FBP2 Antibody, HRP conjugated

What is FBP2 and why is it an important research target?

FBP2 (Fructose-1,6-bisphosphatase 2), also known as muscle FBP, is a 37 kDa protein (339 amino acids) belonging to the FBPase class 1 family. This enzyme plays a crucial role in glycogen synthesis from non-carbohydrates such as lactate, a process known as glyconeogenesis. Beyond its enzymatic functions, FBP2 demonstrates various non-enzymatic roles including regulation of hypoxia-inducible factor 1 (Hif1) stability, cell cycle-dependent events, mitochondrial biogenesis, and organ protection . The significance of FBP2 extends to multiple research areas as its dysregulation has been implicated in various diseases, including metabolic disorders and certain types of cancer. This makes FBP2 a valuable target for investigations in metabolism, diabetes, and cancer research .

To effectively study FBP2, researchers must understand its basic properties:

How does HRP conjugation enhance antibody detection in FBP2 research?

HRP (Horseradish Peroxidase) conjugation significantly enhances antibody detection systems by providing enzymatic signal amplification capabilities. When conjugated to anti-FBP2 antibodies, the HRP enzyme catalyzes oxidation reactions with various substrates to produce colorimetric, chemiluminescent, or fluorescent signals, dramatically improving detection sensitivity compared to direct labeling methods. This signal amplification is particularly valuable when researching FBP2 in tissues where its expression might be limited or in complex biological samples .

The enzymatic nature of HRP provides substantial signal multiplication, as each HRP molecule can process numerous substrate molecules, generating amplified signals that allow for the detection of even low-abundance FBP2 protein. This enzymatic cascade enables researchers to identify subtle changes in FBP2 expression levels that might be missed with less sensitive detection methods, essential for understanding FBP2's role in metabolic regulation and disease progression.

What are the optimal dilution ratios for FBP2 antibody across different applications?

Determining the optimal dilution ratio for FBP2 antibody applications is critical for achieving specific, reproducible results while conserving valuable reagents. Based on experimental validation data, recommended dilution ranges vary significantly depending on the application technique:

These recommendations should serve as starting points for optimization in your specific experimental system. For critical experiments, perform preliminary titration series with your particular samples to determine the optimal signal-to-noise ratio. Note that sample type and preparation method can significantly influence antibody performance, making optimization essential for each new experimental system .

When working with HRP-conjugated FBP2 antibodies specifically, slightly higher dilutions may be appropriate compared to unconjugated primary antibodies due to the signal amplification properties of the HRP enzyme. Always validate the optimal dilution with appropriate positive and negative controls relevant to your experimental tissue or cell type.

How should one optimize antigen retrieval for FBP2 detection in different tissue types?

Antigen retrieval optimization is essential for successful FBP2 detection in fixed tissues, as fixation processes can mask epitopes through protein cross-linking. For FBP2 detection, two primary retrieval methods have demonstrated efficacy:

• Primary Recommended Method: TE buffer (pH 9.0) heat-mediated retrieval

• Alternative Method: Citrate buffer (pH 6.0) heat-mediated retrieval

The choice between these methods depends on tissue type and fixation protocol. Skeletal muscle tissues often yield optimal results with TE buffer (pH 9.0), while other tissue types may require method optimization .

When developing an antigen retrieval protocol for a new tissue type, consider implementing a split-sample approach where serial sections are processed using both methods and compared for signal intensity, specificity, and background levels. Additionally, retrieval duration and temperature should be optimized - typically ranging from 10-30 minutes at 95-100°C for water bath methods or appropriate manufacturer-recommended settings for pressure cooker or microwave-based retrieval systems.

For tissues known to express low levels of FBP2, extending retrieval time may improve detection, but this must be balanced against potential tissue degradation and increased background staining. Always include known positive control tissues (such as skeletal muscle) alongside experimental samples to validate retrieval effectiveness.

How can FBP2 antibodies be effectively used to investigate its non-enzymatic functions?

Investigating FBP2's non-enzymatic functions requires specialized approaches beyond standard expression analysis. Current research indicates that FBP2 plays important roles in hypoxia-inducible factor 1 (Hif1) stability regulation, cell cycle events, mitochondrial biogenesis, and organ protection mechanisms . To effectively study these functions:

• Subcellular Localization Analysis: Implement dual-labeling immunofluorescence with HRP-conjugated FBP2 antibodies alongside organelle markers (mitochondrial, nuclear, etc.) to track FBP2 redistribution under different cellular conditions. This approach is particularly valuable as FBP2 has been observed to relocalize from nucleus to cytoplasm during certain cellular stress conditions, such as viral infection .

• Protein-Protein Interaction Studies: Employ co-immunoprecipitation with FBP2 antibodies followed by mass spectrometry to identify novel interaction partners. This approach has successfully revealed FBP2's interaction with viral RNA elements, suggesting similar methodologies could uncover interactions with cellular factors .

• Functional Impact Assessment: Combine FBP2 knockdown or overexpression with metabolic flux analysis to distinguish between enzymatic and non-enzymatic effects. Compare glycolytic rates, oxygen consumption, and mitochondrial membrane potential changes to isolate specific functional impacts.

For studying FBP2's role in mitochondrial function, consider using permeabilized cell systems where direct substrate addition to mitochondria can bypass glycolytic effects, allowing isolation of non-enzymatic mitochondrial impacts from enzymatic metabolic effects.

How reliable are current FBP2 antibodies for detecting species-specific variants in comparative studies?

• Sequence Homology Assessment: Before selecting an antibody for multi-species studies, analyze FBP2 sequence conservation across target species, particularly in the immunogen region. The primary sequence for human FBP2 (NP_003828.2) should be aligned with orthologues from experimental species to predict potential cross-reactivity .

• Validation Requirements: Each new species application requires independent validation. Western blotting with species-specific positive controls (skeletal muscle tissue is recommended) should be performed to confirm antibody functionality and to identify any molecular weight variations between species.

• Application-Specific Optimization: Even with confirmed cross-reactivity, species-specific protocol optimization is essential. Dilution ratios, incubation times, and blocking conditions often require adjustment when transitioning between species samples.

For novel species applications not previously validated, preliminary testing with multiple antibody clones targeting different FBP2 epitopes is recommended to ensure reliable detection. Additionally, when absolute confirmation is required, consider employing genetic approaches (siRNA knockdown in cell culture models) alongside antibody detection to validate specificity.

What are the most common causes of false negative results when using FBP2 antibodies, and how can they be addressed?

• Inadequate Antigen Retrieval: FBP2 epitopes are particularly susceptible to masking during fixation. If negative results occur in fixed tissues despite presence of FBP2, intensify antigen retrieval by:

  • Switching from citrate buffer (pH 6.0) to TE buffer (pH 9.0)

  • Extending retrieval time (from standard 20 minutes to 30-40 minutes)

  • Ensuring complete specimen submersion during retrieval

• Inappropriate Antibody Dilution: Using overly diluted antibody preparations. Solution: Titrate antibody concentrations, starting with more concentrated preparations than standard recommendations:

  • For IHC: Begin at 1:300 rather than 1:1200

  • For WB: Try 1:2000 before moving to more dilute preparations

• Suboptimal Sample Preparation: Protein degradation during extraction. Solutions:

  • Include protease inhibitors in all extraction buffers

  • Maintain cold chain throughout processing

  • Consider using fresh rather than frozen samples for initial validation

• Buffer Incompatibility: Some extraction buffers may interfere with epitope recognition. Solution:

  • Test multiple extraction methods (RIPA, NP-40, Triton-based buffers)

  • Perform dialysis to remove potential interfering compounds

• Low Target Expression: FBP2 expression varies significantly across tissues. Solution:

  • Include known positive control tissues (skeletal muscle, heart) alongside experimental samples

  • Consider enrichment techniques for low-abundance samples

For persistent false negative results, utilizing alternative detection methods such as mRNA analysis (RT-PCR) can help confirm whether the issue is technical or biological in nature.

How can researchers troubleshoot poor signal-to-noise ratios when using HRP-conjugated FBP2 antibodies?

Poor signal-to-noise ratios represent a significant challenge when using HRP-conjugated antibodies for FBP2 detection. To address this issue systematically:

• Optimize Blocking Conditions:

  • Test alternative blocking agents (5% BSA, 5% milk, commercial blocking buffers)

  • Extend blocking time from standard 1 hour to 2-3 hours at room temperature

  • Consider overnight blocking at 4°C for problematic samples

• Adjust Antibody Concentration:

  • Counterintuitively, sometimes more dilute antibody preparations (1:20000-1:50000 for WB) provide better signal-to-noise ratios than more concentrated ones

  • Perform systematic titration experiments to identify optimal concentration

• Modify Washing Procedures:

  • Increase wash buffer stringency (add 0.1-0.3% Tween-20)

  • Extend wash times and increase wash buffer volumes

  • Implement additional wash steps between antibody incubations

• Substrate Development Optimization:

  • For colorimetric detection: Shorten substrate development time and observe development continuously

  • For chemiluminescent detection: Adjust exposure times to prevent signal saturation

  • Consider using signal enhancers specifically designed for HRP systems

• Reduce Non-Specific Binding:

  • Pre-adsorb antibodies with tissues/cells not expressing the target

  • Add 0.1-0.5% Triton X-100 to antibody diluent to reduce hydrophobic interactions

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

When troubleshooting, modify only one parameter at a time and maintain careful records of all protocol variations to identify the most effective approach for your specific experimental system.

What are the optimal storage conditions for maintaining long-term stability of HRP-conjugated FBP2 antibodies?

Maintaining the stability and activity of HRP-conjugated FBP2 antibodies is critical for experimental reproducibility and reagent cost-effectiveness. Optimal storage practices include:

• Temperature Conditions:

  • Store at -20°C for long-term stability (up to one year after shipment)

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

  • For short-term storage (1-2 weeks), 4°C is acceptable if antibody contains preservatives

• Buffer Composition:

  • Optimal storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

  • Glycerol prevents freeze-thaw damage and maintains protein stability

  • Note: While sodium azide preserves antibody stability, it can inhibit HRP activity at high concentrations

• Aliquoting Strategy:

  • For 20μL antibody preparations containing 0.1% BSA, aliquoting is generally unnecessary for -20°C storage

  • For larger volumes, divide into single-use aliquots of 5-10μL to minimize freeze-thaw cycles

  • Use sterile tubes for aliquoting to prevent microbial contamination

• Alternative Preservation Methods:

  • For field applications or resource-limited settings, consider dry storage methods as described for HRP-conjugated detection systems

  • Lyophilization with appropriate cryoprotectants can maintain antibody function

When determining storage conditions for valuable antibody preparations, validate the activity retention through periodic testing of stored aliquots. This can be performed using simple dot blots with known positive samples to confirm signal generation capability over time.

How can researchers establish an effective quality control system for monitoring FBP2 antibody performance over time?

Establishing a robust quality control (QC) system is essential for ensuring consistent FBP2 antibody performance across experiments. An effective QC program should include:

• Reference Standard Creation:

  • Prepare a master lot of positive control lysate (mouse skeletal muscle or heart tissue)

  • Aliquot and store at -80°C

  • Use this standard for all QC testing to enable direct comparison between tests

• Regular Performance Assessment:

  • Test antibody activity quarterly using standardized conditions

  • Monitor critical parameters:

    • Signal intensity at standard concentration

    • Background levels

    • Specificity (single vs. multiple bands)

    • Lot-to-lot consistency when replacing reagents

• Documentation System:

  • Maintain detailed records including:

    • Antibody dilution used

    • Incubation conditions

    • Detection methods

    • Image acquisition settings

    • Raw and processed data files

  • Document any deviations from expected performance

• Performance Metrics Establishment:

  • Define acceptable ranges for key parameters:

    • Signal-to-noise ratio (minimum 10:1)

    • Background levels (maximum OD value in negative control regions)

    • Coefficient of variation between technical replicates (<15%)

• Remediation Protocol Development:

  • Create decision trees for addressing performance deterioration

  • Include steps for troubleshooting based on specific failure modes

  • Establish criteria for reagent replacement

A well-designed QC system allows researchers to identify performance issues before they impact experimental results, enhancing data reliability and reproducibility in FBP2 research applications.

How can FBP2 antibodies be utilized to investigate its role in viral infection mechanisms?

Recent research has revealed an unexpected role for FBP2 in viral infection, particularly in its interaction with Enterovirus 71 (EV71). FBP2 has been identified as a novel ITAF (IRES trans-acting factor) that interacts with EV71 IRES and negatively regulates viral translation . To investigate FBP2's role in viral infection mechanisms:

• Subcellular Redistribution Analysis:

  • Track FBP2 nuclear-to-cytoplasmic redistribution during viral infection using immunofluorescence microscopy

  • Immunofluorescence assays have demonstrated that FBP2 significantly redistributes from the nucleus to the cytoplasm upon EV71 infection

  • This approach can be adapted to study other viral infections potentially regulated by FBP2

• RNA-Protein Interaction Studies:

  • Implement co-immunoprecipitation assays using anti-FBP2 antibodies to isolate FBP2-viral RNA complexes

  • RT-PCR can then be used to amplify and identify viral RNA elements that interact with FBP2

  • This technique successfully demonstrated FBP2 association with EV71 5' UTR during infection

• Functional Impact Assessment:

  • Combine knockdown and overexpression approaches with viral protein synthesis monitoring

  • Using [35S] methionine pulse-labeling in FBP2-depleted cells showed increased viral protein synthesis, while FBP2-overexpressed cells exhibited decreased viral protein synthesis

  • This approach is valuable for characterizing FBP2's regulatory effect on viral translation

• Competition Binding Mechanism Analysis:

  • Investigate the mechanism by which FBP2 negatively regulates viral translation

  • In vitro competition binding assays revealed that FBP2 competes with positive ITAFs like PTB for IRES binding

  • Similar approaches can be used to study FBP2's interaction with other viral elements

These methodologies provide a framework for investigating FBP2's role in viral pathogenesis and potentially identifying novel antiviral strategies targeting this interaction.

What are the most effective approaches for studying FBP2's role in cancer progression using HRP-conjugated antibodies?

FBP2 dysregulation has been implicated in cancer pathogenesis, making it an important target for oncology research. HRP-conjugated FBP2 antibodies offer powerful tools for investigating its role in cancer progression:

• Tissue Microarray Analysis:

  • Utilize HRP-conjugated FBP2 antibodies for high-throughput IHC screening of tissue microarrays

  • Validated protocols show successful detection in human ovary cancer tissue using TE buffer (pH 9.0) for antigen retrieval

  • Quantify expression patterns across tumor grades, stages, and subtypes to establish clinical correlations

• Metabolic Reprogramming Assessment:

  • Given FBP2's role in regulating gluconeogenesis and glycolysis, investigate how its expression correlates with metabolic reprogramming in cancer cells

  • Combine FBP2 immunostaining with metabolic markers to map metabolic phenotypes within tumor microenvironments

  • This approach can identify specific cancer subtypes potentially vulnerable to metabolic interventions targeting FBP2

• Multi-parametric Analysis:

  • Implement multiplex immunofluorescence including FBP2 alongside markers of:

    • Proliferation (Ki-67)

    • Apoptosis (cleaved caspase-3)

    • Metabolic enzymes (PKM2, LDHA)

    • Hypoxia markers (given FBP2's role in HIF1 regulation)

• Functional Validation in Model Systems:

  • Correlate immunohistochemical findings with functional studies in cell culture models

  • Implement genetic manipulation (siRNA, CRISPR) followed by phenotypic assays to validate observations from human samples

  • Use matched patient-derived xenografts and corresponding primary tumor samples to track FBP2 expression changes during cancer evolution

• Circulating Tumor Cell Analysis:

  • Develop protocols for detecting FBP2 in CTCs as potential biomarkers

  • Optimize fixation and permeabilization protocols for rare cell detection

  • Combine with epithelial-mesenchymal transition markers to characterize CTC subpopulations

These approaches leverage the sensitivity and specificity of HRP-conjugated FBP2 antibodies to advance understanding of FBP2's multifaceted roles in cancer biology, potentially identifying novel therapeutic targets or diagnostic biomarkers.

How can dry storage technologies enhance the utility of HRP-conjugated FBP2 antibodies in resource-limited settings?

Recent advances in dry storage technologies offer promising solutions for extending the utility of HRP-conjugated antibodies, including FBP2 detection systems, in resource-limited settings:

• Long-term Dry Storage Methods:

  • Novel approaches for preserving HRP-conjugated antibodies in dry form have been developed

  • These methods maintain enzyme activity and antibody specificity under elevated temperature conditions

  • Such technologies could substantially extend the shelf-life of FBP2 antibody diagnostics without cold chain requirements

• Integration with Paper-Based Diagnostic Platforms:

  • Two-dimensional paper network formats (2DPNs) using shaped paper with multiple inlets can "program" automated multistep assay sequences

  • These platforms require only a single user activation step, making them suitable for minimally trained personnel

  • Such systems have been successfully implemented for detecting malarial biomarkers and could be adapted for FBP2 detection

• Glass Fiber Pad Preservation:

  • HRP-antibody conjugates can be dried on glass fiber pads and rehydrated to working concentrations when needed

  • This approach eliminates the need for continuous refrigeration

  • For FBP2 antibodies, optimization of rehydration buffers would be necessary to maintain optimal detection sensitivity

• Substrate Stability Enhancement:

  • Colorimetric substrates for HRP can also be preserved in dry form

  • Diaminobenzidine (DAB) has been successfully stabilized for long-term storage

  • Combined with dried antibodies, this creates fully shelf-stable detection systems

These technologies create opportunities for implementing FBP2 detection in field settings, remote laboratories, and resource-constrained environments without compromising analytical performance. The development of such systems would particularly benefit epidemiological studies of metabolic disorders in regions lacking consistent refrigeration infrastructure.

What are the most promising applications of FBP2 antibodies in metabolic disease research?

FBP2's central role in gluconeogenesis and glycolysis positions it as a key target for metabolic disease research. Several promising applications for FBP2 antibodies in this field include:

• Tissue-Specific Metabolic Reprogramming:

  • FBP2 regulation differs across tissues, with unique expression patterns in skeletal muscle and heart

  • HRP-conjugated FBP2 antibodies enable high-sensitivity mapping of expression changes in diseased tissues

  • This approach can identify tissue-specific metabolic adaptations in diabetes, obesity, and related disorders

• Mitochondrial Function Assessment:

  • FBP2's involvement in mitochondrial biogenesis suggests potential applications in mitochondrial dysfunction research

  • Co-localization studies with mitochondrial markers can reveal how FBP2 distribution changes in metabolic diseases

  • This may uncover novel mechanisms connecting glycolytic regulation to mitochondrial health

• Therapeutic Target Validation:

  • As dysregulation of FBP2 has been implicated in metabolic disorders , antibody-based detection provides critical validation for therapeutic development

  • Monitoring FBP2 expression, localization, and post-translational modifications in response to experimental therapeutics

  • This application bridges basic research to translational medicine

• Biomarker Development:

  • Changes in FBP2 expression patterns may serve as diagnostic or prognostic indicators

  • Standardized IHC protocols using optimized antigen retrieval methods enable consistent assessment across clinical samples

  • Such biomarkers could improve stratification of metabolic disease subtypes

• Exercise Physiology Research:

  • Given FBP2's prominence in skeletal muscle metabolism, antibody-based studies can investigate adaptations to exercise interventions

  • This application can illuminate mechanisms underlying exercise benefits in metabolic disorders

  • Time-course studies can track acute versus chronic adaptations in FBP2 regulation

These applications highlight the versatility of FBP2 antibodies in advancing understanding of metabolic disease pathophysiology and identifying potential intervention points for therapeutic development.