FN3K Antibody

What is FN3K and what is its primary function in cellular metabolism?

FN3K (Fructosamine-3-kinase) is an enzyme that catalyzes ATP-dependent phosphorylation of fructosamines, which form when glucose reacts with amines through glycation. Its primary function is to destabilize and remove potentially harmful fructosamines from proteins, protecting them from the detrimental effects of nonenzymatic glycation . FN3K mediates phosphorylation of fructoselysine residues on glycated proteins to generate fructoselysine-3 phosphate adducts, which are unstable and decompose under physiological conditions . This enzymatic process effectively reverses protein glycation, making FN3K a critical component of the cellular protein quality control system . Additionally, FN3K is involved in the response to oxidative stress by mediating deglycation of NFE2L2/NRF2, as glycation impairs NFE2L2/NRF2 function .

Which antibodies are most commonly used for FN3K detection in Western blotting?

Several validated antibodies are available for FN3K detection in Western blotting applications:

When selecting an antibody for Western blotting, researchers should consider specificity, sensitivity, and validated applications reported by the manufacturer . The Proteintech antibody (14293-1-AP) has been extensively validated in multiple sample types including HeLa cells, human brain tissue, human heart tissue, human kidney tissue, and mouse skeletal muscle tissue .

What are the recommended dilutions for FN3K antibodies in various applications?

Optimal antibody dilutions vary by application type and specific antibody:

Manufacturers consistently recommend titrating each antibody in specific testing systems to obtain optimal results . For instance, Proteintech notes that sample-dependent optimization may be necessary and encourages checking their validation data gallery for application-specific guidance .

What are the typical molecular weights observed for FN3K in Western blot analysis?

The calculated and observed molecular weights for human FN3K in Western blot analysis are:

When running Western blots, researchers should expect to see bands in the 30-35 kDa range, although post-translational modifications may cause slight variations in the observed molecular weight . It's important to note that FN3KRP (FN3K-related protein) has a similar molecular weight range (30-34 kDa), which should be considered when analyzing samples that might express both proteins .

What is the relationship between FN3K and protein deglycation?

FN3K plays a central role in protein deglycation through a specific enzymatic mechanism:

• It catalyzes the phosphorylation of fructosamines formed by glycation (nonenzymatic reaction of glucose with primary amines followed by Amadori rearrangement)

• This phosphorylation generates fructoselysine-3 phosphate adducts, which are unstable and decompose under physiological conditions

• The decomposition effectively removes the glycation modification, resulting in deglycation of the protein

• This deglycation process is particularly important for maintaining protein function and preventing the accumulation of advanced glycation end products (AGEs)

FN3K is considered one of the first examples of deglycating enzymes acting on the Amadori product to reverse the early stages of the Maillard reaction in vivo . This function is critical in conditions like diabetes, where abnormal FN3K expression can contribute to complications due to increased protein glycation .

How does FN3K activity modulate Nrf2-mediated antioxidant responses in cancer cells?

FN3K is involved in the deglycation of Nrf2 (Nuclear factor erythroid 2-related factor 2, also known as NFE2L2), a significant regulator of oxidative stress in cancer cells . The relationship functions through the following mechanism:

• Glycation of Nrf2 impairs its function as a transcription factor that regulates antioxidant responses

• FN3K mediates deglycation of Nrf2, which restores its function

• Deglycated Nrf2 predominantly induces antioxidant responses by binding to small MAF proteins (sMAF)

• In cancer cells, this deglycation activity can offer protection against oxidative stress

For researchers studying this pathway, several approaches are recommended:

• Using FN3K inhibitors to maintain Nrf2 in a glycated state, potentially reducing cancer cell protection against oxidative stress

• Implementing combinatorial approaches using phytochemicals like brusatol, topotecan, and platinum derivatives along with kinase modulators

• Monitoring the expression patterns of downstream targets of Nrf2, such as NQO1, HO-1, and their relationship with Keap1

Western blot analysis using antibodies against both FN3K and Nrf2 pathway proteins (Nrf2, NQO1, HO-1, Keap1) can help elucidate this relationship. Primary antibodies for these targets are commercially available: FN3K (Invitrogen, Catalog # PA5-28603), Nrf2 (cell signaling, cat#: 12721), NQO1 (cat#: 62262), HO-1 (Abcam, cat#: ab13248), and Keap1 (Abcam, cat#: ab227828) .

What are the best experimental approaches to study FN3K's role in cellular redox homeostasis?

Based on the research data, FN3K knockout alters redox-sensitive cellular metabolites . To study this relationship, researchers can employ multiple complementary approaches:

Genetic Approaches:

• CRISPR/Cas9-mediated knockout of FN3K in appropriate cell lines (HepG2 cells have been successfully used)

• siRNA or shRNA knockdown for transient reduction of FN3K expression

• Overexpression systems to examine the effects of increased FN3K activity

Pharmacological Approaches:

• Treatment with 1-deoxy-1-morpholinofructose (DMF), a competitive inhibitor of FN3K that can inhibit ~10% of total FN3K activity

• Screening and utilizing anticancer molecules that interact with the catalytic domain of FN3K using computational approaches such as molecular docking

Metabolic Profiling:

• ¹H NMR metabolomics to measure changes in metabolite abundance, as described in published research

• Focus on measuring redox-sensitive metabolites including:

  • Glutathione (increased in FN3K-KO cells)

  • Pantothenate (decreased in FN3K-KO cells)

  • Phosphocreatine/creatine ratio (decreased in FN3K-KO cells)

  • Aspartate, glycine, and serine (all decreased in FN3K-KO cells)

  • Lactate (increased in FN3K-KO cells)

Protein Modification Analysis:

• Monitor glycation status of key redox-regulating proteins

• Assess Nrf2 pathway activation through Western blotting of Nrf2, NQO1, HO-1, and Keap1

• Evaluate nuclear translocation of Nrf2 using immunofluorescence or subcellular fractionation techniques

These approaches provide complementary data on how FN3K modulates the cellular redox state, potentially through its deglycating activity on key proteins involved in oxidative stress responses.

How can researchers accurately quantify FN3K-mediated deglycation of specific protein targets?

To accurately quantify FN3K-mediated deglycation of specific protein targets, researchers should employ a multi-faceted approach:

Mass Spectrometry Approaches:

• LC-MS/MS analysis to identify and quantify glycated vs. deglycated peptides

• MALDI-TOF MS to assess mass shifts associated with glycation/deglycation

• Multiple reaction monitoring (MRM) for targeted analysis of specific glycation sites

Immunological Methods:

• Western blotting using specific antibodies against FN3K (such as Proteintech's 14293-1-AP)

• ELISA-based quantification using sandwich assays like ELK Biotechnology's Human FN3K ELISA Kit (detection range: 0.32-20 ng/mL)

• Immunoprecipitation to isolate specific glycated proteins followed by deglycation assays

Cellular Systems:

• Comparison between wild-type and FN3K knockout/knockdown cells

• Pulse-chase experiments to track glycation/deglycation dynamics

• Use of FN3K inhibitors like DMF as negative controls

Target-Specific Analysis:

• Focus on known FN3K substrates such as Nrf2

• Monitor functional changes in target proteins (e.g., transcriptional activity of Nrf2)

• Correlate deglycation with functional outcomes through reporter assays

For optimal quantification, researchers should consider combining these approaches to provide complementary data on both the extent of deglycation and its functional significance.

What are the key considerations when designing FN3K inhibitor screening experiments?

When designing FN3K inhibitor screening experiments, researchers should consider several critical factors based on published methodologies:

Structural Considerations:

• The 3-dimensional structure of Human FN3K should be modeled using homology modeling techniques, as there is no available crystal structure in PDB

• The amino acid FASTA sequences (Uniprot, 309 AA, accession number: Q9H479) can be used as the starting point

• Specific templates like FN3K from Arabidopsis thaliana (PDB: 6OID) can be used with 'SWISS EXPASY Tools'

Screening Methodology:

• Start with computational approaches:

  • Blind docking followed by targeted docking within specified search spaces

  • LibDock Program for preliminary screening-based docking

  • CDocker approach for detailed evaluation of lead molecules

• Validated compounds to consider including:

  • Synthetic kinase inhibitors: neratinib, AZD1480, sorafenib, ponatinib, gefitinib, tricirbine

  • Natural compounds: Jadomycin B, tamoxifen citrate, EGCG

  • Known FN3K inhibitor: 1-deoxy-1-morpholinofructose (DMF)

Validation Approaches:

• Molecular dynamics simulations using tools like Gromacs 5.0

• ATP binding loop interactions analysis

• In vitro enzymatic assays to confirm computational predictions

• Cell-based assays measuring FN3K activity in the presence of inhibitors

• Western blotting to assess effects on FN3K protein expression and downstream targets

Experimental Readouts:

• Direct measurement of FN3K enzymatic activity

• Assessment of target protein deglycation efficiency

• Effects on Nrf2 pathway activation

• Metabolomic analysis to detect changes in redox-sensitive metabolites

• Cytotoxicity assays in relevant cell lines (BT-474, T-47D have been used in published research)

By incorporating these considerations, researchers can develop robust screening platforms for identifying and validating novel FN3K inhibitors with potential therapeutic applications.

How does the metabolomic profile change in FN3K-deficient cells and what are the implications?

¹H NMR metabolomics analysis has revealed significant differences in metabolite abundance between FN3K knockout and wild-type cells . These changes and their implications include:

Altered Metabolites in FN3K-KO Cells:

Implications:

• Redox Homeostasis: The enrichment of glutathione (a major cellular redox regulator) suggests FN3K plays a role in maintaining redox balance . Increased glutathione in FN3K-KO cells may represent a compensatory mechanism to manage altered redox states.

• Energy Metabolism: Decreased phosphocreatine/creatine ratio indicates potential alterations in ATP production mechanisms . Combined with increased lactate, this suggests a shift in energy metabolism, possibly toward enhanced glycolysis.

• Amino Acid Metabolism: Reductions in aspartate, glycine, and serine, all known to be reactive to cellular redox status, further support FN3K's involvement in redox regulation .

• Potential Therapeutic Implications: These metabolic changes suggest targeting FN3K could simultaneously affect cancer cell metabolism and redox defenses, representing a potential dual-action therapeutic strategy.

• Biomarker Development: The consistent metabolic signature in FN3K-deficient cells suggests these metabolites could serve as biomarkers for effective FN3K inhibition in therapeutic settings.

To fully understand these implications, researchers should consider integrating metabolomic data with other omics approaches (transcriptomics, proteomics) and validate findings across multiple cell types and in vivo models.

What are the optimal protocols for Western blotting detection of FN3K?

For optimal Western blotting detection of FN3K, researchers should follow these validated protocols:

Sample Preparation:

• Use validated positive control samples: HeLa cells, human brain/heart/kidney tissues, or mouse skeletal muscle tissue

• Prepare whole cell lysates using standard lysis buffers containing protease inhibitors

• Include phosphatase inhibitors if phosphorylation status is important

SDS-PAGE Conditions:

• Load 20-40 μg of total protein per lane

• Expect to detect FN3K at approximately 34-35 kDa

• Consider using pre-cast gradient gels (4-20%) for optimal resolution around this molecular weight

Antibody Selection and Dilution:

• Primary antibodies:

  • Proteintech 14293-1-AP: Use at 1:1000-1:4000 dilution

  • Abcepta AM2193a: Use at 1:1000-8000 dilution

  • Santa Cruz sc-271503: Follow manufacturer's recommendation

• Secondary antibodies:

  • Anti-rabbit or anti-mouse HRP-conjugated antibodies depending on primary

  • Dilute according to manufacturer's recommendations

Optimization Strategies:

• Blocking: Use 5% non-fat dry milk or BSA in TBST

• Primary antibody incubation: Overnight at 4°C or 1.5 hours at room temperature

• Include positive and negative controls to validate specificity

• For challenging samples, consider using an m-IgG Fc BP-HRP Bundle (like sc-527252) to reduce background

Detection Methods:

• Enhanced chemiluminescence (ECL) works well for FN3K detection

• For quantitative analysis, consider fluorescent secondary antibodies and imaging systems

• Exposure times may need optimization depending on expression levels

Properly executed Western blotting can reliably detect FN3K in various human and mouse samples, with validation data showing clear bands at the expected molecular weight .

What are the challenges in distinguishing between FN3K and FN3KRP in experimental settings?

FN3K and FN3KRP (FN3K-related protein) share structural and functional similarities that can create challenges in experimental differentiation:

Key Differences:

• Molecular Weight: FN3K is observed at 35 kDa while FN3KRP is observed at 30-34 kDa

• Gene IDs: FN3K (64122) vs. FN3KRP (79672)

• Substrate Specificity: Both phosphorylate ketosamines but with different preferences

Antibody Selection Strategy:

• Use specific antibodies validated for each protein:

  • FN3K: Proteintech 14293-1-AP or other validated antibodies

  • FN3KRP: Proteintech 16312-1-AP

• Verify specificity through knockout/knockdown validation

• Consider using epitope-tagged constructs for overexpression studies

Experimental Design Considerations:

• Run both proteins on the same gel for direct comparison

• Include appropriate positive controls for each protein

• Consider immunoprecipitation followed by mass spectrometry for definitive identification

• When possible, assess enzyme activity with substrate specificity assays

Expression Pattern Analysis:

• Compare expression across tissues (FN3K is prominently expressed in erythrocytes)

• Use RT-PCR with specific primers to distinguish between their mRNA expression

• Consider single-cell analysis techniques to map expression at cellular resolution

By carefully selecting antibodies and experimental approaches that exploit the known differences between these related proteins, researchers can accurately distinguish between FN3K and FN3KRP in their studies.

How should researchers design ELISA-based quantification of FN3K in biological samples?

For accurate ELISA-based quantification of FN3K in biological samples, researchers should consider the following validated approaches:

Commercial Kit Selection:

• ELK Biotechnology's Human FN3K ELISA Kit offers:

  • Sandwich ELISA format for high specificity

  • Detection range: 0.32-20 ng/mL

  • Sensitivity: 0.119 ng/mL

  • Standard concentration: 20 ng/mL

  • Compatible sample types: plasma, tissue homogenates, cell lysates, and other biological fluids

Sample Preparation:

• Proper sample collection and processing is critical:

  • Serum: Allow blood to clot, centrifuge and collect serum

  • EDTA/Heparin plasma: Collect blood with anticoagulant, centrifuge promptly

  • Tissue homogenates: Homogenize in PBS (pH 7.2-7.4), centrifuge to remove debris

  • Cell culture supernatants: Collect and centrifuge to remove particulates

Dilution Optimization:

• Sample dilution is often necessary:

  • Start with manufacturer's recommended dilutions

  • For serum/plasma: Recovery range 81-107% observed across sample types

  • Prepare multiple dilutions to ensure measurements fall within the standard curve

Quality Control Measures:

• Include standard curve on each plate (0.32-20 ng/mL range)

• Run samples in duplicate or triplicate

• Include known positive and negative controls

• Consider spike-recovery experiments to validate accuracy in specific sample types

Performance Expectations:

• Intra-assay precision: CV% <8% (within an assay)

• Inter-assay precision: CV% <10% (between assays)

• Recovery ranges:

  • Serum: 81-95% (average 88%)

  • EDTA plasma: 88-96% (average 92%)

  • Heparin plasma: 95-107% (average 101%)

By following these guidelines, researchers can achieve reliable quantification of FN3K in various biological samples, enabling accurate comparison between experimental conditions and contributing to reproducible research outcomes.

What is the potential role of FN3K in cancer biology and therapeutic development?

Emerging research reveals important connections between FN3K and cancer biology, suggesting several promising therapeutic avenues:

FN3K-Nrf2 Axis in Cancer:

• FN3K is involved in the deglycation of Nrf2, a significant regulator of oxidative stress in cancer cells

• Deglycated Nrf2 predominantly induces antioxidant responses by binding to sMAF proteins

• This activation offers cancer cell protection against oxidative stress

• Targeting FN3K could maintain Nrf2 in a glycated state, potentially sensitizing cancer cells to oxidative stress-inducing therapies

Therapeutic Strategies:

• FN3K inhibition could represent a novel approach to modulate cancer cell redox homeostasis

• Combinatorial approaches using:

  • Phytochemicals like brusatol and topotecan

  • Platinum derivatives such as cisplatin and oxaliplatin

  • Kinase modulators targeting FN3K activity

• Molecular docking studies have identified several potential FN3K inhibitors with anticancer potential, including:

  • Synthetic kinase inhibitors: neratinib, AZD1480, sorafenib, ponatinib, gefitinib, tricirbine

  • Natural compounds: Jadomycin B, tamoxifen citrate, EGCG

Metabolic Targeting:

• FN3K knockout alters metabolites critical for cancer cell survival:

  • Changes in glutathione affect redox defenses

  • Alterations in phosphocreatine/creatine ratio impact energy metabolism

  • Modified lactate levels influence glycolytic activity

• These metabolic vulnerabilities could be exploited alongside existing cancer therapies

Experimental Models:

• Breast cancer cell lines (BT-474, T-47D) have been successfully used in FN3K studies

• HepG2 cells have been used for FN3K knockout experiments

• These models provide platforms for screening therapeutic compounds and studying resistance mechanisms

The emerging understanding of FN3K's role in cancer biology highlights its potential as a therapeutic target, particularly through its influence on Nrf2-mediated antioxidant responses and cancer cell metabolism.

How is FN3K research advancing our understanding of diabetes complications?

While the search results focus primarily on cancer research, FN3K plays a crucial role in diabetes complications through its protein deglycation function:

Functional Significance in Diabetes:

• FN3K catalyzes phosphorylation of fructosamines formed by glycation, which is accelerated in hyperglycemic conditions

• This enzymatic action helps destabilize and ultimately remove potentially harmful fructosamines from proteins

• In diabetes, abnormal FN3K expression or activity can contribute to complications through accumulation of glycated proteins

Research Applications:

• FN3K antibodies enable researchers to:

  • Quantify FN3K expression in diabetic vs. non-diabetic tissues

  • Correlate FN3K levels with markers of glycation damage

  • Evaluate potential therapeutic interventions targeting the glycation pathway

Methodological Approaches:

• ELISA kits with sensitivity of 0.119 ng/mL allow precise quantification of FN3K in patient samples

• Western blotting protocols using validated antibodies permit assessment of FN3K expression in various tissues

• Immunohistochemistry can reveal tissue-specific changes in FN3K distribution in diabetic complications

Therapeutic Implications:

• Modulating FN3K activity could potentially reduce protein glycation damage

• 1-deoxy-1-morpholinofructose (DMF), a competitive inhibitor of FN3K, has been reported to inhibit approximately 10% of total FN3K activity

• FN3K activation might represent a protective mechanism against glycation-induced protein damage

As research progresses, FN3K antibodies will continue to be essential tools for investigating the role of this enzyme in diabetes pathophysiology and developing targeted interventions to mitigate glycation-related complications.