SNCA Recombinant Monoclonal Antibody

What is SNCA and what role does it play in neurological function?

SNCA (Alpha-synuclein) is a neuronal protein that plays several crucial roles in synaptic activity. It regulates synaptic vesicle trafficking and subsequent neurotransmitter release . As a monomer, it participates in synaptic vesicle exocytosis by enhancing vesicle priming, fusion, and dilation of exocytotic fusion pores . Mechanistically, it increases local Ca²⁺ release from microdomains, which is essential for enhancing ATP-induced exocytosis .

In its multimeric membrane-bound state, alpha-synuclein acts as a molecular chaperone, assisting in the folding of synaptic fusion components called SNAREs (Soluble NSF Attachment Protein REceptors) at the presynaptic plasma membrane in conjunction with cysteine string protein-alpha/DNAJC5 . This chaperone activity is important for sustaining normal SNARE-complex assembly during aging .

Additionally, SNCA plays a role in regulating dopamine neurotransmission by associating with the dopamine transporter (DAT1) and modulating its activity . Pathologically, SNCA fibrillar aggregates represent a major component of Lewy body inclusions in Parkinson's disease and the non-Aβ component of Alzheimer's disease amyloid plaques .

What are SNCA recombinant monoclonal antibodies and how do they differ from conventional antibodies?

SNCA recombinant monoclonal antibodies are laboratory-engineered antibodies that specifically target alpha-synuclein protein. Unlike conventional hybridoma-derived antibodies, recombinant antibodies are produced using molecular biology techniques where the genes encoding the antibody are cloned and expressed in defined expression systems.

The key advantages of recombinant SNCA antibodies over conventional ones include:

• Unrivaled batch-to-batch consistency, eliminating the need for same-lot requests

• Increased sensitivity and confirmed specificity (often validated with knockout cell lines)

• High repeatability and sustainable supply (not dependent on immunized animals)

• Animal-free production options for ethically conscious research

For example, the Anti-alpha-synuclein antibody [MJFR1] is a rabbit monoclonal recombinant antibody that has been developed with support from the Michael J. Fox Foundation for Parkinson's Research . It has been extensively validated, including confirmation of specificity using SNCA knockout cell lines .

Similarly, the Mouse Anti-SNCA Recombinant Antibody (TAB-0750CLV) is expressed in mammalian cells with chemically defined culture media and purified by affinity chromatography . Its binding affinities have been rigorously determined using multiple methods including isothermal calorimetry, ELISA, and surface plasmon resonance-based approaches .

What are the common applications of SNCA recombinant monoclonal antibodies in research?

SNCA recombinant monoclonal antibodies serve as versatile research tools with multiple applications in neuroscience and neurodegeneration research:

These antibodies have been validated across multiple species samples including human, mouse, and rat brain tissues and neuronal cell lines . The choice of application should guide antibody selection, as some clones perform better in certain applications than others.

How do different antibody clones recognize various forms of alpha-synuclein?

Different SNCA antibody clones exhibit varying affinities for different conformational states of alpha-synuclein:

The Mouse Anti-SNCA Recombinant Antibody (TAB-0750CLV) demonstrates a remarkable selectivity for fibrillar forms, with an IC50 value of 0.5 nM for fibrils compared to 3.6 μM for monomers – representing a >7,000-fold selectivity . This makes it particularly valuable for studying pathological aggregates.

In contrast, some antibody clones like MJFR1 are designed to recognize epitopes that may be equally accessible in both native and aggregated forms, making them suitable for detecting total alpha-synuclein regardless of conformation .

When analyzing experimental samples:

• Western blots can reveal different molecular weight species (14 kDa monomer, 28 kDa dimer, 40 kDa trimer)

• Immunohistochemistry can distinguish between diffuse cytoplasmic staining (typical of monomeric forms) and punctate inclusions (characteristic of aggregated forms)

For comprehensive studies of alpha-synuclein pathology, researchers often employ multiple antibodies targeting different conformational states to obtain a complete picture of protein behavior in their experimental system.

What validation methods ensure SNCA antibody specificity?

Rigorous validation is essential for ensuring SNCA antibody specificity. Multiple complementary approaches should be employed:

• Genetic validation:

  • Use of SNCA knockout cell lines as negative controls

  • Comparison with SNCA-overexpressing systems

  • siRNA/shRNA knockdown controls

• Immunological validation:

  • Pre-absorption tests with purified recombinant SNCA

  • Epitope mapping to confirm binding specificity

  • Multi-tissue microarray (TMA) validation for IHC applications

• Commercial validation examples:

  • The Anti-alpha-synuclein antibody [MJFR1] specificity was confirmed with SNCA knockout cell line validation and multi-tissue microarray analysis

  • Mouse Anti-SNCA Recombinant Antibody (TAB-0750CLV) binding affinities were determined by multiple methods including isothermal calorimetry, ELISA, and surface plasmon resonance

• Application-specific validation:

  • For Western blot: Confirming expected molecular weight (14 kDa for monomeric alpha-synuclein)

  • For immunostaining: Demonstrating expected subcellular localization patterns in neuronal cultures

Proper validation ensures experimental reliability and reproducibility, particularly important when studying a protein with multiple conformational states like alpha-synuclein.

What are the optimal fixation and antigen retrieval methods for SNCA immunohistochemistry?

Successful detection of alpha-synuclein in tissue sections requires careful consideration of fixation and antigen retrieval methods:

Fixation protocols:

• 4% paraformaldehyde (PFA) fixation has been successfully used for both cell cultures and tissue sections

• For cell cultures, fixation for approximately 15 minutes at room temperature is typically sufficient

• For tissue sections, fixation time may need to be optimized based on tissue thickness and density

Antigen retrieval strategies:

• Heat-induced epitope retrieval (HIER) is commonly employed for formalin-fixed paraffin-embedded (FFPE) tissues

• The Anti-alpha-synuclein antibody [MJFR1] has been validated on the Leica BOND™ RX automated IHC staining platform

Special considerations for pathological alpha-synuclein:

• For detecting aggregated forms, pretreatment with formic acid may enhance immunoreactivity

• When examining Lewy bodies, longer incubation times with primary antibody (e.g., 24 hours at 4°C) may improve detection

• For dual immunofluorescence, compatibility of fixation methods between primary antibodies should be verified

Optimization recommendations:

• Always perform titration experiments to determine optimal antibody concentration

• Include positive control tissues known to express alpha-synuclein

• For human postmortem tissue, account for postmortem interval when designing protocols

These methodological considerations ensure optimal antigen preservation and accessibility for SNCA antibodies.

How can researchers troubleshoot common issues with SNCA antibody performance?

When facing challenges with SNCA antibody performance, systematic troubleshooting can resolve many common issues:

Western blot troubleshooting:

Immunohistochemistry/Immunofluorescence troubleshooting:

General recommendations:

• Always include appropriate positive and negative controls in each experiment

• For critical experiments, validate results with multiple antibody clones

• Consider the impact of post-translational modifications on epitope accessibility

• For studies of aggregated alpha-synuclein, ensure your antibody can recognize the pathological conformation

Systematic optimization of these parameters will significantly improve experimental outcomes with SNCA antibodies.

What technical considerations are important for multiplex immunofluorescence with SNCA antibodies?

Multiplex immunofluorescence with SNCA antibodies requires careful planning and optimization:

Antibody compatibility strategies:

• Select primary antibodies from different host species to avoid cross-reactivity

• For example, mouse anti-SNCA antibodies can be paired with rabbit or chicken antibodies against other targets

• Documented compatible combinations include mouse anti-SNCA with chicken anti-MAP2 for neuronal co-staining

Sample preparation optimization:

• Consistent fixation with 4% formaldehyde for 15 min at room temperature

• Thorough blocking to minimize non-specific binding

• Appropriate counterstains: DAPI for nuclei, phalloidin for F-actin visualization

Successful multiplex combinations from the literature:

• Alpha-synuclein (1:100) + F-Actin (Phalloidin Texas Red) + DAPI

• Alpha-synuclein (1:200) + NeuN (neuronal marker, visualized with Alexa Fluor 647) + DAPI

Image acquisition considerations:

• Sequential scanning for confocal microscopy to minimize bleed-through

• Appropriate filter sets with minimal spectral overlap

• Consistent exposure settings between experimental conditions

Analysis approaches:

• Colocalization analysis to quantify spatial relationships

• Single-channel controls to establish thresholds

• Automated quantification algorithms for unbiased analysis

These technical considerations will ensure reliable results in multiplex studies investigating alpha-synuclein in relation to other cellular markers or structures.

How do epitope locations affect SNCA antibody performance in different applications?

The location of antibody epitopes on the alpha-synuclein protein significantly impacts experimental performance:

Alpha-synuclein structural domains and epitope implications:

Epitope mapping approaches:

• Using recombinant protein constructs comprising different regions of alpha-synuclein

• Analysis of antibody reactivity patterns against panels of alpha-synuclein variants

• Computational analysis of amino acid sequences shared among reactive alpha-synuclein variants

Impact on experimental outcomes:

• Epitopes buried in protein-protein interactions may yield false negatives

• Epitopes affected by post-translational modifications (phosphorylation, ubiquitination) may alter detection

• Cross-reactivity with other synuclein family members (beta- and gamma-synuclein) may occur with certain epitopes

When reporting research findings, explicitly stating which antibody clone was used and its known epitope is important for reproducibility and proper interpretation of results.

What are the latest methodological advances in using SNCA antibodies for neurodegenerative disease research?

Recent methodological advances have expanded the utility of SNCA antibodies in neurodegenerative disease research:

Novel delivery systems:

• Brain-targeted liposomes (BTL) loaded with monoclonal antibodies have been developed to reduce alpha-synuclein aggregation within neurons

• These delivery systems enhance antibody penetration across the blood-brain barrier, addressing a major limitation in therapeutic applications

Innovative antibody formats:

• Intrabodies: Anti-human SNCA intrabodies with various tags ([(D-Arg)9], [+36 GFP], [Tat]) have been developed for specialized applications including functional studies

• The table below summarizes these specialized formats:

Advanced imaging techniques:

• Super-resolution microscopy allows detailed visualization of alpha-synuclein aggregates beyond the diffraction limit

• Proximity ligation assays enable specific detection of alpha-synuclein oligomers

• Multi-label imaging with neuronal markers helps establish pathological context

Biofluid-based detection methods:

• Seed amplification assays using antibodies to detect pathological alpha-synuclein in cerebrospinal fluid

• Immunomagnetic reduction assays for ultrasensitive detection in blood

Therapeutic applications:

• Antibodies as potential therapeutics to target and clear pathological alpha-synuclein

• Example: Anti-Human SNCA Therapeutic (SynO4) Antibody was used to evaluate the ability of brain-targeted liposomes to reduce alpha-synuclein aggregation within neurons

These methodological advances are expanding the utility of SNCA antibodies beyond traditional research applications into potential diagnostic and therapeutic domains.