serac1 Antibody

What is SERAC1 and why is it a significant research target?

SERAC1 (Serine Active Site Containing 1) is a protein that localizes to the outer mitochondrial membrane and functions as a component of the one-carbon cycle. It plays a critical role in facilitating serine transport from the cytosol to the mitochondria by interacting with the mitochondrial serine transporter protein SFXN1 . This protein is essential for phosphatidylglycerol remodeling, which impacts both mitochondrial function and intracellular cholesterol trafficking .

Research interest in SERAC1 has intensified following the discovery that mutations in this gene are associated with multiple neurological disorders, including MEGDHEL syndrome (3-methylglutaconic aciduria with deafness–dystonia, hepatopathy, encephalopathy, and Leigh-like syndrome), juvenile-onset complicated spastic paraplegia, and adult-onset generalized dystonia . More recently, mutations in SERAC1 have also been linked to Canine Multiple System Degeneration in Kerry Blue Terriers and Chinese Crested dogs .

What criteria should researchers use when selecting a SERAC1 antibody for their experiments?

When selecting a SERAC1 antibody for research applications, consider the following criteria:

For SERAC1 specifically, several well-validated antibodies are available. For instance, Proteintech's 25729-1-AP antibody has been validated for Western blotting, immunohistochemistry, immunofluorescence, and ELISA applications, showing reactivity with human and mouse samples . Another example is the Novus Biologicals antibody (catalog #18235337), which has been tested for Western blot applications .

How should researchers optimize Western blot protocols for SERAC1 detection?

Optimizing Western blot protocols for SERAC1 detection requires careful consideration of several factors:

• Sample preparation: Total cell lysates can be prepared using RIPA buffer containing protease inhibitors. For mitochondrial enrichment, follow established mitochondrial isolation protocols as SERAC1 localizes to the outer mitochondrial membrane .

• Protein loading: Load 20-40 μg of total protein per lane. For mitochondrial fractions, 10-20 μg may be sufficient.

• Antibody dilution: Use SERAC1 antibodies at appropriate dilutions:

  • Proteintech 25729-1-AP: 1:500-1:2000

  • Sigma HPA025716: 0.04-0.4 μg/mL

  • Novus Biologicals antibody: Follow manufacturer recommendations (typically 1.0 μg/ml)

• Expected band size: SERAC1 should appear at approximately 70-75 kDa .

• Controls: Include a positive control (e.g., mouse brain tissue, HEK-293 cells, or HeLa cells) and, when available, SERAC1 knockout cells as negative controls .

• Troubleshooting:

  • If no band is detected, verify antibody reactivity with your species and consider using fresh protein samples.

  • Multiple bands may indicate post-translational modifications, splice variants, or non-specific binding.

What methods are recommended for validating the specificity of SERAC1 antibodies?

Validating antibody specificity is critical for reliable research outcomes. For SERAC1 antibodies, implement these validation methods:

A comprehensive validation approach would be similar to protocols used for other proteins, such as the RNA-binding protein TIA1, where knockout cell lines were compared with isogenic parental controls .

How can SERAC1 antibodies be used to study MEGDHEL syndrome pathophysiology?

SERAC1 antibodies are valuable tools for investigating MEGDHEL syndrome pathophysiology through several experimental approaches:

• Protein expression analysis: Western blot analysis can be used to confirm the absence or reduction of SERAC1 protein expression in patient-derived cells. For example, in a study by Identification of a novel splice site mutation in the SERAC1 gene, researchers used Western blot to demonstrate the absence of SERAC1 expression in patient fibroblasts compared to controls .

• Subcellular localization: Immunofluorescence microscopy with SERAC1 antibodies can determine the protein's localization in normal and patient cells, particularly regarding its association with mitochondria and endoplasmic reticulum contact sites.

• Functional studies: Co-immunoprecipitation experiments can identify SERAC1 interaction partners, such as SFXN1, to understand how mutations disrupt protein-protein interactions .

• Therapeutic studies: SERAC1 antibodies can be used to monitor protein expression after treatment interventions, such as nucleotide/nucleoside supplementation, which has been shown to restore mtDNA content and mitochondrial function in SERAC1-deficient models .

• Animal models: In mouse models of MEGDHEL syndrome, antibodies can track SERAC1 expression patterns across tissues and development stages to correlate with disease progression .

What are the significant challenges in detecting endogenous SERAC1 in different tissue samples?

Detecting endogenous SERAC1 presents several challenges that researchers should address:

• Variable expression levels: SERAC1 expression varies across tissues, with potentially higher expression in metabolically active tissues like liver and brain. Adjust protein loading accordingly.

• Mitochondrial localization: As SERAC1 localizes to the outer mitochondrial membrane, standard protein extraction methods may not efficiently solubilize the protein. Consider using specialized extraction buffers containing mild detergents such as digitonin or n-dodecyl β-D-maltoside .

• Post-translational modifications: These may affect antibody recognition. Use antibodies targeting different epitopes if available.

• Cross-reactivity concerns: Some SERAC1 antibodies may cross-react with other proteins. Validate specificity using knockout controls when possible .

• Tissue-specific isoforms: Different splice variants may express in different tissues. Select antibodies that recognize conserved regions.

• Fixation sensitivity: For immunohistochemistry, optimize fixation protocols as some epitopes may be sensitive to certain fixatives .

How can SERAC1 antibodies be applied to investigate mitochondrial lipid metabolism disorders?

SERAC1 antibodies enable sophisticated investigations of mitochondrial lipid metabolism disorders through multiple approaches:

• Cardiolipin analysis: SERAC1 deficiency affects cardiolipin composition. Use antibodies to correlate SERAC1 expression levels with cardiolipin abnormalities detected by LC-MS/MS . This is particularly relevant as SERAC1 mutations change acyl-chain composition of phosphatidylglycerol (PG), a precursor of cardiolipin (CL) .

• Mitochondrial respiratory chain complex analysis: Use Blue Native PAGE analysis of solubilized mitochondrial material combined with SERAC1 immunoblotting to study how SERAC1 deficiency affects respiratory chain supercomplexes .

• One-carbon metabolism: Investigate how SERAC1 deficiency impairs the one-carbon cycle and disrupts nucleotide pool balance through co-localization studies with other one-carbon cycle components .

• Mitochondrial DNA depletion: Use SERAC1 antibodies alongside mtDNA quantification to examine the relationship between SERAC1 expression and mtDNA content in different experimental models .

• Therapeutic screening: Monitor SERAC1 expression in response to treatments targeting mitochondrial function. For example, assess whether nucleoside/nucleotide supplementation restores normal SERAC1 localization and function .

What are the recommended approaches for using SERAC1 antibodies in co-immunoprecipitation studies?

For effective co-immunoprecipitation studies with SERAC1 antibodies:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation applications. Consider using multiple antibodies targeting different epitopes to confirm results.

• Sample preparation:

  • Use mild lysis buffers (e.g., 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% triton X-100) to preserve protein-protein interactions .

  • Include protease and phosphatase inhibitors to prevent degradation.

  • For mitochondrial proteins, use digitonin or n-dodecyl β-D-maltoside instead of harsher detergents.

• Experimental controls:

  • Include IgG control from the same species as the SERAC1 antibody.

  • If available, use SERAC1 knockout cells as negative controls.

  • Consider using tagged SERAC1 constructs as positive controls.

• Detection methods:

  • For Western blot detection of co-immunoprecipitated proteins, use antibodies from different host species to avoid detecting the IP antibody.

  • Consider mass spectrometry for unbiased identification of interaction partners.

• Validation of interactions:

  • Confirm key interactions with reverse co-IP experiments.

  • Validate physiological relevance using proximity ligation assays or FRET-based approaches.

What are common issues researchers encounter when using SERAC1 antibodies and how can they be resolved?

How do different fixation and permeabilization methods affect SERAC1 antibody performance in immunocytochemistry?

The choice of fixation and permeabilization methods significantly impacts SERAC1 antibody performance in immunocytochemistry:

• Fixation methods:

  • Paraformaldehyde (4%): Standard fixation that generally preserves SERAC1 epitopes while maintaining cellular architecture. Recommended initial approach.

  • Methanol: May expose some epitopes better than PFA but can disrupt membrane structures, potentially affecting mitochondrial morphology.

  • Glutaraldehyde: Provides better ultrastructural preservation but may mask epitopes and increase autofluorescence.

• Permeabilization methods:

  • Triton X-100 (0.1-0.5%): Effective for accessing intracellular antigens including mitochondrial proteins.

  • Digitonin (10-50 μg/ml): Selectively permeabilizes plasma membrane while leaving organelle membranes intact; useful for distinguishing outer mitochondrial membrane proteins.

  • Saponin (0.1%): Mild detergent that preferentially extracts cholesterol; may be gentler for preserving membrane protein complexes.

• Epitope retrieval:

  • For formalin-fixed paraffin-embedded tissues, heat-induced epitope retrieval with citrate buffer (pH 6.0) or TE buffer (pH 9.0) may be necessary .

• Recommended protocol optimization:

  • Test multiple fixation/permeabilization combinations with your specific antibody.

  • Include mitochondrial markers (e.g., TOM20) to confirm SERAC1 localization to mitochondria.

  • Use SERAC1-deficient cells as negative controls to confirm specificity.

For SERAC1 specifically, researchers should note that the transmembrane domain (approximately amino acids 33-58) is critical for proper localization , and fixation methods that disrupt membrane integrity may affect antibody binding to this region.

How can SERAC1 antibodies be utilized to explore one-carbon metabolism and its connection to mitochondrial disease?

SERAC1 antibodies can be leveraged to investigate the poorly understood connections between one-carbon metabolism and mitochondrial disease through several innovative approaches:

• Co-localization studies: Use SERAC1 antibodies in combination with antibodies against other one-carbon cycle components (e.g., SHMT2, MTHFD2) to map the spatial organization of this pathway within and around mitochondria.

• Metabolic flux analysis: Combine SERAC1 immunoprecipitation with metabolomics to identify metabolites associated with SERAC1 complexes under normal and pathological conditions.

• Interaction network mapping: Use SERAC1 antibodies for proximity labeling techniques (BioID, APEX) to identify the proximal protein network around SERAC1 at the mitochondrial membrane.

• Disease model comparison: Apply SERAC1 antibodies across different mitochondrial disease models to determine whether SERAC1 dysfunction represents a common pathway in various disorders.

• Therapeutic monitoring: Utilize SERAC1 antibodies to assess treatment efficacy in models supplemented with one-carbon cycle intermediates or nucleosides/nucleotides, which have shown promise in restoring mtDNA content and mitochondrial function in SERAC1-deficient models .

This research direction is particularly promising as the connection between SERAC1, the one-carbon cycle, and nucleotide pool balance may represent a fundamental mechanism underlying mitochondrial DNA depletion syndromes .

What novel techniques combine SERAC1 antibodies with other research tools to advance understanding of mitochondrial membrane dynamics?

Emerging techniques combining SERAC1 antibodies with advanced research tools offer new insights into mitochondrial membrane dynamics:

• Super-resolution microscopy: Combining SERAC1 antibodies with techniques like STORM or PALM can visualize SERAC1 distribution at mitochondria-ER contact sites with nanometer precision, revealing organizational principles not visible with conventional microscopy.

• Live-cell imaging approaches: Using split-GFP systems where one part is attached to SERAC1 and another to interaction partners can visualize dynamic interactions in living cells.

• Correlative light and electron microscopy (CLEM): This technique allows precise localization of SERAC1 at the ultrastructural level by combining antibody-based fluorescence with electron microscopy.

• Mass spectrometry imaging: Coupling SERAC1 immunoprecipitation with spatial metabolomics can map the distribution of phospholipids and other metabolites in relation to SERAC1 localization.

• Single-molecule tracking: Using antibody fragments conjugated to quantum dots can track individual SERAC1 molecules to understand their dynamics and residence time at contact sites.

• Optogenetic approaches: Combining SERAC1 antibodies with optogenetic tools allows researchers to manipulate SERAC1 function with spatiotemporal precision while monitoring consequences for mitochondrial morphology and function.