HCCS Antibody

What is HCCS and why is it relevant to mitochondrial research?

HCCS (Holocytochrome C Synthase) is a mitochondrial enzyme that functions as a heme lyase by catalyzing the covalent attachment of heme to both apocytochrome c and c1, the precursor forms, thereby producing the mature holocytochrome c and c1. These mature forms are critical components of the mitochondrial respiratory chain .

The significance of HCCS in mitochondrial research stems from its crucial role in oxidative phosphorylation (OXPHOS). Without functional HCCS, the conversion of apocytochrome c to its holo form fails, impacting the electron transport chain's efficiency and leading to impaired energy production . Additionally, cytochrome c plays a dual role in both OXPHOS and apoptosis, making HCCS a key protein for studies investigating the intersection of mitochondrial function and cell death pathways .

Validating the specificity of an HCCS antibody requires a multi-faceted approach:

• Positive and negative tissue controls: Use tissues with known HCCS expression levels. For example, HCCS is expressed in mouse small intestine, heart, and skeletal muscle as demonstrated in Western blot analysis .

• Cell line validation: Test the antibody in cell lines with varying HCCS expression levels. HeLa, RAW 264.7, MCF7, and HepG2 cell lines have been used successfully for HCCS detection .

• Molecular weight verification: The predicted molecular weight of HCCS is approximately 31 kDa . Confirm that your antibody detects a band at this size in Western blot.

• Knockout/knockdown controls: If available, use HCCS knockout or knockdown samples as negative controls to confirm antibody specificity.

• Recombinant protein testing: Use recombinant HCCS protein as a positive control. Some suppliers offer recombinant HCCS for this purpose .

What is the optimal protocol for using HCCS antibodies in Western blotting?

Based on research protocols, the following methodology is recommended for optimal Western blot results with HCCS antibodies:

• Sample preparation: Prepare cell or tissue lysates using standard protocols. Various tissue types have been successfully used, including human cell lines (HeLa, MCF7, HepG2) and mouse tissues (small intestine, heart, skeletal muscle) .

• Antibody dilutions:

  • For ab224321: Use at 1/100 dilution

  • For ab234874: Use at 1/1000 dilution

  • For abbexa HCCS antibodies: Use at 1/500 - 1/2000 dilution

• Detection system: Use appropriate secondary antibodies and detection systems. ECL (Enhanced Chemiluminescence) technique has been successfully employed for visualization .

• Expected results: Look for a band at approximately 31 kDa, which is the predicted molecular weight of HCCS .

• Controls: Include positive controls such as RT4 cell lysate, which has been validated for HCCS detection .

How can HCCS antibodies be used to study mutations and their impact on mitochondrial function?

HCCS mutations have been linked to various disorders including MLS (microphthalmia with linear skin defects) . Researchers can use HCCS antibodies to:

• Analyze expression levels: Compare HCCS protein expression in wild-type versus mutant samples using quantitative Western blot analysis.

• Localization studies: Determine if mutations affect HCCS localization using immunofluorescence. Wild-type HCCS localizes to mitochondria, while certain mutants (e.g., C-terminal-truncated Δ197–268) fail to be sorted to mitochondria .

• Functional assays: Combine antibody detection with functional assays to correlate HCCS expression with cytochrome c maturation and mitochondrial respiratory chain activity.

• Complementation studies: As demonstrated in research, HCCS antibodies can be used to confirm expression of HCCS constructs in complementation assays, where wild-type HCCS and mutant variants (R217C and Δ197–268) were tested for their ability to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p .

• Apoptosis investigations: Since cytochrome c is implicated in apoptosis, HCCS antibodies can help study how HCCS mutations affect the balance between apoptosis and necrosis in affected tissues .

What are the considerations for using HCCS antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with HCCS antibodies, consider:

• Fixation method: PFA (paraformaldehyde) fixation has been successfully used for HCCS detection in cell lines .

• Antibody dilutions:

  • For ab224321: Use at 1/20 dilution for paraffin-embedded human tissues

  • For abbexa HCCS antibodies: Use at 1/20 - 1/200 dilution or 5-20 μg/ml

• Tissue types: HCCS antibodies have been validated on human oral mucosa tissue in paraffin sections .

• Antigen retrieval: May be necessary for optimal staining in formalin-fixed, paraffin-embedded tissues, though specific protocols for HCCS are not detailed in the search results.

• Detection systems: Standard secondary antibody detection systems appropriate for rabbit primary antibodies should be used, as most HCCS antibodies are rabbit polyclonal .

• Controls: Include positive control tissues with known HCCS expression and negative controls where primary antibody is omitted.

How does HCCS relate to hepatocellular carcinoma (HCC) research?

While HCCS itself hasn't been directly implicated as a major target in HCC research based on the provided search results, understanding its role can inform broader studies on mitochondrial function in liver cancer:

• Mitochondrial dysfunction in HCC: As HCCS is essential for cytochrome c maturation and oxidative phosphorylation, studying its expression and function in HCC samples could provide insights into mitochondrial dysfunction in liver cancer.

• Apoptotic resistance: Given cytochrome c's role in apoptosis, investigating HCCS in HCC may shed light on mechanisms of apoptotic resistance in liver cancer cells. The search results suggest that "the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis" .

• Complementary to other HCC biomarkers: While antibodies against other targets such as GPC3, anti-p53, and Claudin-1 are more explicitly mentioned in HCC research , HCCS antibodies could be used alongside these to develop a more comprehensive understanding of mitochondrial involvement in HCC.

What controls should be included when using HCCS antibodies?

To ensure reliable and reproducible results with HCCS antibodies, include:

• Positive controls:

  • Cell lines: RT4, U-251 MG, HeLa, RAW 264.7, MCF7, and HepG2 cells have been validated

  • Tissues: Mouse small intestine, heart, and skeletal muscle show HCCS expression

  • Recombinant protein: Use recombinant HCCS protein as a positive control for antibody validation

• Negative controls:

  • Primary antibody omission: Include samples where primary antibody is replaced with buffer or non-immune serum

  • HCCS-deficient samples: If available, use HCCS knockout or knockdown samples

• Specificity controls:

  • Pre-absorption control: Pre-incubate antibody with recombinant HCCS protein before staining

  • Secondary antibody only: Verify absence of non-specific binding by secondary antibody

• Loading controls (for Western blot):

  • Use established housekeeping proteins like β-actin, GAPDH, or tubulin

How can HCCS antibodies be used to investigate mitochondrial disorders?

HCCS antibodies can be valuable tools for investigating mitochondrial disorders:

• Expression analysis: Quantify HCCS protein levels in patient samples compared to healthy controls using Western blot. Altered HCCS expression may indicate mitochondrial dysfunction.

• Localization studies: Use immunofluorescence to determine if HCCS properly localizes to mitochondria in patient cells. Mislocalization could contribute to disease pathology, as seen with the C-terminal-truncated Δ197–268 mutant .

• Functional correlation: Combine HCCS antibody detection with assays measuring cytochrome c levels, respiratory chain activity, and ATP production to establish correlations between HCCS expression and mitochondrial function.

• Genetic disorder investigation: HCCS mutations have been associated with MLS (microphthalmia with linear skin defects) . Antibodies can help validate the impact of these mutations at the protein level.

• Therapeutic evaluation: When testing potential therapies for mitochondrial disorders, HCCS antibodies can be used to monitor changes in expression or localization following treatment.

What are the methodological differences between using HCCS antibodies in different experimental applications?

Different applications require specific methodological considerations:

How can researchers optimize immunofluorescence experiments with HCCS antibodies?

For optimal immunofluorescence results with HCCS antibodies:

• Fixation protocol: Use PFA fixation followed by Triton X-100 permeabilization, as this has been validated for HCCS detection in U-251 MG cells .

• Antibody concentration: Use ab224321 at 4 μg/ml or abbexa antibodies at 5-20 μg/ml .

• Counterstaining: Since HCCS is a mitochondrial protein, co-staining with mitochondrial markers (e.g., MitoTracker or antibodies against other mitochondrial proteins) can help confirm localization.

• Fluorophore selection: Choose secondary antibodies with fluorophores that have minimal spectral overlap with mitochondrial markers if performing co-localization studies.

• Imaging parameters: Use confocal microscopy for optimal resolution of mitochondrial structures. Z-stack imaging may be necessary to fully capture the three-dimensional mitochondrial network.

• Controls: Include cells where HCCS is overexpressed as positive controls and secondary-only stained cells as negative controls.

What are the common challenges and troubleshooting strategies when using HCCS antibodies?

Researchers may encounter several challenges when working with HCCS antibodies:

• Low signal intensity:

  • Solution: Increase antibody concentration, extend incubation time, or use more sensitive detection systems

  • For Western blot: Use enhanced chemiluminescence (ECL) technique as successfully employed with HCCS antibodies

• High background:

  • Solution: Increase blocking time/concentration, reduce primary and secondary antibody concentrations, add additional washing steps

  • Use 3% milk in PBS for blocking as used in ELISA protocols with GPC3 antibodies (which might be applicable to HCCS as well)

• Non-specific bands in Western blot:

  • Solution: Increase blocking stringency, optimize antibody dilution, use gradient gels for better separation

  • Remember that the expected molecular weight for HCCS is 31 kDa

• Poor reproducibility:

  • Solution: Standardize protocols, use freshly prepared reagents, and ensure consistent sample preparation

  • Store antibodies as recommended (e.g., aliquot and store at -20°C, avoid repeated freeze/thaw cycles)

• Mitochondrial localization issues:

  • Solution: Ensure proper cell permeabilization, co-stain with established mitochondrial markers, use confocal microscopy

How do different storage conditions affect HCCS antibody performance?

Proper storage is critical for maintaining antibody performance:

• Storage temperature: Store HCCS antibodies at -20°C as recommended by manufacturers . Avoid storing at 4°C for extended periods.

• Buffer composition: HCCS antibodies are typically provided in buffers containing:

  • PBS, pH 7.3, with 0.02% sodium azide and 50% glycerol

  • 0.01 M PBS, pH 7.4, containing 0.05% Proclin-300, 50% glycerol

  These components help maintain antibody stability.

• Aliquoting: Divide antibodies into small aliquots upon receipt to avoid repeated freeze/thaw cycles, which can degrade antibody performance .

• Thawing protocol: Thaw antibodies on ice or at 4°C, never at room temperature or with heat.

• Working dilution stability: Diluted antibody working solutions generally have reduced stability. Prepare fresh working dilutions for each experiment when possible.

• Shelf life: Most antibodies have a recommended validity period of 12 months under optimal storage conditions .

How can HCCS expression patterns be correlated with cytochrome c function in experimental designs?

To correlate HCCS expression with cytochrome c function:

• Co-immunoprecipitation studies: Use HCCS antibodies to pull down HCCS and associated proteins, then probe for cytochrome c to investigate their physical interaction.

• Dual immunostaining: Perform co-localization studies with antibodies against HCCS and cytochrome c to visualize their spatial relationship in cells.

• Functional assays coupled with expression analysis:

  • Measure cytochrome c oxidase activity and correlate with HCCS expression levels

  • Assess apoptotic response (cytochrome c release from mitochondria) in cells with varying HCCS expression

  • Evaluate electron transport chain efficiency in relation to HCCS protein levels

• Genetic manipulation experiments: Use HCCS knockdown/knockout models or overexpression systems to directly observe the impact on cytochrome c maturation and function.

• Disease model studies: As suggested in research on MLS syndrome, "the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis" . This hypothesis can be tested using HCCS antibodies to confirm HCCS deficiency in experimental models.

What is the significance of HCCS in comparative studies across different species?

HCCS is evolutionarily conserved across species, making comparative studies valuable:

• Sequence homology: The human HCCS gene and the corresponding murine gene share 83% nucleotide sequence identity and 85% amino acid identity . This high conservation suggests important functional roles.

• Cross-reactivity of antibodies: Many HCCS antibodies show cross-reactivity with human, mouse, and rat HCCS , facilitating comparative studies across these species.

• Model organism research: HCCS function has been studied in yeast (Saccharomyces cerevisiae) using the ortholog Cyc3p . Complementation assays with human HCCS in yeast mutants can provide insights into conserved functional domains.

• Evolutionary insights: Comparative studies of HCCS across species can reveal how this essential mitochondrial enzyme has evolved and adapted to different cellular environments.

• Disease modeling: Animal models expressing mutant HCCS variants can help understand human diseases like MLS syndrome. Antibodies that recognize both human and model organism HCCS are valuable tools for translational research.