CLSTN3 Antibody

What is CLSTN3 and why is it an important research target?

CLSTN3 (Calsyntenin 3) is a type I transmembrane protein belonging to the cadherin superfamily. In humans, the canonical protein has 956 amino acid residues with a mass of 106.1 kDa, localizing to the endoplasmic reticulum (ER), Golgi apparatus, and cell membrane . CLSTN3 plays crucial roles in cell adhesion, lipid metabolism, and neuronal function, particularly in cerebellar Purkinje cells . Recent research has highlighted its importance in hepatic steatosis , adaptive thermogenesis , and neurological function , making it a valuable target for studying metabolic and neurological conditions.

What are the main experimental applications for CLSTN3 antibodies?

CLSTN3 antibodies are predominantly used in Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), ELISA, and Co-Immunoprecipitation (Co-IP) applications . These applications enable researchers to detect CLSTN3 expression patterns, subcellular localization, protein-protein interactions, and functional modifications. When selecting an antibody, researchers should verify its validated applications, as some antibodies are optimized for specific techniques (e.g., 13302-1-AP has been validated for WB, IHC, IP, CoIP, ELISA, and IF) .

What species reactivity should be considered when selecting a CLSTN3 antibody?

Most commercially available CLSTN3 antibodies show reactivity against human, mouse, and rat CLSTN3, with some extending to pig, bovine, dog, and other species . For example, the 13302-1-AP antibody has confirmed reactivity with mouse, rat, and pig samples . When designing experiments using animal models, it's critical to select antibodies with validated cross-reactivity to your species of interest, as sequence homology varies between species and may affect antibody binding efficacy.

What are the optimal conditions for Western blot analysis of CLSTN3?

For optimal Western blot detection of CLSTN3:

• Sample preparation: Brain tissue (where CLSTN3 is highly expressed) should be homogenized in RIPA buffer with protease inhibitors

• Gel percentage: Use 8-10% SDS-PAGE gels to properly resolve the 110-130 kDa CLSTN3 protein

• Antibody dilution: Most CLSTN3 antibodies work optimally at 1:500-1:1000 dilution for WB

• Expected band size: Look for bands between 110-130 kDa, which corresponds to the observed molecular weight of CLSTN3

• Blocking: 5% non-fat milk in TBST is typically sufficient for reducing background

Note that post-translational modifications, particularly glycosylation, may affect the observed molecular weight .

How should researchers optimize immunohistochemistry protocols for CLSTN3 detection?

For effective IHC detection of CLSTN3:

• Antigen retrieval: Use TE buffer (pH 9.0) for heat-mediated antigen retrieval; alternatively, citrate buffer (pH 6.0) may be used but might yield reduced signal intensity

• Antibody dilution: Start with 1:50-1:500 dilution range and optimize based on signal-to-noise ratio

• Incubation conditions: Overnight incubation at 4°C typically yields better results than shorter incubations

• Detection system: HRP-based detection systems are commonly used for CLSTN3 visualization

• Controls: Include both positive controls (brain tissue, particularly cerebellum) and negative controls

For mouse brain tissue, paraffin embedding followed by sectioning at 5-8 μm thickness has been validated for CLSTN3 detection .

What methods are available for assessing CLSTN3 knockdown or overexpression efficiency?

Several validated approaches for confirming CLSTN3 genetic manipulation include:

• Quantitative RT-PCR: Primers targeting CLSTN3 exons can detect ~60% reduction in mRNA levels following CRISPR-mediated knockout

• Western blot: Can confirm ~80% reduction in protein levels following knockdown

• Immunofluorescence: Allows visualization of reduced CLSTN3 in cellular membranes following manipulation

• Adenoviral vectors: Ad-Clstn3 has been successfully used for overexpression in liver tissue, with verification by qRT-PCR and protein analysis

For in vivo CRISPR-mediated knockout, targeting exons 2 and 3 of the CLSTN3 gene has proven effective, with sgRNAs delivered via AAV-DJ serotype vectors, particularly for cerebellar Purkinje cells .

How does CLSTN3 contribute to hepatic lipid metabolism, and how can antibodies help investigate this?

CLSTN3 plays a significant role in hepatic lipid metabolism, with decreased expression observed in non-alcoholic fatty liver disease (NAFLD) models . Research shows:

• Expression patterns: CLSTN3 is significantly reduced in HFD, db/db, and ob/ob mice livers compared to controls

• Functional effects: Overexpression of CLSTN3 via Ad-Clstn3 adenovirus improves:

  • Lipid metabolism disorders

  • Glucose tolerance and insulin sensitivity

  • Serum ALT and AST levels (markers of liver function)

  • Reduction of hepatic inflammatory markers (F4/80 and CD11b)

  • Antioxidant gene expression

Researchers can use CLSTN3 antibodies for:

• Western blotting to quantify expression changes in various metabolic conditions

• Immunofluorescence to track subcellular localization changes during lipid metabolism perturbations

• Co-IP to identify novel interaction partners in metabolic pathways

What is CLSTN3B, and how does it differ from standard CLSTN3 in research applications?

CLSTN3B is an adipose-specific isoform of CLSTN3 that plays a key role in adaptive thermogenesis . Important distinctions include:

• Function: CLSTN3B inhibits the activity of CIDEA and CIDEC on lipid droplets, preventing lipid droplet fusion and facilitating lipid utilization

• Mechanism: CLSTN3B promotes ER-to-lipid droplet (LD) phospholipid flow, enhancing LD surface structure

• Structure: CLSTN3B contains an arginine-rich segment (RTRNLRPTRRR) between the ER and LD membrane that facilitates membrane fusion

• Effect on thermogenesis: CLSTN3B promotes sympathetic innervation of thermogenic adipose tissue by driving secretion of neurotrophic factor S100B

When designing experiments targeting CLSTN3B specifically:

• Choose antibodies recognizing the unique regions of CLSTN3B

• Account for its localization to ER and lipid droplets rather than cell membrane

• Use appropriate adipose tissue or adipocyte models for highest expression

How is CLSTN3 involved in neuronal function, particularly in cerebellar circuits?

CLSTN3 shows highly selective expression in cerebellar Purkinje cells, with levels >20-fold higher than CLSTN1 or CLSTN2 . Research using CRISPR-mediated knockout revealed:

• Localization: Predominantly expressed in Purkinje cells with minimal expression in granule cells and some expression in basket and stellate cells

• Function: CRISPR-mediated deletion of CLSTN3 in cerebellar Purkinje cells suppresses synaptic transmission

• Timing: CLSTN3 appears to be involved not only in initial synapse formation but also in ongoing maintenance and remodeling of synaptic connections

For neuronal studies, researchers should:

• Use brain region-specific approaches (e.g., cerebellar slices for Purkinje cells)

• Consider developmental timing when manipulating CLSTN3 expression

• Combine electrophysiological measurements with antibody-based protein detection

What are common challenges when detecting CLSTN3 in Western blots and how can they be addressed?

Researchers may encounter several challenges when detecting CLSTN3 via Western blotting:

• Multiple bands: CLSTN3 undergoes post-translational modifications including glycosylation, ubiquitination, and proteolytic cleavage . To address this:

  • Use deglycosylation enzymes (PNGase F) to confirm glycosylation-related bands

  • Include protease inhibitors during sample preparation

  • Compare band patterns with positive controls

• Weak signal: For enhanced detection:

  • Increase protein loading (50-100 μg total protein)

  • Optimize antibody concentration (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation to overnight at 4°C

  • Use high-sensitivity ECL substrates

• Non-specific bands: To improve specificity:

  • Increase blocking time (2 hours at room temperature)

  • Include 0.1% Tween-20 in wash buffers

  • Consider using milk instead of BSA for blocking (or vice versa)

  • Validate with knockout or knockdown controls when possible

How can tissue-specific expression of CLSTN3 affect antibody selection and experimental design?

CLSTN3 shows distinct expression patterns across tissues, necessitating tailored experimental approaches:

• Brain tissue (particularly cerebellum):

  • Highest expression in Purkinje cells

  • Use antibodies validated for neural tissue

  • Consider fixation effects on epitope accessibility

• Liver tissue:

  • Expression decreases in fatty liver disease models

  • Select antibodies validated for metabolic tissues

  • Consider effects of high lipid content on extraction efficiency

• Adipose tissue:

  • CLSTN3B isoform predominates

  • Choose antibodies that can distinguish CLSTN3 from CLSTN3B

  • Optimize protein extraction from lipid-rich tissues

• General considerations:

  • Use tissue-specific positive controls

  • Adjust protein extraction methods based on tissue type

  • Consider tissue-specific interfering substances

What controls should be included when validating CLSTN3 antibody specificity?

Rigorous validation of CLSTN3 antibody specificity requires multiple controls:

• Positive controls:

  • Brain tissue (especially cerebellum) for wild-type CLSTN3

  • Liver tissue for metabolic studies

  • Recombinant CLSTN3 protein of known concentration

• Negative controls:

  • CRISPR-mediated knockout tissue (60-80% reduction confirmed)

  • siRNA or shRNA knockdown samples

  • Tissues known to express minimal CLSTN3

• Specificity controls:

  • Peptide competition assays with immunizing peptides

  • Secondary antibody-only controls

  • Cross-reactivity assessment with related proteins (CLSTN1, CLSTN2)

• Application-specific controls:

  • For IHC/IF: Include isotype controls and secondary-only controls

  • For IP: Include IgG control pulldowns

  • For WB: Include molecular weight markers

How should researchers interpret CLSTN3 expression changes in metabolic disease models?

When analyzing CLSTN3 expression in metabolic conditions:

• Baseline comparisons:

  • CLSTN3 is significantly decreased in HFD, db/db, and ob/ob mice models compared to controls

  • OA&PA treatment significantly reduces CLSTN3 expression in primary hepatocytes

• Intervention effects:

  • Ad-Clstn3 overexpression improves:

    • Fasting blood glucose levels

    • Glucose, insulin, and pyruvate tolerance

    • Hepatic lipid deposition

    • Serum ALT/AST levels

    • Inflammatory marker expression

• Quantification methods:

  • Combine qRT-PCR data with protein quantification

  • Normalize to appropriate housekeeping genes/proteins

  • Consider both acute and chronic changes in expression

• Physiological correlations:

  • Connect CLSTN3 changes to metabolic parameters like body weight, VO₂, VCO₂, and thermogenesis

  • Assess relationships between CLSTN3 levels and oxidative stress markers

What molecular weight variants of CLSTN3 might be detected, and what do they represent?

CLSTN3 antibodies may detect multiple molecular weight forms that represent different biological states of the protein:

When multiple bands are observed:

• Compare with positive control tissues

• Consider tissue-specific processing differences

• Evaluate possible degradation during sample preparation

• Assess antibody specificity for different domains that might be affected by processing

How can CLSTN3 antibodies be used to investigate its role in protein-protein interactions and signaling pathways?

CLSTN3 antibodies enable investigation of complex molecular interactions through several approaches:

• Co-Immunoprecipitation (Co-IP):

  • CLSTN3 antibodies have been validated for IP and Co-IP applications

  • Can identify novel binding partners in different cellular contexts

  • Useful for confirming predicted interactions from bioinformatic analyses

• Proximity ligation assays:

  • Combine CLSTN3 antibodies with antibodies against suspected interaction partners

  • Provides in situ visualization of protein complexes

  • Yields quantitative data on interaction frequency

• Subcellular localization studies:

  • Immunofluorescence with CLSTN3 antibodies reveals dynamics between ER, Golgi, and cell membrane

  • In adipocytes, can track CLSTN3B localization to lipid droplets

  • Co-staining with organelle markers illuminates trafficking patterns

• Signaling pathway analysis:

  • In liver tissue, correlate CLSTN3 changes with antioxidant gene expression (Nrf2, Sod2, Ho-1)

  • In adipose tissue, assess relationship with thermogenic and sympathetic innervation pathways

  • In neurons, investigate relationship with synaptic proteins

By combining these approaches, researchers can build comprehensive models of CLSTN3's role in diverse cellular pathways and physiological processes.

How are CLSTN3 antibodies being used to investigate its role in neurodegenerative diseases?

Recent research suggests potential connections between CLSTN3 and neurodegenerative conditions:

• Alzheimer's disease connections:

  • CLSTN3 may act as an auxin and regulator of cellular vitamin C uptake

  • It promotes ascorbic acid to reduce oxidative stress

  • It decreases inflammatory factor secretion in Alzheimer's disease models

• Methodological approaches:

  • Immunohistochemistry of brain sections from disease models

  • Co-localization with established disease markers

  • Differential expression analysis across disease progression stages

• Synaptic function investigations:

  • The role of CLSTN3 in maintaining synaptic connections may be relevant to neurodegenerative processes

  • Antibodies enable visualization of synaptic localization changes during disease progression

What are the latest techniques for studying CLSTN3 in the context of metabolic diseases and obesity?

Cutting-edge approaches for investigating CLSTN3 in metabolic disorders include:

• Adenoviral-mediated gene delivery:

  • Ad-Clstn3 (10¹¹ PFU) and ShClstn3 (10⁹ PFU) adenoviruses enable liver-specific overexpression or silencing

  • Effects can be assessed 7 days post-infection

  • Combines with metabolic phenotyping (glucose, insulin, pyruvate tolerance tests)

• Energy expenditure analysis:

  • CLSTN3-overexpressing mice show increased VO₂, VCO₂, and thermogenesis

  • These parameters correlate with reduced hepatic lipid accumulation

• Oxidative stress measurements:

  • ROS measurements combined with qRT-PCR for antioxidant genes (Nrf2, Sod2, Ho-1)

  • CLSTN3 overexpression reduces cellular ROS levels and elevates expression of these genes

• Inflammatory profiling:

  • Immunofluorescence for monocyte inflammatory markers (F4/80, CD11b)

  • Gene expression analysis of inflammatory cytokines

  • CLSTN3 overexpression suppresses these inflammatory markers

How can single-cell approaches enhance CLSTN3 research using antibody-based techniques?

Single-cell techniques offer unprecedented resolution for CLSTN3 research:

• Single-cell RT-PCR with patch-clamping:

  • Validated for measuring CLSTN3 expression in individual Purkinje cells, granule cells, and basket cells

  • Revealed Purkinje cells express >20-fold more CLSTN3 than other cerebellar neurons

• Ribotag isolation with cell-type specific Cre:

  • L7-Cre-dependent ribotag isolation confirmed selective CLSTN3 expression in Purkinje cells

  • Can be combined with antibody-based protein verification

• Single-cell spatial proteomics:

  • Combining fluorescence microscopy with CLSTN3 antibodies enables subcellular localization studies

  • Can reveal heterogeneity in expression and localization within seemingly uniform cell populations

• Mass cytometry with metal-conjugated antibodies:

  • Allows multiplexed protein detection at single-cell resolution

  • Can correlate CLSTN3 levels with multiple other markers simultaneously