Recombinant Mouse Acetolactate synthase-like protein (Ilvbl)

What is Recombinant Mouse Acetolactate synthase-like protein (Ilvbl) and what are its key functions?

Recombinant Mouse Acetolactate synthase-like protein (Ilvbl) is a membrane single-pass protein involved in the biosynthesis of branched-chain amino acids and is essential for cellular metabolism . It functions as an endoplasmic reticulum 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in fatty acid alpha-oxidation in a thiamine pyrophosphate (TPP)-dependent manner . This protein also plays a significant role in the phytosphingosine degradation pathway . Due to its involvement in multiple metabolic pathways, Ilvbl serves as a valuable biomarker for studying metabolic disorders, cancer, and other diseases . The protein is also known by several synonyms including HACL2, AHAS, 2-hydroxyacyl-CoA lyase 2, and IlvB-like protein .

What are the structural properties of Recombinant Mouse Ilvbl protein?

Recombinant Mouse Ilvbl protein has a molecular weight of approximately 42.8 kDa according to some sources , though Western blot analysis with anti-AHAS antibodies shows a predicted band size of 67 kDa . This discrepancy may be due to post-translational modifications or different isoforms of the protein. The protein is characterized as a membrane single-pass protein , indicating it spans the membrane once with portions exposed on both sides. The protein is encoded by the gene identified with UniProt code Q8BU33 and NCBI GenInfo Identifier 30424591 . Structurally, the protein requires thiamine pyrophosphate (TPP) as a cofactor for its enzymatic activity in fatty acid α-oxidation , suggesting the presence of a TPP-binding domain common to other enzymes in this family.

What are the recommended methods for detecting Recombinant Mouse Ilvbl in experimental samples?

Several validated methods are available for detecting Recombinant Mouse Ilvbl in experimental samples. ELISA (enzyme-linked immunosorbent assay) is highly effective for quantitative detection of Ilvbl in mouse serum, plasma, tissue homogenates, cell culture supernatants, and other biological fluids . Commercial ELISA kits offer detection ranges of 0.156-10 ng/mL with sensitivity of 0.083 ng/mL . For protein localization and semi-quantitative analysis, immunoblotting techniques such as Western blot can be employed, typically using antibodies at dilutions around 1/1000 . Flow cytometry is suitable for intracellular detection of Ilvbl in permeabilized cells, with recommended antibody dilutions of approximately 1/10 . When selecting a detection method, researchers should consider the specific experimental requirements, including sensitivity needs, sample type, and whether quantitative or qualitative data is required.

How should researchers prepare and store Recombinant Mouse Ilvbl protein for experimental use?

Recombinant Mouse Ilvbl protein is typically supplied in lyophilized form and requires proper reconstitution and storage for optimal experimental performance. Though specific information for Ilvbl is not provided in the search results, we can infer recommended protocols based on similar recombinant proteins. Reconstitution should generally be performed at a concentration of approximately 100 μg/mL in sterile PBS . For applications where protein purity is critical, carrier-free versions should be selected and reconstituted in sterile PBS without additional protein carriers . If using the protein for cell culture applications or as an ELISA standard, versions with bovine serum albumin (BSA) as a carrier protein may be preferable as they offer enhanced stability and shelf-life . For storage, researchers should use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain protein integrity . When shipping is necessary, the protein can be transported at ambient temperature, but upon receipt, it should be immediately stored at the recommended temperature according to the manufacturer's guidelines .

What are the performance characteristics of ELISA assays for Recombinant Mouse Ilvbl detection?

ELISA assays for Recombinant Mouse Ilvbl detection offer robust performance characteristics that make them suitable for precise quantification in research applications. Commercial sandwich ELISA kits demonstrate a detection range of 0.156-10 ng/mL with a sensitivity threshold of 0.083 ng/mL . The intra-assay coefficient of variation (CV) is approximately 5.4%, while the inter-assay CV is 7.6% , indicating good reproducibility both within and between assays. These kits show specificity for both natural and recombinant mouse Acetolactate synthase-like protein . The assay is validated for multiple sample types including serum, plasma, tissue homogenates, cell culture supernatants, and other biological fluids . Researchers should note that when using ELISA for Ilvbl detection, proper sample preparation is crucial to minimize matrix effects and optimize sensitivity. While the search results don't provide specific information on linearity and recovery parameters for these assays, these metrics are typically evaluated by manufacturers and should be consulted when selecting an appropriate kit for specific experimental needs.

What are the critical factors to consider when designing experiments involving Recombinant Mouse Ilvbl?

When designing experiments involving Recombinant Mouse Ilvbl, researchers must carefully consider several critical factors to ensure valid and reproducible results. First, selecting the appropriate formulation is essential - carrier-free versions are recommended for applications where the presence of bovine serum albumin (BSA) could interfere with experimental outcomes, while BSA-containing formulations may be more suitable for cell or tissue culture applications or as ELISA standards . Second, proper sample size and replication are crucial - avoid the common mistake of attempting statistical analysis with single samples, as this severely limits statistical power and validity . Third, researchers must carefully select appropriate detection methods based on experimental goals - ELISA offers quantitative measurement with detection ranges of 0.156-10 ng/mL , while Western blot and flow cytometry provide information about protein expression patterns and cellular localization . Fourth, appropriate controls must be incorporated, including both positive controls with known Ilvbl expression and negative controls such as isotype antibodies for immunological techniques . Finally, researchers should consider the biological context of Ilvbl function in branched-chain amino acid biosynthesis and fatty acid α-oxidation when designing experiments to investigate its role in specific physiological or pathological processes .

How can researchers optimize antibody-based detection methods for Recombinant Mouse Ilvbl?

Optimizing antibody-based detection methods for Recombinant Mouse Ilvbl requires careful consideration of several technical parameters. For Western blot applications, researchers should determine the optimal antibody dilution through titration experiments, with reported effective dilutions around 1/1000 for primary antibodies and 1/2000 for secondary antibodies such as goat anti-rabbit HRP . Predicted band size for Ilvbl is approximately 67 kDa, which provides a reference point for validation . For flow cytometry applications, permeabilization of cells is necessary since Ilvbl is an intracellular protein, with antibody dilutions around 1/10 reported as effective . In both applications, inclusion of appropriate controls is essential - negative controls should include isotype-matched irrelevant antibodies (e.g., rabbit IgG for rabbit-derived anti-Ilvbl antibodies) . To reduce non-specific binding, optimization of blocking conditions using appropriate blocking buffers is recommended. When developing or optimizing ELISA protocols, researchers should consider the sandwich format which offers higher specificity for Ilvbl detection . Sample preparation techniques should be optimized for each sample type (serum, plasma, tissue homogenates, or cell culture supernatants) to minimize matrix effects and maximize sensitivity. Finally, validation across multiple sample types and experimental conditions is essential to ensure reproducibility and reliability of results.

What are the key considerations for studying Ilvbl protein-protein interactions and pathway analysis?

Studying Ilvbl protein-protein interactions and pathway analysis requires sophisticated experimental approaches that account for its membrane-associated localization and specific biochemical properties. Researchers should first consider that Ilvbl functions as a single-pass membrane protein , necessitating appropriate lysis conditions that effectively solubilize membrane proteins while preserving protein-protein interactions. Detergent selection is critical - mild non-ionic detergents like digitonin or CHAPS may better preserve native interactions compared to stronger detergents like SDS. Since Ilvbl is involved in thiamine pyrophosphate (TPP)-dependent reactions in fatty acid α-oxidation , experimental buffers should consider the potential role of this cofactor in mediating protein-protein interactions. For co-immunoprecipitation experiments, researchers should select antibodies with high specificity and affinity for Ilvbl, preferably validated for immunoprecipitation applications. When analyzing pathway relationships, particular attention should be paid to branched-chain amino acid biosynthesis pathways and phytosphingosine degradation pathways where Ilvbl plays documented roles . Modern proximity-dependent labeling approaches like BioID or APEX may be particularly valuable for capturing transient or weak interactions in the native cellular environment. Finally, computational approaches that integrate experimental interaction data with existing knowledge of metabolic pathways can provide valuable insights into the functional role of Ilvbl within broader cellular networks.

How can Ilvbl be utilized as a biomarker in metabolic disorder and cancer research?

Utilizing Ilvbl as a biomarker in metabolic disorder and cancer research requires systematic validation across multiple experimental systems and clinical samples. Researchers should begin by establishing baseline expression profiles of Ilvbl across relevant tissues and cell types using techniques such as Western blotting, immunohistochemistry, or quantitative PCR. ELISA assays with detection ranges of 0.156-10 ng/mL and sensitivity of 0.083 ng/mL provide a robust method for quantifying Ilvbl in biological fluids and tissue samples . For cancer research, comparative analysis of Ilvbl expression between tumor and adjacent normal tissues can identify potential diagnostic or prognostic value. This should be extended to correlation analyses with clinical parameters such as tumor stage, grade, and patient outcomes to establish clinical relevance. Since Ilvbl is involved in branched-chain amino acid metabolism and fatty acid α-oxidation , researchers should investigate how alterations in these pathways relate to specific cancer metabolic phenotypes, potentially identifying metabolic vulnerabilities that could be therapeutically targeted. For metabolic disorders, particularly those involving disruptions in amino acid metabolism or fatty acid oxidation, quantification of Ilvbl activity rather than just protein levels may provide more functionally relevant information. Longitudinal studies tracking Ilvbl levels during disease progression or treatment response can assess its value as a monitoring biomarker. Finally, integration of Ilvbl data with other established biomarkers and clinical parameters using multivariate statistical approaches can determine whether Ilvbl provides complementary or redundant information, ultimately defining its place in the biomarker landscape for specific conditions.

What are the common challenges in working with Recombinant Mouse Ilvbl and how can they be addressed?

Working with Recombinant Mouse Ilvbl presents several technical challenges that researchers should anticipate and address proactively. Protein stability is a primary concern, as recombinant proteins can lose activity during storage or experimental manipulation. To mitigate this, researchers should consider using formulations that include carrier proteins like BSA, which enhance stability and increase shelf-life . For applications where carrier proteins would interfere, such as certain binding assays or crystallography, carrier-free versions should be used with appropriate stabilizing buffers . Proper reconstitution is critical - typically performed at concentrations around 100 μg/mL in sterile PBS, with or without additional human or bovine serum albumin depending on the specific application . Repeated freeze-thaw cycles significantly reduce protein activity, so researchers should aliquot reconstituted protein for single use and store in manual defrost freezers . For detection challenges, researchers should be aware of potential cross-reactivity with structurally similar proteins, particularly when using antibody-based detection methods. Validation with positive and negative controls is essential, as is careful titration of antibody concentrations to optimize signal-to-noise ratios . The membrane-associated nature of Ilvbl presents additional challenges for solubilization and purification, requiring careful selection of detergents that preserve protein structure and function. Finally, researchers should be aware of potential discrepancies in molecular weight estimations (42.8 kDa vs. 67 kDa predicted band size) , which may reflect post-translational modifications or different detection methods.

How can researchers overcome specificity and sensitivity issues when studying Ilvbl in complex biological samples?

Overcoming specificity and sensitivity issues when studying Ilvbl in complex biological samples requires a thoughtful combination of technical approaches and controls. For antibody-based detection methods, researchers should perform thorough validation using positive controls (samples with known Ilvbl expression) and negative controls (samples with Ilvbl knocked down or from knockout models). Pre-adsorption experiments using purified recombinant Ilvbl can help establish antibody specificity by demonstrating reduction in signal. When working with ELISA assays, researchers should be aware of potential matrix effects from complex biological samples. Sample dilution series can help identify optimal concentrations where matrix effects are minimized while maintaining signal within the quantifiable range (0.156-10 ng/mL) . For Western blot applications, protocol optimization is essential - using gradient gels can improve separation of proteins with similar molecular weights, and transfer conditions should be optimized for membrane proteins like Ilvbl . For low-abundance samples, enrichment techniques such as immunoprecipitation or subcellular fractionation focused on membrane proteins can improve detection sensitivity. When analyzing flow cytometry data, careful gating strategies and appropriate isotype controls (e.g., rabbit IgG for rabbit-derived anti-Ilvbl antibodies) are necessary to distinguish specific signal from background . Finally, complementary methodologies such as mass spectrometry-based proteomics can provide orthogonal confirmation of Ilvbl detection and quantification, particularly valuable in cases where antibody specificity is questionable or when studying novel protein interactions.

What strategies can researchers employ when facing data inconsistencies in Ilvbl research?

When facing data inconsistencies in Ilvbl research, researchers should implement a systematic troubleshooting approach that addresses both technical and biological sources of variability. First, researchers should carefully evaluate experimental reproducibility by performing technical replicates (same sample, multiple measurements) and biological replicates (multiple independent samples) to distinguish random variation from true biological effects . Standardization of protocols is essential - factors such as sample collection, processing time, storage conditions, and assay procedures should be consistent across experiments. When inconsistencies appear between different detection methods (e.g., ELISA vs. Western blot), researchers should consider the fundamental differences in what each method measures: ELISA may detect soluble forms or specific epitopes, while Western blot provides information about protein size and potential modifications . The reported discrepancy in molecular weight estimations for Ilvbl (42.8 kDa vs. 67 kDa) highlights the importance of characterizing the specific protein forms being detected. Researchers should investigate potential post-translational modifications, splice variants, or protein complexes that could explain such differences. For functional studies, inconsistencies may reflect context-dependent roles of Ilvbl in different cellular environments or physiological states. Researchers should carefully document experimental conditions, including cell confluence, passage number, culture conditions, and treatment protocols. When integrating data across multiple studies or platforms, meta-analysis approaches with appropriate statistical methods for handling inter-study heterogeneity can help identify robust patterns despite individual study variations. Finally, researchers should maintain transparency in reporting both consistent and inconsistent results, as apparent contradictions often lead to new hypotheses and deeper understanding of complex biological systems.

How can mouse models be utilized to study Ilvbl function in metabolic disorders?

Mouse models offer powerful systems for studying Ilvbl function in metabolic disorders through genetic manipulation and controlled environmental conditions. Researchers can develop and characterize whole-body or tissue-specific Ilvbl knockout or knockdown models to elucidate its role in branched-chain amino acid metabolism and fatty acid α-oxidation . When designing such models, researchers should consider potential embryonic lethality if Ilvbl is essential for development, possibly necessitating conditional knockout approaches using Cre-lox systems. Phenotypic characterization should include comprehensive metabolic profiling, focusing on branched-chain amino acids and fatty acid metabolites specifically linked to Ilvbl function. Metabolic challenge experiments (e.g., high-fat diet, fasting-refeeding cycles) can reveal phenotypes that may not be evident under standard conditions. Since Ilvbl is implicated in fatty acid α-oxidation as a thiamine pyrophosphate (TPP)-dependent enzyme , researchers should investigate how thiamine deficiency or supplementation affects Ilvbl-dependent processes in these models. For studying potential therapeutic interventions, humanized mouse models expressing human ILVBL can provide more translatable insights. Integration of in vivo findings with ex vivo analyses of tissues and primary cells from these models can provide mechanistic details at multiple levels of resolution. Finally, researchers should consider potential compensatory mechanisms that may mask phenotypes in chronic knockout models, possibly using acute genetic or pharmacological approaches to disrupt Ilvbl function. By systematically characterizing these models under various physiological and pathological conditions, researchers can gain valuable insights into how Ilvbl contributes to metabolic homeostasis and how its dysfunction may lead to disease.

What is the current understanding of Ilvbl's role in cancer metabolism and potential therapeutic implications?

The current understanding of Ilvbl's role in cancer metabolism is still emerging, but its involvement in branched-chain amino acid metabolism and fatty acid α-oxidation suggests potentially significant implications for cancer biology. Cancer cells often exhibit altered metabolism, including changes in amino acid utilization and lipid metabolism, making Ilvbl a potential player in these cancer-associated metabolic reprogramming events . Although the search results don't provide specific information on Ilvbl in cancer, we can infer potential roles based on its biochemical functions. As a protein involved in branched-chain amino acid biosynthesis , Ilvbl may contribute to cancer cells' increased demand for amino acids to support rapid protein synthesis and growth. Its role in fatty acid α-oxidation may be particularly relevant in cancers that rely on altered lipid metabolism for energy production, membrane synthesis, or signaling molecule generation. From a therapeutic perspective, metabolic enzymes like Ilvbl represent potential targets for cancer treatment, particularly in metabolically distinct cancer subtypes. Researchers investigating this angle should first establish expression and activity profiles of Ilvbl across various cancer types compared to normal tissues, then evaluate correlations with clinical outcomes and response to existing therapies. Functional studies using genetic or pharmacological inhibition of Ilvbl in cancer cell lines and xenograft models can help determine whether it represents a metabolic vulnerability that could be therapeutically exploited. Since Ilvbl functions in a thiamine pyrophosphate (TPP)-dependent manner , investigating how thiamine availability affects cancer cell dependency on Ilvbl could reveal interesting metabolic interactions. Finally, researchers should explore potential synergies between Ilvbl inhibition and existing cancer therapies, particularly those targeting other metabolic pathways.

How might targeting Ilvbl function provide new approaches for treating metabolic diseases?

Targeting Ilvbl function represents a potentially novel approach for treating metabolic diseases, particularly those involving dysregulation of branched-chain amino acid metabolism or fatty acid oxidation. As an enzyme involved in the biosynthesis of branched-chain amino acids and cellular metabolism , Ilvbl modulation could influence metabolic flux through these pathways. Several strategic approaches merit investigation by researchers developing therapeutic interventions. First, small molecule inhibitors targeting Ilvbl's enzymatic activity could be developed, with rational design approaches leveraging its dependence on thiamine pyrophosphate (TPP) as a cofactor . Structural studies of the TPP-binding domain could identify unique features for selective targeting. Second, since Ilvbl functions as a 2-OH acyl-CoA lyase in fatty acid α-oxidation , selective modulators of this activity might provide therapeutic benefits in disorders characterized by abnormal accumulation of 2-hydroxy fatty acids. Third, gene therapy approaches to modulate Ilvbl expression might be considered for genetic disorders involving this pathway. Fourth, metabolic bypass strategies that provide alternative routes for processing Ilvbl substrates could potentially circumvent deficiencies in its function. For optimal therapeutic development, researchers should establish comprehensive profiles of Ilvbl expression and activity across relevant tissues in both normal and disease states. Biomarker development using ELISA assays with sensitivity in the range of 0.083 ng/mL could help identify patient populations most likely to benefit from Ilvbl-targeted therapies and monitor treatment efficacy. Finally, potential off-target effects and compensatory mechanisms should be thoroughly investigated, given Ilvbl's involvement in fundamental metabolic processes.

How can new omics technologies advance our understanding of Ilvbl function and regulation?

New omics technologies offer unprecedented opportunities to advance our understanding of Ilvbl function and regulation within complex biological systems. Multi-omics integration approaches that combine proteomics, metabolomics, transcriptomics, and genomics data can provide comprehensive insights into Ilvbl's role in cellular metabolism. Quantitative proteomics using techniques such as tandem mass tag (TMT) labeling or SILAC (stable isotope labeling with amino acids in cell culture) can map Ilvbl protein-protein interactions and post-translational modifications that regulate its activity. Researchers should pay particular attention to potential phosphorylation, acetylation, or ubiquitination sites that might modulate Ilvbl function or stability. Metabolomics approaches using high-resolution mass spectrometry can characterize changes in metabolite profiles upon Ilvbl modulation, with particular focus on branched-chain amino acids and fatty acid intermediates where Ilvbl plays documented roles . For analyzing Ilvbl gene regulation, ChIP-seq (chromatin immunoprecipitation sequencing) and ATAC-seq (assay for transposase-accessible chromatin using sequencing) can identify transcription factors and chromatin states associated with Ilvbl expression changes under different physiological or pathological conditions. Single-cell technologies across these omics platforms can reveal cell-type-specific functions and heterogeneity in Ilvbl expression and activity that might be masked in bulk analyses. Spatial omics approaches like spatial transcriptomics or imaging mass spectrometry can map Ilvbl expression and activity within tissue microenvironments, providing context for its function in complex organs. Finally, computational integration of these multi-omics datasets using machine learning or network analysis approaches can identify emergent properties and generate testable hypotheses about Ilvbl's role in health and disease.

What novel experimental approaches could provide deeper insights into Ilvbl structure-function relationships?

Novel experimental approaches leveraging recent technological advances could substantially deepen our understanding of Ilvbl structure-function relationships. Cryo-electron microscopy (cryo-EM) represents a powerful approach for determining the high-resolution structure of Ilvbl, particularly advantageous for membrane proteins like Ilvbl that may be challenging to crystallize. This technique could reveal critical structural features including the thiamine pyrophosphate (TPP) binding domain essential for its enzymatic activity in fatty acid α-oxidation . AlphaFold2 and other AI-based structure prediction tools can complement experimental approaches by generating testable structural models, particularly valuable for exploring conformational dynamics or protein-protein interaction interfaces. To map structure-function relationships at high resolution, researchers should employ systematic mutagenesis approaches coupled with enzymatic assays, focusing on predicted functional domains and evolutionarily conserved residues. CRISPR-based saturation mutagenesis techniques can generate comprehensive libraries of Ilvbl variants for high-throughput functional screening. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can provide insights into protein dynamics and conformational changes upon substrate or cofactor binding. For studying Ilvbl's membrane association , advanced biophysical techniques such as solid-state NMR, neutron reflectometry, or electron paramagnetic resonance (EPR) spectroscopy can characterize its membrane insertion and topology. Time-resolved studies using approaches such as temperature-jump or stopped-flow techniques coupled with spectroscopic methods can capture transient conformational states during catalysis. Finally, integrating structural biology with systems biology approaches can place atomic-level insights into broader biological contexts, connecting structural features to physiological functions in branched-chain amino acid metabolism and fatty acid α-oxidation pathways .

How might computational approaches enhance Ilvbl research and therapeutic development?

Computational approaches offer transformative potential for Ilvbl research and therapeutic development by integrating diverse data types and enabling predictions across biological scales. Molecular modeling and simulation techniques, including molecular dynamics simulations and quantum mechanical calculations, can provide atomic-level insights into Ilvbl's catalytic mechanism, particularly its thiamine pyrophosphate (TPP)-dependent reactions in fatty acid α-oxidation . These approaches can identify key residues involved in substrate binding and catalysis, informing rational design of specific inhibitors or activity modulators. Virtual screening of chemical libraries against the Ilvbl structure can accelerate identification of potential therapeutic compounds, with molecular docking and free energy calculations predicting binding affinities and selectivity profiles. Systems biology approaches using genome-scale metabolic models can predict how Ilvbl perturbations propagate through metabolic networks, identifying potential compensatory mechanisms or synthetic lethal interactions that could be therapeutically exploited. Machine learning algorithms trained on multi-omics datasets can identify complex patterns in Ilvbl expression or activity across diseases, potentially revealing unexpected therapeutic applications. For personalized medicine approaches, computational methods can integrate genetic variation in Ilvbl and related pathway components with clinical data to stratify patients for targeted interventions. Network pharmacology approaches can identify potential drug combinations that synergistically modulate Ilvbl-dependent pathways while minimizing off-target effects. Pharmacokinetic/pharmacodynamic (PK/PD) modeling can optimize dosing regimens for Ilvbl-targeted therapeutics, accounting for tissue-specific expression patterns . Finally, text mining and knowledge graph approaches can synthesize information across the scientific literature, identifying connections between Ilvbl and other biological entities that might not be apparent from individual studies, thus generating novel hypotheses for experimental validation.