MCCC1 Antibody

What is MCCC1 and what is its biological function?

MCCC1 (methylcrotonoyl-CoA carboxylase 1) is the large subunit of 3-methylcrotonyl-CoA carboxylase. This enzyme functions as a heterodimer and catalyzes the carboxylation of 3-methylcrotonyl-CoA to form 3-methylglutaconyl-CoA . MCCC1 has multiple protein aliases, including 3-methylcrotonyl-CoA carboxylase biotin-containing subunit, 3-methylcrotonyl-CoA:carbon dioxide ligase subunit alpha, and MCCase subunit alpha . Beyond its metabolic role, MCCC1 has been identified as a positive regulator in virus-induced immune responses, particularly in the enhancement of interferon (IFN) and inflammatory cytokine expression through the NF-κB signaling pathway .

What are the optimal dilution ratios for MCCC1 antibody in different applications?

For MCCC1 antibody applications, optimal dilution ratios vary depending on the specific experimental technique. For Western Blot (WB) applications, a dilution range of 1:1000-1:4000 is recommended for detecting MCCC1 . When performing immunohistochemistry (IHC), researchers should use dilutions between 1:200-1:800 . For immunofluorescence (IF) or immunocytochemistry (ICC) applications, the recommended dilution range is 1:50-1:500 . It is important to note that these ranges are starting points, and researchers should validate optimal dilutions in their specific experimental systems as results may be sample-dependent.

How should MCCC1 antibodies be stored to maintain effectiveness?

MCCC1 antibodies should be stored at -20°C to maintain effectiveness . The antibody is typically provided in a storage buffer consisting of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability . When stored properly, the antibody remains stable for one year after shipment. For the 30081-1-AP antibody specifically, aliquoting is unnecessary for -20°C storage, simplifying laboratory management . It's worth noting that some preparations (20μl sizes) contain 0.1% BSA, which may need to be considered in certain experimental designs.

What molecular weight should be expected when detecting MCCC1 protein by Western blot?

When detecting MCCC1 using Western blot, researchers should be aware of the discrepancy between the calculated and observed molecular weights. The calculated molecular weight of MCCC1 is 76 kDa, while the observed molecular weight in experimental conditions is typically around 70 kDa . This difference may be due to post-translational modifications, protein processing, or structural characteristics affecting migration in SDS-PAGE. When validating a new MCCC1 antibody, researchers should include appropriate positive controls such as HepG2 cells, mouse brain tissue, mouse heart tissue, or rat brain tissue, which have been confirmed to express detectable levels of MCCC1 .

Which cell lines and tissues are suitable as positive controls for MCCC1 antibody validation?

Several cell lines and tissues have been validated as positive controls for MCCC1 antibody testing. For Western blot applications, HepG2 cells, mouse brain tissue, mouse heart tissue, and rat brain tissue have demonstrated positive MCCC1 detection . For immunohistochemistry (IHC), human ovary cancer tissue, human intrahepatic cholangiocarcinoma tissue, and human stomach cancer tissue have shown positive results . When performing immunofluorescence (IF) or immunocytochemistry (ICC), U-251 and MCF-7 cells serve as reliable positive controls . Including these validated positive controls in experimental designs helps ensure antibody functionality and provides appropriate reference points for comparing experimental samples.

How can MCCC1 antibodies be used to investigate the role of MCCC1 in antiviral immunity?

MCCC1 has been identified as an important component in virus-triggered innate immunity, specifically in the regulation of type I interferons and inflammatory cytokines through the NF-κB signaling pathway . When designing experiments to investigate this role, researchers should implement a multi-faceted approach. First, utilize MCCC1 antibodies in co-immunoprecipitation experiments to confirm interactions with MAVS and components of the MAVS signalosome after viral infection . Second, employ immunofluorescence with MCCC1 antibodies to visualize mitochondrial localization and potential relocalization during viral infections, as MCCC1 is a mitochondrial protein and MAVS is located on mitochondria .

For functional studies, combine MCCC1 knockdown (validated by Western blot using MCCC1 antibodies) with reporter assays measuring IFN-β, IFN-λ1, and NF-κB promoter activities in response to virus infection . Quantitative PCR should be used to measure the expression of IFNs and pro-inflammatory cytokines (IL-6, IL-8, IL-1β, and TNFα) in both MCCC1-overexpressing and MCCC1-knockdown cells infected with RNA viruses such as influenza A virus (IAV), human enterovirus 71 (EV71), or vesicular stomatitis virus (VSV) . MCCC1 antibodies are essential for confirming successful overexpression or knockdown in these experimental systems.

What are the methodological considerations for using MCCC1 antibodies in immunoprecipitation studies to identify interaction partners?

When using MCCC1 antibodies for immunoprecipitation (IP) studies to identify interaction partners, several methodological considerations are essential for obtaining reliable results. First, validate the efficiency of the MCCC1 antibody in IP applications using known MCCC1-expressing cell lines before proceeding with interaction studies. Second, carefully optimize lysis conditions to preserve protein-protein interactions while effectively extracting MCCC1 from mitochondrial membranes, as MCCC1 is a mitochondrial protein .

During experimental design, include appropriate controls: IgG isotype control for non-specific binding, input controls (5-10% of lysate) for expression level comparison, and where possible, reciprocal IPs with antibodies against suspected interaction partners. For detecting MAVS-MCCC1 interactions specifically, consider crosslinking approaches before cell lysis to capture transient interactions that may occur during viral signaling .

For analyzing MCCC1's role in the MAVS signalosome, combine IP with Western blotting to detect co-precipitated components such as TRAF3 and TRAF6, which are known to be recruited by MAVS during antiviral signaling . To comprehensively identify novel interaction partners, IP followed by mass spectrometry can be employed, with subsequent validation of interactions using targeted co-IP and Western blot analysis with MCCC1 antibodies.

What approaches should be used to investigate MCCC1's role in NF-κB signaling using MCCC1 antibodies?

Investigating MCCC1's role in NF-κB signaling requires a systematic approach combining multiple techniques in which MCCC1 antibodies play a crucial role. First, establish baseline experiments using reporter assays to confirm that MCCC1 enhances virus-triggered activation of NF-κB but not ISRE promoters, as indicated by previous research . Use MCCC1 antibodies in Western blot analysis to detect changes in phosphorylation of the IκB kinase (IKK) complex and NF-κB inhibitor-α (IκBα) in MCCC1-overexpressing or MCCC1-knockdown cells following viral infection .

To visualize NF-κB nuclear translocation, employ immunofluorescence techniques with dual staining using MCCC1 antibodies and antibodies against NF-κB p65 subunit. Time-course experiments are recommended to track the kinetics of NF-κB activation in relation to MCCC1 expression levels . For mechanistic studies, combine MCCC1 knockdown with reconstitution experiments using wild-type and mutant MCCC1 constructs, verified by Western blot with MCCC1 antibodies, to identify domains crucial for NF-κB activation.

Additionally, chromatin immunoprecipitation (ChIP) assays can be performed to assess NF-κB binding to the promoters of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, and TNFα) in the presence or absence of MCCC1. This comprehensive approach will provide detailed insights into how MCCC1 regulates the NF-κB signaling pathway during viral infections.

How can researchers troubleshoot cross-reactivity issues when using MCCC1 antibodies across different species?

Cross-reactivity issues when using MCCC1 antibodies across different species require systematic troubleshooting approaches. Start by examining sequence homology; the MCCC1 antigen sequence shows varying identity across species (mouse and rat orthologs share 72% identity with human) . When cross-reactivity problems arise, consider testing alternative antibodies raised against more conserved epitopes or antibodies specifically validated for the species of interest.

For Western blot applications, optimize blocking conditions (try both BSA and milk-based blockers) and increase stringency of washing steps. Testing a range of antibody dilutions beyond the recommended 1:1000-1:4000 may help identify optimal conditions for each species . For immunohistochemistry applications where cross-reactivity is observed, optimize antigen retrieval methods—both TE buffer pH 9.0 and citrate buffer pH 6.0 have been found effective for MCCC1 detection .

When validating antibody specificity across species, include appropriate controls: lysates from MCCC1-knockout cells/tissues (if available), pre-absorption controls using recombinant MCCC1 protein, and comparison of staining patterns with alternative MCCC1 antibodies recognizing different epitopes. For particularly challenging cross-reactivity issues, consider using species-specific secondary antibodies with minimal cross-reactivity and performing peptide competition assays to confirm binding specificity.

What are the best experimental designs to study MCCC1 function using both antibody detection and genetic manipulation approaches?

Investigating MCCC1 function comprehensively requires integrating antibody-based detection with genetic manipulation approaches. Begin with baseline characterization using MCCC1 antibodies to determine endogenous expression patterns across relevant cell types and tissues by Western blot, immunohistochemistry, and immunofluorescence . This establishes the foundation for subsequent functional studies.

For loss-of-function studies, implement both transient siRNA-mediated knockdown and stable shRNA or CRISPR-Cas9-mediated knockout approaches. Validate knockdown/knockout efficiency using MCCC1 antibodies in Western blot analysis, ideally at multiple time points to capture the temporal dynamics of protein depletion . For gain-of-function studies, generate cell lines stably expressing wild-type MCCC1 or domain-specific mutants, tagged with small epitopes that don't interfere with protein function. Verify expression levels by Western blot using both MCCC1 antibodies and tag-specific antibodies.

To investigate MCCC1's antiviral functions specifically, design viral infection experiments with RNA viruses such as influenza A virus, enterovirus 71, or vesicular stomatitis virus in cells with modified MCCC1 expression . Measure viral replication parameters (viral RNA levels, viral protein expression) alongside immune parameters (IFN expression, inflammatory cytokine production) at various time points post-infection.

For mechanistic studies of MCCC1's interaction with the MAVS signalosome, combine co-immunoprecipitation using MCCC1 antibodies with mutagenesis approaches targeting specific domains of MCCC1 to map interaction regions . This comprehensive experimental design allows for detailed characterization of MCCC1's functional roles in both metabolic and immune signaling pathways.

What are the optimal conditions for performing Western blot analysis with MCCC1 antibodies?

For optimal Western blot analysis with MCCC1 antibodies, follow these methodological guidelines for consistent and specific detection. Begin by extracting proteins using a lysis buffer containing protease inhibitors to prevent degradation of MCCC1. Since MCCC1 is a mitochondrial protein, ensure your lysis protocol effectively solubilizes mitochondrial membranes . When separating proteins, use 8-10% polyacrylamide gels to achieve good resolution around the expected 70 kDa band for MCCC1 .

For the transfer step, semi-dry transfer systems work well, but wet transfer may provide better results for this relatively large protein. After transfer, block membranes with either 5% non-fat milk or 3-5% BSA in TBST for 1 hour at room temperature. Apply MCCC1 antibody at recommended dilutions (1:1000-1:4000) , and incubate overnight at 4°C for optimal binding. For challenging detections, consider using signal enhancers or more sensitive detection systems.

When troubleshooting detection issues, note that the observed molecular weight of MCCC1 (70 kDa) differs from the calculated weight (76 kDa) , which may cause confusion in band identification. Include positive controls such as HepG2 cell lysates to confirm correct band identification. If multiple bands appear, optimize antibody dilution and washing steps, or consider using different blocking agents. For verification of specificity, MCCC1 knockdown/knockout samples serve as essential negative controls.

How should researchers design immunofluorescence experiments with MCCC1 antibodies for subcellular localization studies?

Designing immunofluorescence experiments for MCCC1 subcellular localization requires careful attention to several methodological aspects. Start with cell preparation on appropriate coverslips, using cell types known to express MCCC1 such as U-251 or MCF-7 cells . After fixation with 4% paraformaldehyde (10-15 minutes), perform membrane permeabilization using 0.1-0.2% Triton X-100 in PBS for 10 minutes, as this is critical for allowing antibody access to the mitochondrially-located MCCC1 protein .

For optimal staining, block with 1-5% BSA in PBS for 30-60 minutes at room temperature before applying MCCC1 antibody at dilutions ranging from 1:50-1:500 . Incubate primary antibody overnight at 4°C in a humidified chamber to enhance specific binding. For co-localization studies, combine MCCC1 antibody with established mitochondrial markers such as MitoTracker or antibodies against other mitochondrial proteins like TOMM20.

Include appropriate controls with each experiment: a secondary-only control to assess background fluorescence, a positive control using cell lines with confirmed MCCC1 expression, and when possible, a negative control using MCCC1-knockdown cells. For challenging detection scenarios, consider signal amplification methods such as tyramide signal amplification. When analyzing results, use confocal microscopy with z-stack imaging to accurately assess the three-dimensional co-localization of MCCC1 with mitochondrial markers, which is essential for confirming its expected mitochondrial localization .

What controls are essential when using MCCC1 antibodies in virus-infection studies?

When using MCCC1 antibodies in virus-infection studies, implementing comprehensive controls is crucial for generating reliable and interpretable data. First, include uninfected controls alongside virus-infected samples to establish baseline MCCC1 expression and localization patterns . This comparison allows for assessment of infection-induced changes in MCCC1 expression, post-translational modifications, or subcellular redistribution.

For functional studies exploring MCCC1's role in antiviral responses, parallel experiments with MCCC1-overexpressing and MCCC1-knockdown cells are essential . Validate the efficacy of overexpression or knockdown using Western blot with MCCC1 antibodies prior to viral infection. Include rescue experiments where MCCC1 expression is restored in knockdown cells to confirm that observed phenotypes are specifically due to MCCC1 depletion rather than off-target effects .

When investigating MCCC1's interaction with MAVS and its involvement in NF-κB signaling, include MAVS-knockdown controls to confirm the MAVS-dependency of MCCC1-mediated effects . For reporter assays measuring IFN promoter or NF-κB activation, include positive controls (such as cells stimulated with TNFα for NF-κB activation) and negative controls (dominant-negative constructs of key signaling components).

Time-course experiments are particularly important in virus-infection studies, as the kinetics of MCCC1's contribution to antiviral signaling may vary with different viral pathogens (IAV, EV71, VSV) . Finally, when using multiple virus strains, implement suitable biosafety controls appropriate to the risk level of each pathogen, ensuring both experimental integrity and laboratory safety.

How can researchers interpret discrepancies in MCCC1 detection between different antibody-based techniques?

When researchers encounter discrepancies in MCCC1 detection across different antibody-based techniques, systematic analysis and interpretation are required. First, consider technique-specific factors: Western blot detects denatured proteins and may miss conformational epitopes; IHC involves fixation that can mask epitopes; while IF/ICC preserves some spatial context but may suffer from autofluorescence . The recommended dilution ranges vary significantly between techniques (1:1000-1:4000 for WB, 1:200-1:800 for IHC, and 1:50-1:500 for IF/ICC), suggesting different sensitivity thresholds .

For discrepancies between calculated (76 kDa) and observed (70 kDa) molecular weights in Western blot , consider post-translational modifications, proteolytic processing, or splice variants. When immunostaining results differ from Western blot, evaluate fixation and antigen retrieval methods; MCCC1 detection in IHC benefits from different antigen retrieval approaches (TE buffer pH 9.0 or citrate buffer pH 6.0) , which may impact epitope accessibility.

Cross-validation with alternative detection methods is essential: complement antibody-based techniques with mRNA detection via RT-qPCR or RNA-seq data. For definitive validation, implement genetic approaches (siRNA knockdown, CRISPR knockout) and demonstrate corresponding reduction in signal across all detection methods . When interpreting subcellular localization discrepancies, prioritize results from techniques with higher spatial resolution (super-resolution microscopy) over conventional methods, particularly for mitochondrial proteins like MCCC1 .

What approaches can be used to quantitatively analyze MCCC1 expression in response to viral infection?

Quantitative analysis of MCCC1 expression in response to viral infection requires multiple complementary approaches for comprehensive assessment. For protein-level quantification, Western blot analysis with MCCC1 antibodies provides a direct measure of expression changes . Use densitometric analysis normalized to appropriate loading controls (β-actin for total cell lysates, mitochondrial markers like VDAC or COX IV for mitochondrial fractions) to accurately quantify changes. When performing these analyses, include time-course experiments (0, 6, 12, 24, 48 hours post-infection) to capture dynamic expression changes.

For higher throughput analysis, develop ELISA-based assays using MCCC1 antibodies for quantitative measurement across multiple samples and conditions. This approach is particularly valuable when screening multiple viral strains or doses. At the transcriptional level, quantify MCCC1 mRNA expression using RT-qPCR to determine whether viral infection affects MCCC1 at the transcriptional or post-transcriptional level .

To assess spatial changes alongside expression levels, combine immunofluorescence with quantitative image analysis. Measure mean fluorescence intensity of MCCC1 staining in infected versus uninfected cells, and analyze potential changes in subcellular distribution using colocalization coefficients with mitochondrial markers. For the most comprehensive analysis, implement proteomics approaches such as stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry to quantify changes in MCCC1 and interacting partners simultaneously. This multi-modal quantitative approach provides a robust framework for characterizing how viral infection impacts MCCC1 expression and function.

How can researchers use MCCC1 antibodies to investigate post-translational modifications affecting MCCC1 function?

Investigating post-translational modifications (PTMs) of MCCC1 requires specialized approaches centered around strategic use of MCCC1 antibodies. Begin by enriching MCCC1 protein through immunoprecipitation with validated MCCC1 antibodies , followed by Western blot analysis with antibodies specific to common PTMs such as phosphorylation (phospho-serine/threonine/tyrosine), acetylation, ubiquitination, and SUMOylation. For phosphorylation studies specifically, treat lysates with phosphatase inhibitors during preparation and consider lambda phosphatase treatment controls to confirm phosphorylation-specific bands.

To identify specific modified residues, combine immunoprecipitation using MCCC1 antibodies with mass spectrometry analysis. This approach allows for unbiased identification of modification sites and can reveal novel PTMs. For functional studies, generate site-specific mutants of predicted modification sites (changing serine/threonine to alanine for phosphorylation sites, lysine to arginine for ubiquitination/acetylation) and assess the impact on MCCC1's ability to enhance virus-induced NF-κB activation using reporter assays .

To study dynamic changes in MCCC1 modifications during viral infection, perform time-course experiments collecting samples at multiple time points post-infection. Compare modification patterns between control and virus-infected cells through Western blot or mass spectrometry. For mechanistic studies, identify potential modifying enzymes through literature mining and protein interaction databases, then validate their interaction with MCCC1 using co-immunoprecipitation with MCCC1 antibodies . This systematic approach will provide insights into how PTMs regulate MCCC1's metabolic and immunological functions.

What strategies should be employed when using MCCC1 antibodies in high-throughput screening applications?

Employing MCCC1 antibodies in high-throughput screening applications requires careful optimization and validation strategies to ensure reliable, reproducible results across large sample sets. Begin by selecting MCCC1 antibodies with proven specificity and sensitivity , and thoroughly validate them in your experimental system before scaling up. Optimize antibody concentration through checkerboard titration experiments to determine the minimum concentration that provides robust signal while minimizing background and reagent usage.

For cell-based high-throughput screens, develop automated immunofluorescence protocols with MCCC1 antibodies at validated dilutions (1:50-1:500) , combined with high-content imaging systems. Standardize fixation, permeabilization, and washing steps to ensure consistency across microplates. Include positive (known MCCC1-expressing cells like MCF-7 or U-251) and negative controls (MCCC1-knockdown cells) on each plate for quality control and data normalization.

For biochemical assays, develop ELISA-based detection systems using MCCC1 antibodies as capture or detection reagents. When screening compound libraries for modulators of MCCC1 function, include counterscreens to identify compounds that directly interfere with antibody binding. For screens investigating MCCC1's role in antiviral responses, design reporter-based assays measuring NF-κB activation that can be adapted to high-throughput formats.

Implement robust data analysis pipelines incorporating appropriate statistical methods for hit identification and validation. Follow primary screens with secondary validation assays, including dose-response relationships and orthogonal approaches to confirm findings. This comprehensive strategy maximizes the value of high-throughput screening with MCCC1 antibodies while minimizing false positives and negatives.

How can researchers investigate the relationship between MCCC1's metabolic and immunological functions using antibody-based approaches?

Investigating the dual role of MCCC1 in metabolism and immunity requires integrated experimental approaches centered around antibody-based detection methods. Begin with subcellular fractionation to isolate mitochondria, followed by Western blot analysis with MCCC1 antibodies to confirm mitochondrial localization and assess potential redistribution during immune activation . Implement co-immunoprecipitation studies with MCCC1 antibodies to identify protein interaction partners in both resting cells and cells undergoing immune responses to viral infection .

To connect metabolic and immune functions, measure 3-methylcrotonyl-CoA carboxylase enzymatic activity in cell lysates under various conditions (resting, immune-activated), while simultaneously assessing NF-κB activation using reporter assays or phospho-specific antibodies for IKK and IκBα . Design domain-specific mutants of MCCC1 that selectively disrupt either enzymatic activity or MAVS interaction, then use MCCC1 antibodies to confirm expression of these mutants in reconstitution experiments.

Implement metabolic profiling (metabolomics) in conjunction with immune phenotyping in cells with normal, overexpressed, or knocked-down MCCC1 levels (validated by Western blot with MCCC1 antibodies) . This approach can reveal whether MCCC1's immunoregulatory functions are dependent on or independent of its metabolic role. For in vivo relevance, analyze tissue samples from appropriate disease models using MCCC1 immunohistochemistry (at 1:200-1:800 dilution) alongside markers of inflammation and metabolic state.

Use proximity ligation assays combining MCCC1 antibodies with antibodies against metabolic enzymes or immune signaling components to visualize and quantify protein interactions in situ. This comprehensive approach integrates antibody-based detection with functional readouts to elucidate how MCCC1 coordinates metabolic processes with immunological responses to viral infections.

What are the future research directions for MCCC1 antibody applications in viral immunity studies?

Future research directions for MCCC1 antibody applications in viral immunity studies should focus on several promising avenues. First, developing conditional knockout models validated by MCCC1 antibodies will enable tissue-specific and temporal control over MCCC1 expression, allowing researchers to dissect its role in different phases of viral infection and immune response . Second, engineering highly specific monoclonal antibodies targeting distinct domains of MCCC1 will facilitate more precise studies of structure-function relationships, particularly in distinguishing between MCCC1's metabolic and immunological roles.

The advancement of super-resolution microscopy techniques combined with MCCC1 antibodies will provide unprecedented insights into the spatial organization of MCCC1 within mitochondrial structures and its dynamic redistribution during antiviral signaling . Development of phospho-specific antibodies against MCCC1 will enable monitoring of activation-dependent modifications that may regulate its function in antiviral immunity. Additionally, applying MCCC1 antibodies in single-cell analyses will reveal cell-to-cell variability in MCCC1 expression and its correlation with differential antiviral responses.