FBP2 Antibody, Biotin conjugated

What is FBP2 and what biological functions does it have?

FBP2 (Fructose-1,6-Bisphosphatase isozyme 2) is an enzyme that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate in the presence of divalent cations. This enzyme plays a critical role in glycogen synthesis from carbohydrate precursors, such as lactate. FBP2 is primarily expressed in muscle tissue, which distinguishes it from its isozyme FBP1 that is predominantly expressed in the liver . The enzyme is involved in multiple biological processes including metabolic regulation, signal transduction, and has been implicated in cancer research. Understanding FBP2's functions is essential as it represents an important metabolic control point in gluconeogenesis and glycolysis pathways, providing cells with adaptive capabilities for varying energy demands and substrate availability.

What are the key applications for FBP2 antibodies in research?

FBP2 antibodies have multiple research applications that help scientists investigate this enzyme's role in cellular processes. The primary applications include ELISA (Enzyme-Linked Immunosorbent Assay) for quantitative detection of FBP2 in samples, immunohistochemistry on both frozen and paraffin-embedded tissue sections to visualize the cellular and tissue distribution of FBP2, and Western blotting for protein expression analysis . Additionally, these antibodies are used in immunofluorescence studies to determine subcellular localization patterns, which is particularly important because FBP2 can exhibit both nuclear and cytoplasmic localization depending on cellular conditions . Co-immunoprecipitation experiments using FBP2 antibodies also help identify interacting protein partners, such as the demonstrated interaction with Hif1α, providing insights into FBP2's role in signaling networks and disease mechanisms .

What sample types can be analyzed using FBP2 antibodies?

FBP2 antibodies can be used to analyze various sample types depending on the research question and application. For protein expression studies, cell lysates from cultured cell lines (particularly those derived from muscle tissue, cancer cells, or metabolically relevant model systems) are commonly used in Western blotting and ELISA applications . Tissue sections from humans, mice, rats, and other research models can be examined using immunohistochemistry to visualize tissue-specific expression patterns and changes in disease states . Cell cultures subjected to different treatments can be analyzed with immunofluorescence to track changes in FBP2 subcellular localization. Additionally, protein fractions from subcellular compartmentation studies and immunoprecipitated protein complexes can be probed to investigate FBP2's interactions with other proteins like Hif1α . When using biotin-conjugated antibodies, researchers should consider potential tissue-specific endogenous biotin that might interfere with specific detection.

How can FBP2 antibodies be used to investigate the Reverse Warburg Effect?

The Reverse Warburg Effect, a metabolic phenomenon where stromal cells undergo aerobic glycolysis and provide nutrient metabolites to neighboring cancer cells, can be investigated using FBP2 antibodies through several sophisticated approaches. Researchers can employ dual immunofluorescence labeling using biotin-conjugated FBP2 antibodies along with markers of glycolysis (such as LDHA) to examine the spatial relationship between cells exhibiting different metabolic profiles within the tumor microenvironment . Co-localization studies with Hif1α are particularly valuable as the interaction between FBP2 and Hif1α has been documented through co-immunoprecipitation experiments . To study intercellular metabolic coupling, researchers can combine FBP2 immunostaining with microvesicle tracking using techniques like the PKH67 Green Fluorescent Cell Linker Kit to visualize transfer of metabolic enzymes between stromal and cancer cells . Additionally, fluorescent in situ hybridization (FISH) can be performed in parallel with FBP2 immunodetection to simultaneously analyze mRNA and protein expression patterns, providing insights into transcriptional and translational regulation of FBP2 in the context of the Reverse Warburg Effect.

What are the challenges in detecting nuclear versus cytoplasmic FBP2 and how can they be overcome?

Detecting the differential localization of FBP2 between nuclear and cytoplasmic compartments presents several technical challenges. FBP2 can shuttle between these compartments depending on cellular conditions, with research showing cytoplasmic enrichment during specific states like viral infection . One major challenge is the preservation of subcellular structures during sample preparation, which can be addressed by using optimized fixation protocols—4% paraformaldehyde fixation followed by careful permeabilization steps is recommended to maintain spatial integrity . When using biotin-conjugated antibodies, researchers should employ nuclear counterstains that are spectrally distinct from detection fluorophores to clearly delineate nuclear boundaries. For quantitative assessment of FBP2 localization, confocal microscopy with z-stack acquisition should be used rather than standard epifluorescence to accurately resolve nuclear versus cytoplasmic signals. Cell fractionation followed by Western blotting provides complementary biochemical validation of imaging data but requires careful optimization of lysis conditions to prevent cross-contamination between fractions. Additionally, using positive controls of known nuclear and cytoplasmic proteins in parallel samples helps validate the fractionation efficiency and antibody specificity for each compartment.

How can FBP2 antibodies be optimized for co-immunoprecipitation studies with potential interaction partners?

Optimizing FBP2 antibodies for co-immunoprecipitation (Co-IP) studies requires careful consideration of several parameters to ensure specific detection of genuine protein-protein interactions. Based on published research protocols, successful Co-IP of FBP2 with partners like Hif1α involves incubating the target proteins (0.25 μM of each) in buffer containing BSA (0.6 mg/mL) and divalent cations like MgCl₂ (2 mM) overnight at 4°C with rotation . When using biotin-conjugated antibodies, researchers should first determine whether the biotin moiety interferes with the antibody's binding to the epitope region involved in protein-protein interactions. Pre-clearing lysates with Protein G agarose before adding the biotin-conjugated antibody reduces non-specific binding. For detection of transient or weak interactions, chemical crosslinking with membrane-permeable crosslinkers before cell lysis may help stabilize complexes. Researchers should also optimize antibody concentrations—using 0.5 μM of antibody has been effective in published protocols . Multiple washing steps (at least three washes with PBS) are critical to remove non-specifically bound proteins while preserving genuine interactions. Additionally, reciprocal Co-IP experiments (pulling down with anti-Hif1α and blotting for FBP2, then vice versa) provide stronger evidence for specific interactions as demonstrated in the literature .

What is the relationship between FBP2 and viral infection processes?

Research has revealed an unexpected role for FBP2 in viral infection processes, particularly as an internal ribosomal entry site trans-acting factor (ITAF) that regulates viral translation. Studies using biotinylated RNA-affinity chromatography and proteomic approaches identified FBP2, also known as KSRP or KHSRP (K homology splicing regulatory protein), as a far upstream element binding protein that interacts with the Enterovirus 71 (EV71) internal ribosomal entry site (IRES) . Interestingly, FBP2 acts as a negative regulator of viral translation—knockdown of FBP2 in infected cells resulted in increased viral protein synthesis and enhanced IRES activity, while overexpression of FBP2 decreased IRES activity . A notable cellular response observed during viral infection is the relocalization of FBP2 from the nucleus (where it is predominantly found in uninfected cells) to the cytoplasm where viral replication occurs . This suggests that viruses may induce changes in FBP2's subcellular distribution as part of their replication strategy. Researchers investigating this phenomenon can employ biotin-conjugated FBP2 antibodies in immunofluorescence studies to track this relocalization during infection, while also using co-immunoprecipitation to identify viral components that interact with FBP2.

What are the optimal conditions for using biotin-conjugated FBP2 antibodies in ELISA applications?

For optimal ELISA performance with biotin-conjugated FBP2 antibodies, several methodological factors require careful consideration. Based on the technical specifications of commercially available biotin-conjugated FBP2 antibodies, the following protocol elements are critical for sensitivity and specificity. The coating buffer should be optimized—typically a 50 mM carbonate/bicarbonate buffer at pH 9.6 works well for most capture antibodies. Sample diluent should contain 50% glycerol with PBS (pH 7.4) to maintain antibody stability, as specified in product documentation . Blocking solution requires careful optimization—a buffer containing 0.03% Proclin 300 as a preservative prevents microbial contamination without affecting antibody activity . For detection systems, streptavidin-HRP conjugates at dilutions of 1:5000 to 1:10000 typically provide the best signal-to-noise ratio with biotin-conjugated antibodies. Washing steps should be performed with PBS containing 0.05% Tween-20, with at least four wash cycles between steps. The assay temperature should be maintained at a constant 25°C throughout the procedure, as temperature fluctuations can affect binding kinetics. To determine optimal antibody concentration, a checkerboard titration experiment should be performed testing various dilutions against known positive and negative control samples.

How should sample preparation be optimized for detecting FBP2 in different tissues and cell types?

Sample preparation methods must be tailored to different tissues and cell types for optimal FBP2 detection. For cell culture samples, homogenization in a buffer containing 100 mM Tris/HCl, 2% SDS, 50 mM DTT at pH 8.0, with incubation for 10 minutes at 99°C has proven effective . For tissue samples, fresh freezing is preferable for preserving FBP2 enzymatic activity, while formalin fixation and paraffin embedding require antigen retrieval steps—typically heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes. When preparing subcellular fractions, nuclear and cytoplasmic separation should use gentle lysis conditions to prevent artificial redistribution of FBP2 between compartments. For immunoprecipitation experiments, cell lysis in buffers containing phosphatase and protease inhibitors is critical since post-translational modifications may affect FBP2 interactions with partners like Hif1α . Microvesicle isolation from culture medium requires differential centrifugation followed by verification of purity before analysis of FBP2 content . For each sample type, validation with known positive controls is essential—muscle tissue typically expresses high levels of FBP2 and serves as an appropriate positive control for antibody validation.

What strategies can minimize background and non-specific binding when using biotin-conjugated FBP2 antibodies?

Minimizing background and non-specific binding is crucial when working with biotin-conjugated FBP2 antibodies. Several effective strategies are recommended based on research protocols. Firstly, endogenous biotin blocking is essential, particularly in biotin-rich tissues like liver, kidney, and brain—commercially available avidin/biotin blocking kits should be used prior to antibody application . For immunohistochemistry applications, pretreating tissues with 3% hydrogen peroxide to quench endogenous peroxidase activity before applying biotin-conjugated antibodies prevents false-positive signals with HRP detection systems. Using Protein A-purified antibodies, as mentioned in the antibody specifications (>95% Protein G purified), reduces non-specific binding due to contaminating proteins . Including 0.01-0.05% Tween-20 in wash buffers disrupts weak non-specific interactions while preserving specific antibody binding. For tissue sections, blocking with species-specific normal serum (5-10%) matching the host species of secondary detection reagents reduces background. When performing multiplex immunofluorescence, careful selection of fluorophores with minimal spectral overlap and the use of appropriate absorption controls for each channel prevents misinterpretation of signals. Additionally, including isotype control antibodies in parallel reactions helps distinguish specific from non-specific binding patterns.

What are the best practices for validating FBP2 antibody specificity in research applications?

Rigorous validation of FBP2 antibody specificity is essential for generating reliable research data. A comprehensive validation approach includes multiple complementary methods. Western blotting with positive controls (muscle tissue lysates) and negative controls (tissues with known low FBP2 expression) should demonstrate a single band at the expected molecular weight (approximately 37 kDa for human FBP2) . Knockdown or knockout validation provides compelling evidence for specificity—comparing antibody signals in cells with normal FBP2 expression versus cells where FBP2 expression has been silenced using shRNA, as described in research protocols . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, should abolish specific signals if the antibody is truly specific. Cross-reactivity testing against the highly homologous FBP1 is particularly important—the antibody should distinguish between these isozymes despite their structural similarities . For immunoprecipitation applications, mass spectrometry analysis of immunoprecipitated proteins can confirm the presence of FBP2 and identify potential cross-reacting proteins. When possible, comparing results from antibodies targeting different epitopes of FBP2 provides additional validation of signal specificity. Researchers should also verify species reactivity experimentally, even when predicted reactivity is indicated in product information .

How should researchers interpret unexpected changes in FBP2 subcellular localization?

Unexpected changes in FBP2 subcellular localization can provide valuable insights into cellular processes but require careful interpretation. Research has established that FBP2 can shuttle between nuclear and cytoplasmic compartments under specific conditions—during viral infection, for example, FBP2 becomes enriched in the cytoplasm where viral replication occurs, whereas it predominantly localizes to the nucleus in uninfected cells . When interpreting such changes, researchers should first confirm the observation with multiple technical approaches. Complementing immunofluorescence with subcellular fractionation and Western blotting provides biochemical validation of microscopy observations. Temporal analysis is critical—examining multiple time points can reveal whether the localization change is transient or persistent, potentially correlating with specific cellular events. Researchers should consider post-translational modifications that might regulate FBP2 trafficking between compartments. Co-localization studies with markers of specific subcellular structures (nucleoli, mitochondria, stress granules) can provide functional context for the observed relocalization. Additionally, examining whether the localization change correlates with alterations in FBP2 enzymatic activity or protein-protein interactions provides mechanistic insights into the biological significance of the observation.

What factors might cause variability in FBP2 detection between different experimental replicates?

Variability in FBP2 detection between experimental replicates can arise from multiple sources that require systematic troubleshooting. Cell culture conditions significantly impact FBP2 expression—variations in cell density, passage number, and serum batches can alter metabolic states and consequently FBP2 levels. Technical variability in sample preparation, particularly inconsistent cell lysis procedures, can result in different extraction efficiencies of FBP2 from various subcellular compartments, as FBP2 has been documented to localize to both nuclear and cytoplasmic fractions . Antibody quality and consistency between lots may introduce variability—researchers should record lot numbers and standardize antibody concentrations using calibration curves with recombinant FBP2 protein. Storage conditions of both samples and antibodies are critical—repeated freeze-thaw cycles can reduce protein integrity and antibody activity, with product documentation recommending avoiding repeated freeze-thaw cycles for biotin-conjugated antibodies . For biotin-conjugated antibodies specifically, the age of the conjugate affects detection efficiency as biotin-streptavidin binding may deteriorate over time. Environmental factors during the experiment, such as temperature fluctuations and inconsistent incubation times, can also contribute to variability. Standardization through the inclusion of internal controls and reference samples in each experimental batch helps normalize data and identify potential sources of variation.

How can researchers distinguish between true FBP2 signals and artifacts when using biotin-conjugated antibodies?

Distinguishing between true FBP2 signals and artifacts when using biotin-conjugated antibodies requires implementation of rigorous controls and validation steps. Multiple negative controls are essential: (1) omission of primary antibody while maintaining all other reagents reveals background from the detection system; (2) isotype control antibodies from the same host species and of the same immunoglobulin class (rabbit IgG for most FBP2 antibodies) identify non-specific binding; (3) pre-adsorption controls where the antibody is pre-incubated with recombinant FBP2 protein should abolish specific signals. For tissues with high endogenous biotin content, comparing detection patterns with biotin-conjugated versus unconjugated FBP2 antibodies followed by biotinylated secondary antibodies can identify endogenous biotin interference. Signal specificity can be further confirmed by gene silencing approaches—comparing signals in cells with normal FBP2 expression versus cells where FBP2 has been silenced using shRNA techniques as described in research protocols . Using multiple antibodies targeting different epitopes of FBP2 should produce concordant patterns in truly positive samples. Cross-validation with non-antibody detection methods such as fluorescent in situ hybridization (FISH) for FBP2 mRNA provides independent confirmation of expression patterns . Additionally, confirming that the observed signal distribution is consistent with known biology of FBP2—such as its reported nuclear-cytoplasmic shuttling during specific cellular conditions—increases confidence in signal authenticity.

How can FBP2 antibodies be utilized in research on metabolic reprogramming in cancer?

FBP2 antibodies represent powerful tools for investigating metabolic reprogramming in cancer, particularly in studies of the Reverse Warburg Effect. This phenomenon involves metabolic coupling between cancer cells and surrounding stromal cells, where FBP2 plays regulatory roles . Researchers can use biotin-conjugated FBP2 antibodies in multiplex immunofluorescence studies to simultaneously visualize FBP2 distribution alongside glycolytic markers (GLUT1, LDHA) and mitochondrial markers in tumor tissue sections, providing spatial context for metabolic compartmentation. Laser capture microdissection combined with immunohistochemical staining for FBP2 allows isolation of specific cell populations for downstream molecular analysis. Co-immunoprecipitation studies using FBP2 antibodies can identify novel interaction partners in cancer cells versus normal cells, potentially revealing cancer-specific signaling networks. The documented interaction of FBP2 with Hif1α is particularly relevant for cancer research, as Hif1α is a master regulator of the hypoxic response and metabolic adaptation in tumors . Chromatin immunoprecipitation (ChIP) assays with FBP2 antibodies can investigate potential roles in transcriptional regulation of metabolic genes. For functional studies, comparing FBP2 expression and localization before and after treatment with metabolic inhibitors or hypoxia can reveal regulatory mechanisms of metabolic adaptation. Additionally, analyzing FBP2 in microvesicles secreted by cancer cells provides insights into intercellular metabolic communication within the tumor microenvironment .

What are the emerging applications of FBP2 antibodies in viral infection research?

Emerging applications of FBP2 antibodies in viral infection research build upon the discovery that FBP2 functions as an internal ribosomal entry site trans-acting factor (ITAF) that negatively regulates viral translation . Researchers can employ biotin-conjugated FBP2 antibodies in time-course immunofluorescence studies to track the dynamic relocalization of FBP2 from the nucleus to the cytoplasm during viral infection, providing insights into viral manipulation of host factors . RNA immunoprecipitation (RIP) assays using FBP2 antibodies can identify viral RNA sequences that directly interact with FBP2, complementing previous findings from biotinylated RNA-affinity chromatography studies . Co-immunoprecipitation experiments can reveal interactions between FBP2 and viral proteins, potentially identifying mechanisms by which viruses counteract FBP2's negative regulatory effect on viral translation. Comparative proteomics of FBP2-associated protein complexes in infected versus uninfected cells can uncover infection-induced changes in FBP2's interaction network. For functional studies, combining FBP2 knockdown or overexpression with viral infection models allows assessment of how FBP2 levels affect viral replication kinetics across different virus families. High-resolution microscopy using biotin-conjugated FBP2 antibodies can visualize potential co-localization with viral replication complexes within infected cells. Additionally, examining post-translational modifications of FBP2 during viral infection may reveal regulatory mechanisms that alter its function as host defense or viral target.

What are the recommended experimental designs for studying FBP2 interactions with Hif1α?

Research has established that FBP2 interacts with Hif1α, a master regulator of hypoxic response, suggesting important functions in metabolic adaptation and signaling . To comprehensively study this interaction, a multi-faceted experimental approach is recommended. Co-immunoprecipitation experiments should follow validated protocols using recombinant proteins (0.25 μM of each protein) in buffers containing 0.6 mg/mL BSA and 2 mM MgCl₂, with overnight incubation at 4°C with rotation as described in the literature . Reciprocal co-immunoprecipitation (using anti-FBP2 to pull down Hif1α and vice versa) provides stronger evidence for specific interaction. The Duolink® in situ proximity ligation assay offers visualization of endogenous FBP2-Hif1α interactions within intact cells, using rabbit anti-Hif1α (1:500) and mouse anti-FBP2 (1:500) primary antibodies as specified in published protocols . For functional analysis, researchers should examine how hypoxic conditions affect the interaction by comparing normoxic versus hypoxic treatments. Deletion mutants of both FBP2 and Hif1α can map specific interaction domains. To understand functional consequences, researchers should assess how the interaction affects Hif1α transcriptional activity using reporter assays with hypoxia response elements. Chromatin immunoprecipitation assays can determine whether FBP2 is recruited to Hif1α target genes. Additionally, examining how the interaction impacts FBP2 enzymatic activity provides insights into metabolic consequences of the association.

How can FBP2 antibodies contribute to studies of intercellular communication via microvesicles?

FBP2 antibodies offer valuable tools for investigating the emerging role of FBP2 in intercellular communication via microvesicles. Research has demonstrated that FBP2 can be transferred between cells through microvesicles, suggesting a mechanism for metabolic coordination in tissues . Immunoblotting of isolated microvesicles using biotin-conjugated FBP2 antibodies can quantify FBP2 content in vesicles produced under different cellular conditions. To visualize this transfer, researchers can use a combination of PKH67-stained microvesicles and immunofluorescent detection of FBP2 as described in published protocols . Flow cytometry of isolated microvesicles using fluorescently labeled FBP2 antibodies provides quantitative assessment of FBP2-positive vesicle populations. Immuno-electron microscopy with gold-conjugated detection systems offers high-resolution visualization of FBP2 localization within microvesicle structures. For functional studies, researchers can apply purified microvesicles from donor cells to recipient cells and track changes in FBP2 localization and metabolic parameters over time. Comparison of microvesicle content between normal and pathological states (such as cancer or inflammation) may reveal disease-specific alterations in FBP2 packaging. Co-immunoprecipitation of FBP2 from isolated microvesicles can identify associated proteins that might be co-delivered to recipient cells. Additionally, studying how microvesicle-delivered FBP2 differs functionally from endogenously expressed FBP2 in recipient cells could reveal novel regulatory mechanisms in intercellular metabolic coordination.