigorous statistical approaches tailored to the experimental design:

These statistical approaches ensure robust and reproducible analysis of visfatin expression data across experimental conditions, enhancing the reliability and interpretability of research findings.

How can researchers integrate visfatin antibody-based findings with other molecular and cellular techniques for comprehensive pathway analysis?

Comprehensive pathway analysis requires integration of visfatin antibody-based findings with complementary techniques:

• Multi-omics integration:

  • Combine antibody-based protein detection with transcriptomic analysis

  • Research has demonstrated the value of this approach, revealing 1225 differentially expressed genes in response to visfatin treatment

  • Correlate protein-level changes (detected by antibodies) with mRNA expression patterns

  • Add metabolomic profiling to understand downstream functional consequences

  • Use systems biology approaches to integrate these multi-omics datasets

• Functional validation through genetic manipulation:

  • Complement antibody-based detection with gene knockdown/knockout studies

  • Use siRNA, shRNA, or CRISPR-Cas9 to modulate visfatin expression

  • Assess rescue experiments with recombinant visfatin

  • Determine phenotypic outcomes of genetic manipulation on cellular processes

• Live-cell imaging techniques:

  • Combine fixed-cell antibody imaging with live-cell reporters

  • Use fluorescent fusion proteins to monitor real-time dynamics

  • Implement FRET-based reporters to detect protein-protein interactions

  • Correlate static antibody-based imaging with dynamic cellular processes

• Signaling pathway dissection:

  • Use specific pathway inhibitors (e.g., AG490 for JAK2/STAT3, Bay 7082 for NF-κB)

  • Apply phospho-specific antibodies to detect activated signaling components

  • Implement kinase activity assays to measure functional outcomes

  • Time-course experiments to establish causality in signaling cascades

• Computational modeling:

  • Develop predictive models based on experimental data

  • Use machine learning approaches to identify patterns in complex datasets

  • Implement network analysis to map protein-protein interactions

  • Validate computational predictions with targeted experiments

• Translational relevance:

  • Connect in vitro findings to in vivo models

  • Compare cell culture results with tissue samples

  • Evaluate clinical correlations where appropriate

  • Consider species differences when interpreting results across models

This integrated approach provides a comprehensive understanding of visfatin's role in cellular pathways, moving beyond simple protein detection to mechanistic insights and functional consequences in complex biological systems.

How can visfatin antibodies be utilized in studying metabolic diseases beyond traditional applications?

Visfatin research is expanding beyond established paradigms into novel areas of metabolic disease investigation:

• Non-alcoholic fatty liver disease (NAFLD) mechanisms:

  • Use visfatin antibodies to track expression changes during NAFLD progression

  • Study hepatocyte-specific visfatin signaling using co-localization with cell-type markers

  • Research has established that visfatin can induce inflammation and insulin resistance in hepatocytes via the NF-κB and STAT3 pathways, making it a potential therapeutic target

• Metabolic inflammation intersection:

  • Investigate the "meta-inflammation" concept by studying visfatin's dual role in metabolic and inflammatory pathways

  • Use dual immunostaining to identify cells expressing both visfatin and inflammatory markers in metabolic tissues

  • Apply tissue clearing techniques with visfatin antibodies for 3D visualization of expression patterns in intact organs

• Circadian rhythm impacts:

  • Apply visfatin antibodies to study time-dependent expression patterns

  • Correlate with circadian clock gene expression

  • Examine metabolic consequences of circadian disruption on visfatin signaling

• Extracellular vesicle (EV) research:

  • Use visfatin antibodies to study its packaging into EVs

  • Investigate intercellular communication via visfatin-containing EVs

  • Examine tissue-specific EV uptake and resulting signaling changes

• Gut-liver axis studies:

  • Examine how gut microbiome changes affect visfatin expression

  • Study enteric nervous system regulation of visfatin production

  • Investigate portal circulation as a mediator of visfatin effects between gut and liver

These emerging research directions leverage visfatin antibodies to explore novel aspects of metabolic disease pathophysiology, potentially revealing new therapeutic targets and biomarkers for conditions like NAFLD, metabolic syndrome, and type 2 diabetes.

What are the most promising technical advances for improving visfatin detection sensitivity and specificity?

Recent technical innovations offer significant opportunities to enhance visfatin detection:

• Single-cell analysis techniques:

  • Implement mass cytometry (CyTOF) with visfatin antibodies for high-dimensional single-cell profiling

  • Apply single-cell Western blotting to detect visfatin heterogeneity within populations

  • Use imaging mass cytometry for spatial resolution of visfatin expression in tissues

• Advanced microscopy methods:

  • Super-resolution microscopy (STORM, PALM) for nanoscale visualization of visfatin localization

  • Expansion microscopy to physically enlarge samples for improved resolution

  • Light-sheet microscopy for rapid 3D imaging of visfatin in cleared tissues

• Proximity labeling approaches:

  • BioID or APEX2 proximity labeling to identify visfatin interaction partners

  • Combine with mass spectrometry for unbiased discovery of protein complexes

  • Validate interactions using traditional co-immunoprecipitation with visfatin antibodies

• Antibody engineering advances:

  • Nanobodies/single-domain antibodies for improved tissue penetration

  • Bispecific antibodies targeting visfatin and pathway components simultaneously

  • Site-specific conjugation techniques for optimal reporter attachment

• Microfluidic technologies:

  • Droplet-based single-cell proteomics

  • Organ-on-chip models with integrated antibody-based detection

  • Microfluidic Western blotting for enhanced sensitivity with minimal sample requirements

These technological advances enable researchers to detect visfatin with unprecedented sensitivity, specificity, and spatial resolution, providing deeper insights into its localization, interaction partners, and functional heterogeneity across different cellular contexts.

What key considerations should researchers keep in mind when designing visfatin antibody-based experiments?

When planning visfatin antibody-based experiments, researchers should consider several critical factors to ensure robust and reproducible results:

• Experimental context alignment:

  • Match the experimental system to your research question

  • Consider the dual nature of visfatin in inflammation – both pro-inflammatory and anti-inflammatory effects have been documented depending on context

  • Account for time-dependent effects, as visfatin's impact changes significantly across different timepoints (6h, 12h, 18h, 24h)

• Comprehensive controls:

  • Include appropriate positive controls (cell lines known to express visfatin)

  • Implement negative controls (primary antibody omission, isotype controls)

  • Use treatment controls (e.g., LPS stimulation) when studying inflammatory responses

  • Consider pathway inhibitor controls when investigating signaling mechanisms

• Multi-methodological validation:

  • Verify findings using complementary techniques (Western blot, immunocytochemistry, ELISA)

  • Validate antibody specificity in your specific experimental system

  • Combine protein-level detection with mRNA analysis

  • Implement functional assays to connect expression changes to biological outcomes

• Pathway-specific investigations:

  • Design experiments that distinguish between different signaling pathways

  • Consider the involvement of both JAK2/STAT3 and IKK/NF-κB pathways in visfatin's effects

  • Use specific inhibitors to delineate pathway contributions

  • Monitor both immediate signaling events and downstream gene expression changes

• Translational considerations:

  • Connect in vitro findings to relevant in vivo models where possible

  • Consider species differences when interpreting results across experimental systems

  • Evaluate potential clinical relevance of findings

By carefully addressing these considerations, researchers can design more rigorous visfatin antibody-based experiments that yield reliable, reproducible, and physiologically relevant results.

What are the most significant unanswered questions in visfatin research that antibody-based approaches could help address?

Despite significant progress in understanding visfatin biology, several fundamental questions remain that could be addressed using antibody-based approaches:

• Cell type-specific functions:

  • How does visfatin signaling differ between cell types?

  • What is the relative contribution of autocrine versus paracrine visfatin signaling?

  • Are there tissue-specific visfatin interactomes that could be identified using antibody-based pull-down approaches?

• Receptor-mediated signaling:

  • Is the insulin receptor truly a direct receptor for visfatin, or are other mediators involved?

  • How do visfatin-receptor interactions differ between inflammatory and metabolic contexts?

  • Could proximity labeling with visfatin antibodies help identify novel binding partners?

• Post-translational regulation:

  • How is visfatin function modified by post-translational modifications?

  • Do different cellular compartments contain distinctly modified visfatin pools?

  • Could phospho-specific visfatin antibodies reveal activation-dependent signaling events?

• Pathological mechanisms:

  • What is the precise role of visfatin in insulin resistance development?

  • How does visfatin contribute to the progression from simple steatosis to non-alcoholic steatohepatitis?

  • Is there a causal relationship between visfatin dysregulation and metabolic disease?

• Therapeutic targeting:

  • Could neutralizing visfatin antibodies serve as therapeutic tools?

  • Are there specific visfatin domains that could be selectively targeted?

  • How would pathway-specific inhibition of visfatin signaling affect metabolic versus inflammatory outcomes?

Antibody-based approaches, particularly when combined with advanced technologies like proximity labeling, single-cell analysis, and in vivo imaging, are uniquely positioned to address these questions and advance our understanding of visfatin biology in health and disease.