CLSTN3 Antibody, HRP conjugated

What is CLSTN3 and why is it important in research?

CLSTN3 (Calsyntenin-3) is a transmembrane protein that functions primarily as a postsynaptic adhesion molecule, mediating both excitatory and inhibitory synapse formation by binding to presynaptic neurexins. Recent research has revealed that CLSTN3 plays significant roles beyond neural tissue. It is routinely expressed in human white adipose tissue (WAT) and is predominantly enriched in the adipocyte fraction, localizing to the plasma membrane of these cells . CLSTN3 has emerged as an important research target because:

• It interacts with amyloid precursor protein (APP) in WAT and can increase APP accumulation in mitochondria, which impairs adipose mitochondrial function and promotes obesity

• The variant rs7296261 in the CLSTN3 locus is associated with an increased risk of obesity, with its risk allele linked to increased CLSTN3 expression in human WAT

• An adipose-specific isoform, CLSTN3β, plays a key role in adaptive thermogenesis by inhibiting lipid droplet fusion and facilitating lipid utilization

What are the advantages of using HRP-conjugated CLSTN3 antibodies over unconjugated antibodies?

HRP (Horseradish Peroxidase)-conjugated CLSTN3 antibodies offer several methodological advantages over unconjugated antibodies:

• Direct detection capability: HRP-conjugated antibodies enable direct detection without requiring secondary antibody incubation, which simplifies protocols and reduces experiment time

• Elimination of cross-species reactivity: Direct detection with HRP-conjugated primary antibodies eliminates potential cross-reactivity issues that can occur with secondary antibodies, enhancing specificity particularly in complex tissue samples

• Enhanced sensitivity: The enzymatic amplification of the HRP signal allows for detection of low-abundance CLSTN3 protein, which is crucial when studying its expression in tissues where it may be present at lower levels compared to neural tissue

• Versatility across applications: HRP-conjugated CLSTN3 antibodies can be used across multiple applications including ELISA, Western blotting, and immunohistochemistry, allowing consistent methodology across experimental platforms

• Quantitative analysis: HRP enzymatic activity produces a colorimetric readout that can be measured spectrophotometrically, enabling more precise quantification of CLSTN3 levels compared to fluorescent methods in certain applications

What are the optimal applications for CLSTN3 antibody, HRP conjugated?

Based on the technical specifications and research literature, HRP-conjugated CLSTN3 antibodies demonstrate optimal performance in the following applications:

The optimal application selection depends on your research question:

• For protein expression quantification across different experimental conditions, ELISA provides the most reliable quantitative results

• For molecular weight confirmation and post-translational modification studies, Western blotting is preferred

• For studying the spatial distribution of CLSTN3 in tissue context, particularly in white adipose tissue, immunohistochemistry is most suitable

How should I optimize the experimental protocol when using HRP-conjugated CLSTN3 antibody for ELISA?

When optimizing ELISA protocols with HRP-conjugated CLSTN3 antibodies, consider the following methodological approach:

• Antibody titration:

  • Perform a checkerboard titration starting with the manufacturer's recommended dilution (typically 1:500-1:2000)

  • Test at least three different concentrations above and below the recommended dilution to determine optimal signal-to-noise ratio

• Buffer optimization:

  • Use phosphate-buffered saline (PBS) with 0.02% sodium azide as a base buffer which has been shown to maintain antibody stability

  • For blocking, use 1-5% BSA in PBS to reduce background and enhance specificity

  • Consider adding 0.05% Tween-20 to washing buffers to reduce non-specific binding

• Substrate selection:

  • TMB (3,3',5,5'-Tetramethylbenzidine) is recommended for high sensitivity detection

  • ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) can be used for applications requiring a stable endpoint

• Critical parameters to monitor:

  • Temperature: Maintain consistent reaction temperature (22-25°C) throughout the protocol

  • Timing: Standardize incubation times to ensure reproducibility

  • pH: Optimal activity of HRP occurs at pH 6.0-6.5 for substrate conversion

• Validation controls:

  • Include a standard curve using recombinant CLSTN3 protein

  • Run negative controls using isotype-matched HRP-conjugated antibodies

  • For tissue samples where CLSTN3 expression is known to vary (e.g., adipose tissue vs. brain tissue), include positive control samples to verify antibody performance

How can I address weak signal issues when using CLSTN3 antibody, HRP conjugated?

Weak signal when using HRP-conjugated CLSTN3 antibody may result from several factors. Here's a systematic approach to address this issue:

• Antibody concentration optimization:

  • Increase antibody concentration incrementally (e.g., from 1:2000 to 1:1000, then 1:500)

  • Evaluate signal-to-noise ratio at each concentration to prevent non-specific binding

• Sample preparation improvement:

  • For tissues known to express CLSTN3 (adipose tissue, brain), ensure proper extraction protocols that preserve protein integrity

  • Consider enrichment techniques if CLSTN3 levels are low in your sample

  • For adipose tissue samples, implement adipocyte fraction isolation to enhance detection, as CLSTN3 is predominantly expressed in mature adipocytes

• Enhanced detection methods:

  • Implement signal amplification systems like tyramide signal amplification (TSA)

  • Extend substrate incubation time while monitoring background levels

  • Use high-sensitivity substrates specifically designed for low-abundance targets

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat dry milk, commercial blockers)

  • Increase blocking time or concentration if background is not an issue

• Protein denaturation verification:

  • Ensure proper denaturation for Western blotting applications

  • Test both reducing and non-reducing conditions as protein folding may affect epitope accessibility

• Storage and handling assessment:

  • Verify antibody has been stored properly (-20°C or -80°C as recommended)

  • Avoid repeated freeze-thaw cycles that can diminish activity

  • Check expiration date and proper handling procedures

What strategies can address non-specific binding with CLSTN3 antibody, HRP conjugated?

Non-specific binding is a common challenge with HRP-conjugated antibodies. Here's a methodological approach to minimize this issue:

• Optimize blocking conditions:

  • Increase blocking time from standard 1 hour to 2 hours

  • Test different blocking agents: 5% BSA in PBS may be more effective than milk-based blockers for certain applications

  • Consider adding 0.1-0.3% Triton X-100 for membrane permeabilization in IHC/IF applications

• Adjust antibody dilution and incubation parameters:

  • Try higher dilutions (1:2000-1:5000) to reduce non-specific interactions

  • Perform antibody incubation at 4°C overnight instead of room temperature

  • Add 0.1% BSA to antibody diluent to compete for non-specific binding sites

• Implement more stringent washing protocols:

  • Increase washing steps from 3 to 5 times

  • Extend wash duration to 10 minutes per wash

  • Use PBS-T with higher Tween-20 concentration (0.1% instead of 0.05%)

• Pre-adsorption technique:

  • Incubate the antibody with the lysate from cells not expressing CLSTN3

  • This pre-adsorption step can remove antibodies that bind non-specifically

• Consider cross-reactivity with related proteins:

  • CLSTN3 is part of a family that includes CLSTN1 and CLSTN2

  • Verify the specificity of your antibody against these related proteins

  • Use lysates from cells expressing only CLSTN1 or CLSTN2 as negative controls

How can I effectively use CLSTN3 antibody, HRP conjugated, to study the relationship between CLSTN3 and adipose tissue metabolism?

To investigate CLSTN3's role in adipose tissue metabolism using HRP-conjugated antibodies, implement the following methodological approach:

• Tissue-specific expression profiling:

  • Use immunohistochemistry (IHC) with HRP-conjugated CLSTN3 antibody to compare expression patterns in different adipose depots (subcutaneous, visceral, brown adipose tissue)

  • Quantify staining intensity across different metabolic states (fasting, fed, cold-exposed) to correlate with functional changes

• Co-localization studies with metabolic markers:

  • Perform dual staining using HRP-conjugated CLSTN3 antibody and other metabolic markers (UCP1, PPAR-γ)

  • Use substrate combinations that produce differently colored precipitates for simultaneous detection

  • This approach can reveal relationships between CLSTN3 expression and thermogenic capacity or adipocyte differentiation state

• Stimulation response analysis:

  • Treat adipocyte cultures with isoproterenol (which has been shown to increase CLSTN3 expression) and quantify changes using ELISA with HRP-conjugated antibodies

  • Compare responses across different adipocyte types (white, beige, brown) to understand tissue-specific regulation

• CLSTN3-APP interaction studies:

  • Use HRP-conjugated antibodies in co-immunoprecipitation experiments to detect CLSTN3-APP complexes in adipose tissue

  • Quantify changes in complex formation under different metabolic conditions

  • This approach can help elucidate the mechanism by which CLSTN3 increases APP accumulation in mitochondria, impairing adipose mitochondrial function

• Genetic variant analysis:

  • Apply HRP-conjugated CLSTN3 antibodies to quantify protein expression in adipose tissue samples from individuals with different rs7296261 genotypes

  • Correlate protein levels with metabolic parameters to validate the proposed mechanism linking the risk allele to increased CLSTN3 expression and obesity risk

How can I integrate CLSTN3 antibody, HRP conjugated, in studies examining the neuro-adipose junction?

CLSTN3's role in the neuro-adipose junction represents an exciting frontier in metabolic research. To incorporate HRP-conjugated CLSTN3 antibodies in this research area:

• Visualization of neuro-adipose interfaces:

  • Develop a dual-staining protocol using HRP-conjugated CLSTN3 antibody and neuronal markers (tyrosine hydroxylase for sympathetic neurons)

  • Implement DAB (3,3′-diaminobenzidine) and Vector SG (blue-gray) substrate systems for HRP to distinguish between the two staining patterns

  • This technique can reveal the spatial relationship between CLSTN3-expressing adipocytes and sympathetic nerve terminals

• Functional assessment of sympathetic innervation:

  • Use HRP-conjugated CLSTN3 antibodies to quantify changes in CLSTN3 expression following cold exposure or β-adrenergic stimulation

  • Correlate these changes with markers of sympathetic activity and thermogenic capacity

  • This approach can help establish the relationship between sympathetic stimulation, CLSTN3 expression, and thermogenic response

• Synaptogenic function analysis:

  • Examine the co-localization of CLSTN3 with synaptic markers at neuro-adipose junctions

  • Quantify CLSTN3 levels in relation to synaptic density parameters

  • This can provide insights into CLSTN3's potential role in mediating functional connections between neurons and adipocytes

• Lipolysis regulation studies:

  • Use HRP-conjugated CLSTN3 antibodies in conjunction with lipolysis assays to correlate CLSTN3 expression with catecholamine-stimulated lipolysis

  • Implement both in vivo and ex vivo approaches to comprehensively assess this relationship

  • This can help elucidate the mechanism by which CLSTN3 attenuates catecholamine-stimulated lipolysis

• Mitochondrial function correlation:

  • Combine immunohistochemistry using HRP-conjugated CLSTN3 antibodies with mitochondrial functional assays

  • Assess the relationship between CLSTN3 levels, APP accumulation in mitochondria, and parameters of mitochondrial respiration

  • This approach can provide mechanistic insights into how CLSTN3 impacts adipose tissue metabolism through mitochondrial pathways

What are the key considerations when using CLSTN3 antibody, HRP conjugated, across different model systems?

When applying HRP-conjugated CLSTN3 antibodies across different experimental models, consider these model-specific methodological adjustments:

For each model system, verification steps should include:

• Western blot confirmation of appropriate molecular weight (protein size varies by species and isoform)

• Positive control tissues with known high expression

• Negative controls using isotype-matched HRP-conjugated antibodies

How should I modify protocols when analyzing CLSTN3 expression in adipose tissues from obese versus lean subjects?

When comparing CLSTN3 expression in adipose tissues between obese and lean subjects, implement these methodological refinements: