MCCC1 Antibody, HRP conjugated

What is MCCC1 and what biological pathways involve this protein?

MCCC1 functions as the large subunit (alpha) of 3-methylcrotonyl-CoA carboxylase, which catalyzes the carboxylation of 3-methylcrotonyl-CoA to form 3-methylglutaconyl-CoA . This enzyme operates as a heterodimer and plays a critical role in leucine catabolism. Recent research has revealed that MCCC1 also participates in autophagy regulation through the mTORC1 pathway, where its depletion has been shown to activate autophagy in multiple cell types including HeLa and neuronal cells . The molecular consequences of MCCC1 dysfunction extend to metabolic disorders, as mutations in this gene are associated with 3-Methylcrotonylglycinuria, an autosomal recessive disorder .

What applications are supported by MCCC1 antibodies?

MCCC1 antibodies have been validated for multiple experimental applications with varying recommended dilutions:

HRP-conjugated MCCC1 antibodies are particularly optimized for ELISA applications, offering enhanced sensitivity and direct detection capabilities without requiring secondary antibodies . The reactivity profile typically includes human and mouse samples, though researchers should verify species specificity for their particular antibody .

How should researchers optimize Western blot protocols for MCCC1 detection?

When designing Western blot experiments for MCCC1 detection, researchers should note that the calculated molecular weight is 76 kDa, but the observed molecular weight typically ranges between 70-76 kDa . For optimal results, consider the following protocol adaptations:

• Sample preparation: Effective protein extraction from mitochondria-rich tissues (liver, heart) has shown reliable detection of MCCC1 .

• Protein loading: 20-40 μg of total protein per lane is typically sufficient for detection.

• Transfer conditions: Use standard PVDF or nitrocellulose membranes with standard transfer protocols for proteins >50 kDa.

• Blocking: 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature.

• Primary antibody incubation: For unconjugated antibodies, dilute 1:500-1:2400 in blocking buffer; for HRP-conjugated antibodies, follow manufacturer's specific recommendations.

• Detection: For HRP-conjugated antibodies, proceed directly to chemiluminescent detection after washing steps.

Positive controls using mouse liver or heart tissue lysates are recommended as these have been validated for consistent MCCC1 expression .

What strategies can enhance the sensitivity and specificity of immunohistochemistry with MCCC1 antibodies?

For immunohistochemical applications, researchers should implement the following methodological approaches:

• Antigen retrieval: Use TE buffer at pH 9.0 as the preferred method, though citrate buffer at pH 6.0 may serve as an alternative .

• Antibody dilution: Begin with a 1:50 dilution and optimize as needed within the 1:20-1:200 range.

• Incubation conditions: Overnight incubation at 4°C typically yields optimal results.

• Positive control tissues: Human liver tissue or liver cancer tissue samples have demonstrated reliable MCCC1 expression patterns .

• Negative controls: Include secondary-only controls and, when possible, MCCC1-knockdown samples.

For HRP-conjugated antibodies specifically, researchers should consider peroxidase blocking steps prior to primary antibody incubation to minimize background signal.

How can MCCC1 antibodies be employed to investigate autophagy regulation?

Research has established that MCCC1 plays a significant role in autophagy regulation through mTORC1 pathway modulation . When designing experiments to investigate this relationship:

• Autophagy markers: Monitor LC3-II levels, which increase following MCCC1 depletion .

• Flux analysis: Combine MCCC1 antibody detection with autophagy flux measurements using Bafilomycin A1 (BAF) treatment.

• mTORC1 activity: Assess phosphorylation status of S6 and S6K1, which decrease with MCCC1 knockdown .

• Visualization approaches: Quantify autophagosome and autolysosome formation using fluorescent reporters like mRFP-GFP-LC3 .

Experimental data has shown that MCCC1 knockdown increased autophagosome biogenesis, WIPI2-positive structures, and functional autolysosomes, demonstrating a clear relationship between MCCC1 function and autophagy regulation that researchers can explore with appropriate antibodies .

What methods can effectively validate MCCC1 antibody specificity?

Rigorous validation of antibody specificity is critical for reliable experimental outcomes. Researchers should implement:

• Genetic controls: Compare antibody signal between wild-type samples and MCCC1 knockdown or knockout models.

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm signal specificity.

• Multiple antibody approach: Use antibodies targeting different epitopes of MCCC1 (e.g., AA 1-200, AA 397-425, AA 526-725) to confirm consistent detection patterns .

• Cross-reactivity assessment: Test antibody against recombinant proteins with similar domains.

• Multiple technique validation: Confirm results across different applications (WB, IHC, IP) when possible.

For HRP-conjugated antibodies specifically, include enzyme activity controls to distinguish true signal from potential enzymatic artifacts.

What are common challenges with MCCC1 antibody applications and their solutions?

When working with MCCC1 antibodies, researchers frequently encounter these challenges:

For HRP-conjugated antibodies specifically, substrate choice and development time significantly impact signal-to-noise ratio; enhanced chemiluminescent substrates typically provide optimal sensitivity.

How should researchers store and handle MCCC1 antibodies for maximum stability?

To maintain antibody performance over time:

• Storage temperature: Store unconjugated antibodies at -20°C, and aliquot to avoid freeze-thaw cycles .

• Buffer composition: MCCC1 antibodies are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• HRP-conjugated antibodies: These may have special storage requirements; refer to manufacturer guidelines.

• Working solutions: Prepare fresh dilutions for each experiment and avoid repeated freeze-thaw cycles.

• Stability assessment: Perform periodic validation experiments to confirm maintained sensitivity over time.

Most MCCC1 antibodies remain stable for one year after shipment when properly stored, but HRP-conjugated versions may have different stability profiles that should be verified with the supplier .

How are MCCC1 antibodies being utilized in neurodegenerative disease research?

Recent studies have revealed connections between MCCC1 and neurological pathways:

• α-synuclein toxicity: MCCC1 depletion has been shown to rescue A53T mutant α-synuclein-induced cell toxicity, suggesting therapeutic implications .

• Neuronal autophagy: MCCC1 knockdown increases autophagy in neuronal cells, including primary murine neurons and SH-SY5Y human neuroblastoma cells .

• mTORC1 signaling: MCCC1 appears to modulate mTORC1 activity in neuronal cells, affecting autophagy regulation and potentially protein aggregation dynamics .

Researchers investigating neurodegenerative conditions may benefit from employing MCCC1 antibodies to examine these pathways, with HRP-conjugated versions offering advantages for quantitative assays and protein interaction studies.

What considerations should be made when using MCCC1 antibodies for metabolic research?

When investigating metabolic pathways:

• Tissue selection: Highest MCCC1 expression is observed in metabolically active tissues such as liver and heart .

• Metabolic state: Consider how fasting, feeding, or metabolic stress might affect MCCC1 expression and localization.

• Subcellular localization: MCCC1 is primarily mitochondrial, requiring appropriate fractionation techniques for enriched detection.

• Pathway integration: Consider co-detection of related metabolic enzymes to establish pathway relationships.

• Functional correlation: Combine antibody detection with metabolomic approaches to correlate protein levels with metabolite profiles.

HRP-conjugated antibodies may be particularly valuable for metabolic pathway studies requiring quantitative ELISA approaches to measure changes in MCCC1 levels under different metabolic conditions.