MOCS2 Antibody

What is MOCS2 and why is it significant in research?

MOCS2 (molybdenum cofactor synthesis 2) is a protein that serves as the catalytic subunit of the molybdopterin synthase complex. It catalyzes the conversion of precursor Z into molybdopterin by mediating the incorporation of two sulfur atoms from thiocarboxylated MOCS2A to generate a dithiolene group. This process is critical in the biosynthesis pathway of molybdenum cofactor (Moco) . The MOCS2 gene is bicistronic, producing both MOCS2A and MOCS2B subunits through a ribosomal leaky scanning mechanism, with the two proteins forming a functional heterotetramer .

The significance of MOCS2 extends beyond its enzymatic role. Mutations in the MOCS2 gene lead to Molybdenum Cofactor Deficiency (MoCD) type B, a severe metabolic disorder affecting approximately one-third of all MoCD patients . Additionally, recent studies have revealed that MOCS2 may have moonlighting functions in signaling, metabolism, and translation regulation, with potential implications for neurodegenerative diseases . This multifunctional nature makes MOCS2 an important target for researchers investigating both enzymatic mechanisms and broader cellular processes.

What applications are MOCS2 antibodies suitable for?

MOCS2 antibodies have been validated for multiple research applications, providing researchers with versatile tools for investigating this protein. Based on current data, MOCS2 antibodies are suitable for:

• Western Blotting (WB): MOCS2 antibodies can detect the protein in cell lysates with a predicted band size of approximately 21 kDa. Recommended dilutions typically range from 1:100-1000 for polyclonal antibodies .

• Immunoprecipitation (IP): MOCS2 can be successfully immunoprecipitated from cell lysates, allowing researchers to study protein-protein interactions and post-translational modifications .

• Immunohistochemistry on Paraffin-embedded Sections (IHC-P): MOCS2 antibodies work effectively on paraffin-embedded tissues like adrenal gland and kidney at typical dilutions of 1:100 .

• Immunocytochemistry/Immunofluorescence (ICC/IF): For subcellular localization studies, MOCS2 antibodies can be used at dilutions around 1:50-500 with appropriate fluorescent secondary antibodies .

• Enzyme-Linked Immunosorbent Assay (ELISA): MOCS2 antibodies can be employed in ELISA applications at dilutions ranging from 1:500-3000 .

When designing experiments, researchers should consider that most MOCS2 antibodies have been validated in human samples, with cross-reactivity to mouse and rat samples in some cases . For each application, optimization of antibody concentration, incubation conditions, and detection methods may be necessary depending on the specific experimental setup and sample type.

How should MOCS2 antibody specificity be validated?

Validating the specificity of MOCS2 antibodies is crucial for ensuring reliable experimental results. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines or tissues known to express MOCS2 (such as Jurkat, HeLa, A549, or HepG2 cells) as positive controls . For negative controls, consider using tissues where MOCS2 expression is minimal or cells where MOCS2 has been knocked down using siRNA/shRNA.

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide before application to the sample. If the antibody is specific, the signal should be significantly reduced or eliminated.

• Multiple detection methods: Confirm findings using different techniques (e.g., if results are obtained by Western blot, verify with immunofluorescence or immunohistochemistry) .

• Molecular weight verification: The predicted band size for MOCS2 is approximately 21 kDa. Verify that the observed band corresponds to this size in Western blotting .

• Knockout/knockdown validation: Compare results between wild-type samples and those where MOCS2 has been genetically modified or suppressed.

• Cross-reactivity assessment: If working with non-human samples, verify that the antibody recognizes the target species through sequence homology analysis and experimental verification. Some MOCS2 antibodies have been reported to cross-react with mouse and rat samples .

• Comparison with alternative antibodies: When possible, compare results using different antibodies targeting distinct epitopes of MOCS2.

Thorough validation ensures that observed signals are genuinely attributable to MOCS2 rather than non-specific interactions, preventing misinterpretation of experimental results.

How can MOCS2 antibodies be applied to study MOCS2A versus MOCS2B?

Studying MOCS2A and MOCS2B presents unique challenges due to their encoding by a single gene through a complex ribosomal leaky scanning mechanism. Developing a methodological approach requires careful consideration of their structural and functional differences:

• Epitope-specific antibodies: For differential study of MOCS2A (88 residues encoded by exons 1-3) and MOCS2B (encoded by exons 3-7), researchers should select antibodies recognizing unique epitopes to each subunit . The small size of MOCS2A and the 77 bp overlap in exon 3 encoding the C-terminal end of MOCS2A and N-terminus of MOCS2B makes this particularly challenging.

• Western blot optimization: When detecting both subunits simultaneously, use gradient gels (10-20%) to effectively separate the smaller MOCS2A from the larger MOCS2B. Expected molecular weights are approximately 10 kDa for MOCS2A and 21 kDa for MOCS2B .

• Co-immunoprecipitation studies: To examine the formation of the heterotetramer, use antibodies specific to one subunit for immunoprecipitation and probe for the other subunit in Western blot analysis. This approach can verify the interaction between MOCS2A and MOCS2B and identify additional binding partners .

• Immunofluorescence co-localization: Double immunofluorescence labeling using antibodies specific to each subunit can reveal subcellular locations where the heterotetramer forms versus where individual subunits might have independent functions.

• Mutation-specific detection: For research on Molybdenum Cofactor Deficiency, consider designing antibodies that can distinguish between wild-type and mutant forms of MOCS2A and MOCS2B, particularly focusing on the 31 known MOCS2 mutations associated with disease .

Understanding the distinct roles of these subunits is essential, as the catalytic activity depends on the thiocarboxylated C-terminus of MOCS2A transferring sulfides to the precursor molecule, while MOCS2B provides the structural scaffold for this reaction .

What methodological approaches optimize MOCS2 detection in different cell types?

Optimizing MOCS2 detection across different cell types requires tailored approaches to account for varying expression levels, cellular compartmentalization, and potential interference from related proteins:

• Cell-specific protocol optimization table:

• Sample preparation enhancements: For cells with low MOCS2 expression, consider enrichment through subcellular fractionation, focusing on cytosolic fractions where MOCS2 predominantly localizes. Alternatively, implement signal amplification methods such as tyramide signal amplification for immunohistochemistry.

• Cross-linking optimization: For immunoprecipitation studies, experiment with different cross-linking agents (DSS, formaldehyde, or DTBP) at varying concentrations and incubation times to preserve protein-protein interactions involving MOCS2.

• Detection system selection: For Western blotting, compare ECL, fluorescent, and infrared detection systems to determine which provides optimal signal-to-noise ratio for your specific cell type. HRP-conjugated light chain specific secondary antibodies work well for immunoprecipitation studies of MOCS2 .

• Antigen retrieval methods: For tissue sections, compare heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) versus Tris-EDTA (pH 9.0) to determine which better exposes MOCS2 epitopes in your specific tissue type.

By systematically optimizing these parameters for each cell type, researchers can achieve consistent and reliable MOCS2 detection across diverse experimental systems.

How can researchers investigate the non-canonical roles of MOCS2 beyond Moco biosynthesis?

Recent research indicates MOCS2 has functions beyond its well-established role in molybdenum cofactor biosynthesis, particularly in signaling, metabolism, and translation regulation . To investigate these non-canonical roles, researchers can employ the following methodological approaches:

• Protein-protein interaction network analysis: Use co-immunoprecipitation with MOCS2 antibodies followed by mass spectrometry to identify novel interaction partners outside the molybdopterin synthase complex. Compare these interactomes between different cell types and under various stress conditions to identify context-specific interactions .

• Chromatin association studies: Given the involvement of MoaE (the bacterial homolog of MOCS2B) in the Ada2a-containing (ATAC) histone acetyltransferase complex in Drosophila, investigate potential chromatin association of MOCS2B using chromatin immunoprecipitation (ChIP) followed by sequencing. This can reveal if MOCS2 directly or indirectly influences gene expression .

• Transcriptome analysis in MOCS2 knockdown/knockout models: Compare RNA-seq data between wild-type and MOCS2-deficient cells to identify genes and pathways affected by MOCS2 beyond the established Moco biosynthesis pathway. Look specifically for changes in genes involved in MAPK signaling, as research suggests connections between MOCS2 and stress-activated protein kinases .

• Subcellular localization under stress conditions: Use immunofluorescence to track MOCS2 localization under various stress conditions (oxidative stress, osmotic stress, ER stress) to determine if MOCS2 translocates to different cellular compartments, potentially indicating stress-response functions .

• Phosphoproteomics analysis: Investigate potential phosphorylation of MOCS2 by MAPKs and other signaling kinases, and determine how these modifications might affect MOCS2 function beyond its enzymatic role.

• Translation regulation studies: Explore potential roles of MOCS2 in translation regulation using polysome profiling in the presence and absence of MOCS2, focusing particularly on mRNAs encoding proteins involved in neurodegenerative processes .

By systematically applying these approaches, researchers can decouple MOCS2's canonical enzymatic function from its emerging roles in cellular signaling and gene regulation, potentially revealing new therapeutic targets for diseases like dementia and neurodegeneration .

How to address non-specific binding issues with MOCS2 antibodies?

Non-specific binding is a common challenge when working with MOCS2 antibodies, potentially leading to misleading results and interpretation. Here are methodological strategies to address this issue:

• Optimize blocking conditions: Test different blocking agents beyond standard BSA or milk. For MOCS2 detection, consider:

  • Fish gelatin (1-3%) for reduced background in immunohistochemistry

  • Casein (0.5-2%) for Western blots with high background

  • Commercial blocking reagents specifically designed for phosphoprotein detection if studying phosphorylated forms of MOCS2

• Adjust antibody parameters:

  • Titrate antibody concentrations systematically (recommended range: 1:100-1000 for WB; 1:500-3000 for ELISA; 1:50-500 for IF/IHC)

  • Include 0.1-0.3% Triton X-100 in antibody diluent for better penetration in ICC/IF

  • Increase incubation time but decrease antibody concentration (e.g., overnight at 4°C with 1:1000 dilution instead of 1 hour at room temperature with 1:500)

• Pre-adsorption protocols: To reduce non-specific binding:

  • Pre-incubate diluted antibody with tissues/cells known not to express MOCS2

  • For cross-reactivity concerns between species, pre-adsorb with tissue lysates from non-target species

  • Use a peptide competition assay with the immunizing peptide as a control for specificity

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity

  • For immunoprecipitation studies, employ HRP-conjugated light chain specific secondary antibodies to avoid detecting the heavy chain which may overlap with your target

  • Consider fluorescent secondaries with non-overlapping spectra for multi-color immunofluorescence experiments

• Sample preparation refinements:

  • Extend fixation time for challenging tissues

  • Try different antigen retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Include additional washing steps with increasing salt concentration (150mM to 300mM NaCl) to disrupt low-affinity non-specific interactions

By systematically implementing these techniques and carefully documenting the outcomes, researchers can significantly reduce non-specific binding issues and improve the reliability of MOCS2 antibody-based experiments.

What strategies can resolve conflicting results between different MOCS2 detection methods?

Conflicting results between different detection methods when studying MOCS2 can significantly complicate data interpretation. A systematic troubleshooting approach is necessary:

• Cross-validation protocol:

  Implement a step-by-step cross-validation strategy using at least three independent methods:

  • Compare Western blot, immunofluorescence, and ELISA results from the same sample

  • Document the specific antibody dilutions, incubation times, and detection systems used

  • For each method, include positive controls (Jurkat, HeLa, A549, or HepG2 cells) and negative controls

• Epitope accessibility analysis:

  Different methods may expose epitopes differently, explaining discrepancies:

  • For samples showing MOCS2 by Western blot but not by IHC: Test different antigen retrieval methods for IHC

  • For samples positive by IHC but negative by Western blot: Consider native versus denatured protein conformations affecting epitope recognition

  • Identify the specific epitope recognized by your antibody and its conservation/accessibility across experimental conditions

• Sample preparation harmonization:

  Standardize sample preparation across methods:

  • Use the same lysis buffer composition for both Western blot and immunoprecipitation

  • Apply consistent fixation protocols for cells analyzed by both flow cytometry and immunofluorescence

  • Process samples in parallel to minimize technical variation

• Isoform and post-translational modification consideration:

  MOCS2 exists as both MOCS2A and MOCS2B forms with potential modifications:

  • Determine which isoform your antibody recognizes (check if it targets exons 1-3 for MOCS2A or exons 3-7 for MOCS2B)

  • Consider potential post-translational modifications masking epitopes in certain assays

  • Test phosphatase treatment prior to Western blot if phosphorylation is suspected to alter antibody binding

• Quantitative reconciliation approach:

  When methods show different quantitative results:

  • Establish standard curves for each method using recombinant MOCS2 protein

  • Calculate relative detection efficiency factors between methods

  • Apply correction factors when comparing quantitative data across platforms

By methodically addressing these factors, researchers can often reconcile conflicting results and develop a more complete understanding of MOCS2 expression and function in their experimental system.

How can MOCS2 antibodies contribute to understanding Molybdenum Cofactor Deficiency?

Molybdenum Cofactor Deficiency (MoCD) is a severe metabolic disorder with approximately one-third of patients classified as having MoCD type B due to mutations in the MOCS2 gene . MOCS2 antibodies offer powerful tools for investigating this condition through several methodological approaches:

• Mutation-specific antibody applications:

  Researchers can develop and apply antibodies that specifically recognize mutant forms of MOCS2 found in patients:

  • Generate antibodies against common pathogenic variants (e.g., those affecting MOCS2A's double glycine motif or MOCS2B's catalytic domain)

  • Use these to screen patient samples for specific mutations at the protein level

  • Compare expression and localization patterns between wild-type and mutant MOCS2 in cellular models

• Functional impact assessment:

  MOCS2 antibodies enable detailed analysis of the functional consequences of MoCD-causing mutations:

  • Examine heterodimerization efficiency between MOCS2A and MOCS2B in patient-derived cells using co-immunoprecipitation

  • Investigate subcellular localization shifts in mutant proteins using immunofluorescence

  • Quantify expression levels of upstream and downstream Moco biosynthesis pathway components in response to MOCS2 mutations

• Diagnostic and prognostic applications:

  Develop standardized immunoassays for clinical applications:

  • Create ELISA-based tests using MOCS2 antibodies to screen high-risk populations

  • Develop immunohistochemical protocols to analyze MOCS2 expression in patient biopsies

  • Establish correlation between MOCS2 protein levels and disease severity

• Therapeutic monitoring protocol:

  For emerging therapies targeting MoCD type B:

  • Use MOCS2 antibodies to monitor treatment efficacy at the protein level

  • Track changes in MOCS2 complex formation following therapeutic intervention

  • Correlate biochemical improvements with clinical outcomes

• Model systems investigation:

  Apply MOCS2 antibodies in research models to better understand disease mechanisms:

  • Compare MOCS2 expression and function across cellular and animal models harboring different MOCS2 mutations from the 31 known pathogenic variants

  • Analyze downstream effects on sulfite oxidase and xanthine dehydrogenase activities

  • Investigate potential moonlighting functions of MOCS2 in the nervous system that might contribute to neurological symptoms of MoCD

These approaches can significantly advance our understanding of MoCD pathophysiology and potentially lead to improved diagnostic tools and therapeutic strategies for this devastating disorder.

How can researchers explore the connection between MOCS2 and neurodegenerative diseases?

Recent research suggests that MOCS2 may play roles beyond molybdenum cofactor biosynthesis, potentially linking to neurodegenerative processes . Researchers can explore this emerging connection using MOCS2 antibodies through several methodological approaches:

• Comparative expression profiling:

  Systematically analyze MOCS2 expression across neurodegenerative disease models:

  • Use immunohistochemistry with MOCS2 antibodies on brain tissue sections from patients with different neurodegenerative conditions

  • Compare MOCS2 expression patterns between affected and unaffected brain regions

  • Correlate MOCS2 levels with disease progression markers

• Stress response pathway investigation:

  Given MOCS2's potential role in stress signaling, examine its behavior under neurodegenerative-relevant stressors:

  • Track MOCS2 localization changes in response to oxidative stress, protein misfolding, and excitotoxicity using immunofluorescence

  • Analyze MOCS2 post-translational modifications under stress conditions

  • Investigate interaction between MOCS2 and stress-activated MAPKs using co-immunoprecipitation and proximity ligation assays

• Protein aggregation studies:

  Explore potential connections between MOCS2 and protein aggregation:

  • Perform double immunofluorescence labeling to assess co-localization of MOCS2 with disease-specific protein aggregates (e.g., Aβ, tau, α-synuclein)

  • Examine whether MOCS2 levels correlate with aggregate burden

  • Investigate if MOCS2 modulates aggregate formation or clearance

• Transcriptional and translational regulation analysis:

  Based on MOCS2's potential involvement in gene expression regulation:

  • Use ChIP-seq with MOCS2 antibodies to identify genomic regions where MOCS2 may influence transcription

  • Investigate whether MOCS2 affects translation of neurodegeneration-related mRNAs

  • Examine if MOCS2 interacts with RNA-binding proteins involved in neurodegeneration

• Therapeutic target exploration:

  Assess MOCS2 as a potential therapeutic target:

  • Develop assays to screen compounds that modulate MOCS2 function

  • Evaluate effects of MOCS2 modulation on neuronal survival in disease models

  • Track MOCS2-related biomarkers in response to experimental therapeutics

By systematically applying these approaches, researchers can elucidate the potentially significant role of MOCS2 in neurodegenerative processes, possibly opening new avenues for diagnostic and therapeutic development.