SV2C Antibody

What is SV2C and why is it significant in neuroscience research?

SV2C is one of three synaptic vesicle glycoprotein 2 paralogs (SV2A, SV2B, and SV2C) belonging to the SLC22B family of solute carriers. Unlike SV2A and SV2B which have ubiquitous expression throughout the brain, SV2C has enriched expression in the basal ganglia, particularly in dopaminergic neurons . SV2C's significance stems from:

• Its role in enhancing vesicular storage of dopamine

• Association with Parkinson's disease pathology as identified in multiple genome-wide association studies (GWAS)

• Function as a modifier of nicotine's protective effect against developing Parkinson's disease

• Potential as a modifier of GBA-associated Parkinson's disease risk and patients' response to L-DOPA

SV2C research is particularly valuable for understanding dopaminergic neuron function and vulnerability in neurodegenerative conditions.

How do I confirm the specificity of an SV2C antibody?

Confirming antibody specificity is critical for reliable results. Based on established protocols, a multi-step validation approach is recommended:

• Immunoblotting with transfected cells: Test the antibody against cells transfected with SV2C and related family members (SV2A and SV2B). The antibody should only detect SV2C and not cross-react with SV2A or SV2B .

• Antigen blocking: Pre-incubate the antibody with the immunizing peptide ("antigen-blocking") and confirm ablation of immunoreactivity .

• shRNA knockdown: Perform shRNA knockdown of SV2C in cells that endogenously express SV2C (such as Neuro-2a cells) and confirm reduction in antibody immunoreactivity .

• Knockout validation: Use tissue from SV2C knockout mice as a negative control, which should show no reactivity with the antibody .

• Comparison with established staining patterns: Compare your staining pattern with published results showing SV2C preferential expression in limited nuclei, including strong immunoreactivity in the ventral pallidum, substantia nigra pars compacta, and ventral tegmental area .

What are the optimal sample preparation methods for SV2C detection?

Sample preparation varies by application and tissue type:

For Western Blotting:

• For cell culture: Collect in RIPA buffer, sonicate, centrifuge, and discard the nuclear fraction

• For tissue: Perform unilateral striatal dissections, homogenize, and use differential centrifugation to achieve a crude synaptosomal protein preparation

• Protein loading: Use 20μg of protein for SDS-PAGE

For Immunohistochemistry:

• Suggested antigen retrieval with TE buffer pH.9.0 (alternatively, citrate buffer pH 6.0 may be used)

• For fixed tissue sections, follow standard protocols with particular attention to antigen retrieval

For Immunofluorescence:

• Blocking solution: PBS with 10% normal goat serum, 0.1% triton-x, and 1% bovine serum albumin

• Primary antibody incubation: Overnight at 4°C with gentle agitation

• Secondary antibody incubation: 1 hour at room temperature

What are the recommended dilutions for SV2C antibodies in different applications?

Based on validated protocols, the following dilutions are recommended:

It is critical to titrate these antibodies for each specific experimental system to achieve optimal results .

How can I optimize SV2C antibody performance for detection in the dopaminergic system?

SV2C detection in dopaminergic neurons requires careful optimization:

• Co-labeling strategy: SV2C is highly colocalized with tyrosine hydroxylase (TH) in the striatum and substantia nigra. Use anti-TH antibodies (1:1,000, Millipore AB152) as a dopaminergic marker alongside SV2C antibodies for confirmation .

• Tissue-specific considerations: Strong SV2C immunoreactivity is found in:

  • Ventral pallidum

  • Substantia nigra pars compacta

  • Ventral tegmental area

  • Cell bodies in the midbrain

  • SV2C-positive fibers in the substantia nigra pars reticulata, dorsal striatum, and nucleus accumbens

• Control experiments: Include brain regions with known high expression (basal ganglia) and low expression as internal controls.

• Age considerations: Expression patterns may vary with age and disease state, so age-matched controls are essential.

• Signal amplification: For weak signals, consider using biotin-streptavidin amplification systems while maintaining specificity.

What are common technical challenges when using SV2C antibodies?

Researchers frequently encounter these challenges with SV2C antibodies:

• Antibody specificity issues: Many commercial SV2C antibodies have cross-reactivity concerns. According to one study, "There is no acceptable SV2C antibody currently commercially available" , which led researchers to design custom polyclonal antibodies.

• Variable molecular weight detection: The calculated molecular weight of SV2C is 82 kDa, but observed weights range from 80-95 kDa , potentially due to post-translational modifications including glycosylation.

• Tissue-specific background: The enriched expression in specific brain nuclei can make distinguishing specific signal from background challenging, especially in areas with low expression.

• Protocol sensitivity: SV2C detection is particularly sensitive to fixation methods and antigen retrieval conditions. Optimization of these parameters is essential for reproducible results.

• Species differences: While most SV2C antibodies detect human, mouse, and rat SV2C, there are sequence differences that may affect antibody binding affinity across species.

How can SV2C antibodies be used to study interactions with α-synuclein in Parkinson's disease models?

SV2C antibodies have revealed important interactions with α-synuclein relevant to Parkinson's disease:

• Co-immunoprecipitation approach:

  • α-synuclein co-immunoprecipitates with SV2C in striatal preparations from wild-type mice

  • Use SV2C antibodies bound to resin columns for immunoprecipitation, followed by α-synuclein detection via immunoblotting

  • Include appropriate controls: neither TH nor DAT co-immunoprecipitate with SV2C (negative control), while synaptotagmin-1 does (positive control)

• Expression pattern analysis in disease models:

  • SV2C-KO animals show altered α-synuclein expression profiles:

    • Reduced monomeric (∼15 kD) α-synuclein (69.1 ± 6.17% of WT)

    • Significantly increased high molecular weight (multimeric, ∼90 kD) α-synuclein (3,020 ± 561% of WT)

  • In A53T-OE mice (α-synuclein mutation model), disrupted SV2C immunoreactivity patterns are observed, not mirrored by changes in SV2A expression

• Imaging analysis:

  • Double immunolabeling with SV2C and α-synuclein antibodies can visualize their co-localization in normal and pathological conditions

  • SV2C-positive puncta patterns differ from α-synuclein aggregation patterns in disease models

These approaches can provide insights into SV2C's potential role in Parkinson's disease pathogenesis and as a therapeutic target.

How can I use SV2C antibodies to investigate vesicular dopamine dynamics?

SV2C antibodies can be valuable tools for studying vesicular dopamine dynamics through several experimental approaches:

• False fluorescent neurotransmitter (FFN) assays:

  • Use SV2C antibodies to correlate SV2C expression with FFN206 uptake and retention

  • SV2C promotes the uptake and retention of FFN206 within vesicles

  • Comparative studies between wild-type and SV2C knockout models show differences in dopamine vesicular dynamics

• Radiolabeled dopamine uptake studies:

  • Use SV2C antibodies to verify SV2C expression in vesicle preparations

  • Compare radiolabeled dopamine retention in vesicles isolated from:

    • SV2C-expressing cell lines vs. control cells

    • Wild-type vs. SV2C knockout mouse brain

• Co-localization with vesicular monoamine transporter 2 (VMAT2):

  • Double immunolabeling with SV2C and VMAT2 antibodies

  • Protocol: Use mouse anti-SV2C (1:500, Sigma MABN367) and rabbit anti-VMAT2 (1:500) with appropriate fluorescent secondary antibodies

  • Although a direct protein-protein interaction between VMAT2 and SV2C has not been identified, these proteins appear to function together to modulate dopamine dynamics

• Toxicant uptake studies:

  • SV2C enhances vesicular storage of neurotoxicants like MPP+

  • Compare neurotoxicant storage between SV2C-expressing and non-expressing systems

How do I interpret conflicting SV2C antibody data in different experimental systems?

When faced with contradictory SV2C antibody results across different experimental platforms, consider these analytical approaches:

• Antibody validation status:

  • Different antibodies target different epitopes of SV2C, which may affect specificity and sensitivity

  • Custom antibodies against mouse SV2C (sequence STNQGKDSIVSVGQPKG) and human SV2C (sequence SMNQAKDSIVSVGQPKG) have been validated extensively

  • Commercial antibodies may vary in validation quality; check if knockout/knockdown controls were used

• Expression level variations:

  • SV2C expression can be modulated by experimental conditions:

    • miR-96 negatively regulates SV2C expression

    • Lipopolysaccharide (LPS) treatment reduces SV2C expression in mice

    • VMAT2 knockout in norepinephrine neurons leads to enriched SV2C expression

• Technical factors:

  • Sample preparation differences (fresh-frozen vs. fixed tissue)

  • Antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Detection systems (fluorescent vs. chromogenic)

• Biological variables:

  • Brain region-specific expression patterns

  • Age-dependent expression changes

  • Disease state alterations (e.g., Parkinson's disease models show disrupted SV2C patterns)

• Quantification approach:

  • Normalize SV2C immunoreactivity to appropriate loading controls

  • For Western blots, GAPDH or Actin are suitable housekeeping proteins

  • For immunohistochemistry, consider area-based normalization or reference to internal controls

How can SV2C antibodies be used to study microRNA regulation of SV2C expression?

Recent studies have identified microRNA regulation of SV2C, opening new research avenues:

• miR-96 regulation of SV2C:

  • miR-96 negatively regulates SV2C expression through direct binding

  • Experimental approach:

    • Dual-luciferase reporter gene assay with wild-type and mutated SV2C binding sites

    • Luciferase activity in SV2C WT is inhibited by miR-96 mimic, whereas SV2C MUT is unaffected

• Validation methodology:

  • RT-qPCR detection of SV2C mRNA expression after miR-96 alteration:

    • miR-96 agomir decreases SV2C mRNA expression

    • miR-96 antagomir increases SV2C mRNA expression

  • Western blot analysis of SV2C protein expression:

    • LPS treatment reduces SV2C protein levels

    • miR-96 antagomir increases SV2C protein in LPS-treated mice

    • miR-96 agomir decreases SV2C protein

• Functional implications:

  • miR-96-mediated reduction of SV2C leads to depression-like behavior and memory impairment in mice

  • Therapeutic targeting of this pathway may have relevance for neuropsychiatric conditions

This research direction connects SV2C to broader neuropsychiatric conditions beyond Parkinson's disease.

What are the considerations for using SV2C antibodies in neurotoxicity studies?

SV2C antibodies provide valuable tools for investigating neurotoxicity mechanisms, particularly in dopaminergic neurons:

• MPTP/MPP+ toxicity models:

  • SV2C enhances the ability of vesicles to store the neurotoxicant MPP+

  • Genetic ablation of SV2C results in enhanced MPTP-induced vulnerability in mice

  • Use SV2C antibodies to:

    • Verify SV2C expression/deletion in experimental models

    • Track SV2C localization during neurotoxicant exposure

    • Correlate SV2C levels with toxicity outcomes

• Experimental design considerations:

  • Timing: Assess SV2C levels before, during, and after neurotoxicant exposure

  • Localization: Compare SV2C distribution in vulnerable vs. resistant neuronal populations

  • Co-markers: Pair SV2C antibodies with TH antibodies to specifically track dopaminergic neurons

• Quantification approaches:

  • For immunohistochemistry: Measure SV2C-positive puncta density

  • For Western blot: Normalize SV2C levels to housekeeping proteins

  • For stereology: Count SV2C/TH double-positive neurons

• Mechanistic investigations:

  • Use SV2C antibodies in conjunction with markers of:

    • Vesicular integrity

    • Oxidative stress

    • Neurodegeneration

  • Compare wild-type vs. SV2C knockout models in response to various neurotoxicants

How can SV2C antibodies contribute to understanding therapeutic approaches for Parkinson's disease?

SV2C antibodies can facilitate research into novel therapeutic approaches for Parkinson's disease:

• Nicotine neuroprotection studies:

  • SV2C was identified as a genetic modifier of nicotine's protective effect against developing Parkinson's disease

  • SV2C genetic ablation alters the effect of nicotine on dopamine transmission

  • Research methodology:

    • Use SV2C antibodies to verify expression in experimental models

    • Compare nicotine effects between wild-type and SV2C knockout systems

    • Investigate changes in SV2C expression/localization after nicotine treatment

• L-DOPA response modulation:

  • SV2C was identified as a modifier of Parkinson's disease patients' response to L-DOPA

  • Experimental approaches:

    • Compare SV2C expression in L-DOPA responders vs. non-responders

    • Use SV2C antibodies to track changes in SV2C localization during L-DOPA treatment

    • Correlate SV2C levels with L-DOPA efficacy in animal models

• α-Synuclein aggregation modulation:

  • SV2C interacts with α-synuclein, and SV2C knockout affects α-synuclein aggregation profiles

  • Research strategy:

    • Use SV2C antibodies for co-immunoprecipitation studies with α-synuclein

    • Image SV2C/α-synuclein co-localization in disease models

    • Investigate compounds that modulate this interaction

• Vesicular dopamine enhancement:

  • SV2C enhances vesicular dopamine storage and reduces cytosolic dopamine toxicity

  • Therapeutic angle:

    • Screen compounds that enhance SV2C expression/function

    • Use SV2C antibodies to verify target engagement

    • Correlate changes in SV2C levels with improved dopamine handling and neuroprotection

This research could lead to novel therapeutic strategies targeting SV2C for Parkinson's disease management.