CLPX1 Antibody

What is CPLX1 and why is it significant in neuroscience research?

Complexin 1 (CPLX1) is a member of the complexin/synaphin gene family, comprising cytosolic proteins that play crucial roles in synaptic vesicle exocytosis. These proteins are highly enriched in neurons where they colocalize with key synaptic proteins including syntaxin and SNAP-25. CPLX1 exhibits weak binding to syntaxin when isolated but demonstrates significant interactions within the complete SNARE complex formation . The protein's calculated molecular weight is approximately 15 kDa, though it typically appears between 15-20 kDa on Western blots due to post-translational modifications . CPLX1 is encoded by the CPLX1 gene (Gene ID: 10815) and has been extensively studied for its involvement in neurotransmitter release and synaptic plasticity.

The significance of CPLX1 in neuroscience research stems from its critical involvement in regulating synaptic transmission, which underlies all brain functions from basic sensory processing to complex cognitive operations. Dysregulation of CPLX1 has been implicated in various neurological disorders, making it an important target for investigating disease mechanisms. Understanding CPLX1 function provides insights into fundamental neurobiological processes including vesicle fusion, neurotransmitter release, and synaptic efficacy modulation. Researchers typically use CPLX1 antibodies to study its expression patterns, subcellular localization, protein interactions, and functional roles in normal and pathological states.

What experimental applications are validated for CPLX1 antibody?

CPLX1 antibody has been validated for multiple experimental applications with specific optimization parameters for each technique. Western blotting (WB) is the most extensively validated application, with published literature supporting its use at dilutions ranging from 1:2000 to 1:10000 . For immunohistochemistry (IHC) applications, CPLX1 antibody has been validated at dilutions of 1:500 to 1:2000, with positive detection specifically confirmed in mouse brain tissue . Researchers should note that antigen retrieval conditions impact staining quality, with TE buffer at pH 9.0 being the recommended protocol, though citrate buffer at pH 6.0 has been described as an alternative option .

Immunofluorescence (IF) and immunocytochemistry (ICC) applications have been validated at dilutions ranging from 1:200 to 1:800, with positive detection confirmed in SH-SY5Y cells . Additionally, CPLX1 antibody has been validated for ELISA applications, though specific dilution recommendations vary by experimental setup. It is important to note that optimal antibody dilutions should be determined empirically for each specific experimental system, as tissue processing methods, detection systems, and sample types can significantly influence antibody performance. The antibody has been documented in at least 6 publications for Western blot applications, 1 publication for IHC, and 1 publication for IF, demonstrating its utility across multiple research contexts .

What species reactivity can be expected with CPLX1 antibody?

CPLX1 antibody (10246-2-AP) has been experimentally validated to react with human, mouse, and rat CPLX1 proteins . This cross-species reactivity makes it particularly valuable for comparative studies and translational research that bridge findings between animal models and human applications. The antibody has been specifically tested and confirmed to detect CPLX1 in mouse brain tissue, rat brain tissue, and human cell lines including SH-SY5Y neuroblastoma cells . This multi-species reactivity provides researchers with flexibility in experimental design and facilitates cross-validation of findings across different model systems.

In addition to the experimentally validated reactivity, the antibody has been cited in published literature for applications involving human, mouse, and macaque samples . When working with species not explicitly listed in the validated reactivity profile, researchers should perform preliminary validation experiments to confirm antibody performance. The antibody's immunogen design (CPLX1 fusion protein Ag0387) likely contributes to its cross-species reactivity by targeting conserved epitopes within the CPLX1 protein sequence. Sequence homology analysis between target species can help predict potential cross-reactivity, though empirical validation remains the gold standard for confirming antibody performance in untested species.

What are the recommended storage and handling protocols for CPLX1 antibody?

Optimal storage of CPLX1 antibody (10246-2-AP) requires maintaining the reagent at -20°C, where it remains stable for one year after shipment when properly handled . The antibody is supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles . Importantly, aliquoting is unnecessary for -20°C storage with this specific formulation, which simplifies laboratory workflows and minimizes handling-related degradation. The 20µl size preparations contain 0.1% BSA as an additional stabilizing agent to protect antibody integrity .

When working with the antibody, researchers should avoid prolonged exposure to room temperature and minimize freeze-thaw cycles to preserve antibody activity. Always centrifuge the antibody vial briefly before opening to collect the liquid at the bottom of the tube and ensure accurate pipetting. When diluting the antibody for experimental applications, use fresh buffers free of microbial contamination and consider adding protein carriers (such as BSA or normal serum) to diluted working solutions to prevent non-specific adsorption to plastic surfaces. If any precipitate is observed in the antibody solution, gentle warming and mixing can usually restore homogeneity without compromising antibody performance. Researchers should always document lot numbers and maintain records of antibody performance across different experimental conditions to track potential lot-to-lot variations.

How can specificity and sensitivity of CPLX1 antibody be validated in experimental protocols?

Validation of CPLX1 antibody specificity requires implementation of multiple complementary approaches to establish confidence in experimental findings. The gold standard approach involves comparing antibody reactivity in wild-type tissues against CPLX1 knockout models or CPLX1-depleted samples using RNAi or CRISPR-based approaches. Researchers should observe the expected 15-20 kDa band in Western blots of brain tissue samples, with subsequent absence or significant reduction of signal in knockout or knockdown conditions . Peptide competition assays provide an additional validation method, where pre-incubation of the antibody with the immunizing peptide should abolish specific staining in all applications, confirming epitope-specific binding.

For immunohistochemistry and immunofluorescence applications, anatomical distribution patterns of CPLX1 staining should be carefully compared with known expression patterns from literature and validated with orthogonal methods such as in situ hybridization. Cross-validation using different antibodies targeting distinct epitopes of CPLX1 provides strong evidence for specificity when concordant staining patterns are observed. Sensitivity validation requires careful titration experiments across a range of antibody concentrations (1:200 to 1:10000, depending on application) to determine the optimal signal-to-noise ratio . Quantitative Western blot analysis using recombinant CPLX1 protein standards can establish the lower limits of detection and linearity range for quantitative applications. Researchers should always include positive controls (mouse or rat brain tissue) and negative controls (tissues known not to express CPLX1) to evaluate both specificity and sensitivity in each experimental run .

What methodological considerations are critical when using CPLX1 antibody across different experimental techniques?

For Western blotting applications, sample preparation significantly impacts CPLX1 antibody performance. Researchers should use fresh tissue extracts or properly stored frozen samples, with brain tissue yielding the strongest and most specific signals . Complete protein denaturation is essential, typically achieved with SDS and reducing agents combined with heating at 95°C for 5 minutes. Transfer efficiency should be optimized for low molecular weight proteins (15-20 kDa range), typically using PVDF membranes with 0.2μm pore size rather than 0.45μm. Blocking conditions require careful optimization, with 5% non-fat dry milk in TBST typically providing good results, though for phospho-specific applications, BSA-based blocking may be preferable.

For immunohistochemistry, tissue fixation methodology critically influences epitope accessibility and antibody performance. Paraformaldehyde fixation (4%) for 24-48 hours followed by paraffin embedding represents the standard approach, with antigen retrieval being absolutely essential. TE buffer at pH 9.0 is specifically recommended for CPLX1 antibody, though citrate buffer at pH 6.0 provides an alternative . For immunofluorescence applications in cultured cells, fixation with 4% paraformaldehyde for 15-20 minutes at room temperature, followed by permeabilization with 0.1-0.3% Triton X-100, typically yields optimal results. Signal amplification strategies such as tyramide signal amplification may be beneficial for detecting low abundance CPLX1 in certain cell types or subcellular compartments. In multiplex immunofluorescence applications, careful selection of compatible primary antibodies from different host species is essential to avoid cross-reactivity with secondary antibodies. Confocal microscopy with appropriate controls for spectral overlap is recommended for colocalization studies involving CPLX1 and other synaptic proteins.

What are the optimal dilution protocols for CPLX1 antibody in different applications?

The following table summarizes the recommended dilution ranges for CPLX1 antibody (10246-2-AP) across various experimental applications:

When establishing optimal dilution protocols for a new experimental system, researchers should perform a systematic titration series across the recommended dilution range . For Western blotting, start with a moderate dilution (1:5000) and adjust based on signal intensity and background levels. For immunohistochemistry applications, antigen retrieval conditions significantly impact optimal antibody concentration, with more aggressive retrieval methods potentially requiring higher dilutions to prevent background staining . The optimal antibody concentration often represents a balance between sensitivity (ability to detect low abundance targets) and specificity (minimizing non-specific background).

Sample type also influences optimal dilution protocols, with endogenous expression levels varying significantly across tissue types and cell lines. Brain tissue typically shows strong CPLX1 expression and may allow for higher antibody dilutions, while tissues with lower expression might require more concentrated antibody solutions . Incubation conditions, including temperature and duration, interact with antibody concentration to determine staining outcomes. For Western blotting, overnight incubation at 4°C allows for higher dilutions compared to shorter room temperature incubations. For immunohistochemistry and immunofluorescence, longer incubation periods (overnight at 4°C) with more dilute antibody often produces better signal-to-noise ratios than shorter incubations with more concentrated antibody. Researchers should systematically document optimized conditions for each experimental system to ensure reproducibility.

How does CPLX1 antibody performance compare across different tissue types and experimental conditions?

CPLX1 antibody performance exhibits significant tissue-dependent variation corresponding to the known expression patterns of the target protein. The strongest and most specific signals are consistently observed in neural tissues, with mouse and rat brain samples showing robust detection in Western blotting applications . Within brain tissue, region-specific expression patterns can be observed, with enrichment in areas with high synaptic density. Non-neural tissues generally show minimal to no CPLX1 detection, consistent with its neuronal-enriched expression pattern. Cell line models show variable CPLX1 expression, with neuroblastoma lines such as SH-SY5Y demonstrating detectable levels suitable for immunofluorescence applications .

Experimental conditions significantly impact antibody performance across different applications. For Western blotting, sample preparation methodology critically influences results, with fresh or properly flash-frozen tissues preserved with protease inhibitors yielding optimal results. Detection sensitivity can be enhanced through chemiluminescent substrates of varying sensitivities, with modern high-sensitivity ECL substrates potentially allowing for higher antibody dilutions. For immunohistochemistry applications, fixation duration and conditions represent critical variables, with overfixation potentially masking epitopes and reducing antibody accessibility. The choice between chromogenic and fluorescent detection systems introduces another variable, with fluorescence-based methods generally offering higher sensitivity and better signal-to-noise ratios for CPLX1 detection.

Temperature and timing conditions during antibody incubation steps significantly impact performance across all applications. For Western blotting, overnight incubation at 4°C typically produces cleaner results compared to shorter room temperature incubations. In IHC and IF applications, the microdomain environment of the tissue section influences antibody penetration and binding kinetics. Primary antibody incubation for these applications benefits from longer incubation periods (overnight at 4°C) with gentle agitation to ensure even antibody distribution. Researchers should systematically optimize and document ideal conditions for each specific tissue type and application to ensure reproducible results.

What controls should be included when using CPLX1 antibody in various experimental setups?

Implementing comprehensive controls is essential for establishing the validity of experimental findings with CPLX1 antibody. For Western blotting applications, positive controls should include mouse or rat brain tissue lysates, which consistently demonstrate strong CPLX1 expression with the expected 15-20 kDa band pattern . Negative controls should include tissues or cell lines known not to express CPLX1, such as non-neuronal tissues. Loading controls using housekeeping proteins (β-actin, GAPDH, or tubulin) are essential for normalization and quantitative comparisons. When feasible, CPLX1 knockout or knockdown samples provide the gold standard negative control to verify antibody specificity.

For immunohistochemistry and immunofluorescence applications, technical negative controls should include primary antibody omission (to assess non-specific binding of secondary reagents) and isotype controls (using non-immune rabbit IgG at equivalent concentration) . Biological controls should include brain tissue sections from CPLX1 knockout animals when available, or tissues known to lack CPLX1 expression. Absorption controls, where the primary antibody is pre-incubated with the immunizing peptide before application to the tissue, provide additional evidence for binding specificity. For immunofluorescence applications, single-color controls are essential when performing multi-label experiments to assess spectral overlap and establish appropriate compensation settings for confocal microscopy.

How can researchers effectively troubleshoot inconsistent results with CPLX1 antibody?

Inconsistent results with CPLX1 antibody can stem from multiple sources that require systematic investigation. Sample quality issues frequently underlie variable performance, particularly in Western blotting applications. Researchers should verify protein integrity through Ponceau S staining of membranes prior to antibody incubation and ensure complete protein denaturation for accurate CPLX1 detection . Degraded samples often show smeared bands or aberrant molecular weight patterns. For tissue-based applications, fixation variables including duration, fixative composition, and post-fixation processing can dramatically affect epitope preservation and accessibility. Standardizing fixation protocols across experimental groups is essential for consistent results.

Antibody handling represents another critical variable. Repeated freeze-thaw cycles can degrade antibody function over time, leading to diminished signal intensity. Working aliquots for frequent use can help preserve the master stock, though for CPLX1 antibody (10246-2-AP), aliquoting is not required for -20°C storage according to manufacturer guidelines . Antibody dilution preparation timing can impact results, with freshly prepared working solutions generally providing more consistent performance compared to stored dilutions. Buffer composition, particularly pH and ionic strength, influences antibody-epitope interactions. For CPLX1 antibody, PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 represents the optimal storage buffer, while application-specific buffers require careful optimization .

Technical variables in detection systems frequently contribute to inconsistent results. For Western blotting, transfer efficiency for low molecular weight proteins like CPLX1 (15-20 kDa) requires optimization, with methanol concentration in transfer buffer and transfer duration being critical parameters . For immunohistochemistry, antigen retrieval conditions significantly impact epitope accessibility, with TE buffer at pH 9.0 specifically recommended for CPLX1 antibody . Detection reagent quality, including secondary antibody specificity and substrate freshness, must be systematically evaluated when troubleshooting. Instrument variables, such as microscope settings for fluorescence detection or imager settings for chemiluminescent detection, should be standardized across experiments. When inconsistent results persist despite addressing these variables, comparison with alternative CPLX1 antibodies targeting different epitopes can help determine whether the issue is antibody-specific or related to the biological system under investigation.

How can CPLX1 antibody be effectively used in multiplex immunoassays?

Multiplex immunoassays involving CPLX1 antibody require careful optimization to achieve reliable co-detection with other markers of interest. For immunofluorescence applications, successful multiplex staining depends on selecting primary antibodies from different host species to enable specific secondary antibody detection without cross-reactivity. Since CPLX1 antibody (10246-2-AP) is rabbit-derived, companion antibodies should ideally be from mouse, goat, or chicken hosts . Sequential staining protocols can be employed when using multiple rabbit antibodies, with complete blocking between rounds using anti-rabbit Fab fragments or microwave treatment to prevent cross-detection.

Spectral considerations are paramount in multiplex fluorescence applications. CPLX1 typically localizes to synaptic regions, which may require high-resolution imaging techniques such as confocal or super-resolution microscopy for accurate co-localization analysis with other synaptic proteins. Fluorophore selection should account for potential spectral overlap, expression level differences between targets, and subcellular localization patterns. For targets with vastly different expression levels, fluorophore brightness should be matched accordingly, with brighter fluorophores assigned to lower abundance targets. When analyzing CPLX1 colocalization with other synaptic proteins, appropriate controls for random overlap must be implemented, particularly in synapse-dense regions where spatial proximity may not indicate true biological interaction.

For multiplexed Western blotting applications, several approaches can be employed. Sequential probing with stripping between antibody applications works well when target proteins have distinct molecular weights, as is often the case when examining CPLX1 (15-20 kDa) alongside larger synaptic proteins . Alternatively, multiplexed detection using spectrally distinct fluorescent secondary antibodies allows simultaneous visualization of multiple targets without stripping. This approach requires careful validation to ensure that signal detection falls within the linear range for each target. For protein interaction studies, co-immunoprecipitation using CPLX1 antibody at a 1:50 dilution can be performed, followed by Western blotting for interaction partners . When designing multiplexed experiments, researchers should consider the biological relationships between targets and interpret colocalization or co-expression patterns within the appropriate physiological context of synaptic function.

How should researchers interpret varying band patterns when using CPLX1 antibody in Western blotting?

Sample preparation conditions significantly influence band patterns. Insufficient denaturation often results in higher molecular weight aggregates or incomplete migration patterns. Conversely, excessive heating or harsh detergent conditions may cause protein degradation, resulting in lower molecular weight fragments. Sample degradation during storage or processing can produce multiple bands below the expected molecular weight range. When interpreting CPLX1 blots from different species, researchers should account for potential species-specific differences in protein size due to sequence variations, though human, mouse, and rat CPLX1 show high sequence homology and similar molecular weights .

What considerations should be made when quantifying CPLX1 expression levels?

Accurate quantification of CPLX1 expression requires careful attention to methodological details across different experimental platforms. For Western blot quantification, researchers should verify that detection falls within the linear range of both the antibody binding and detection system response curves. Standard curves using recombinant CPLX1 protein can help establish this range. Signal normalization strategies should account for potential variability in loading and transfer efficiency, with total protein normalization (using stain-free technology or Ponceau S staining) often providing more reliable normalization than single housekeeping proteins, which may themselves be regulated under experimental conditions. When analyzing multiple immunoreactive bands, researchers should establish consistent rules for which bands to include in quantification based on validated molecular weight patterns .

For immunohistochemistry and immunofluorescence quantification, standardized image acquisition parameters are essential for comparative analyses. Fixed exposure settings, consistent threshold determination methods, and standardized region-of-interest selection criteria help minimize technical variability. Background subtraction methodology should be consistently applied across all samples and explicitly documented. For CPLX1 synaptic localization studies, puncta analysis requires careful consideration of size, intensity, and density parameters, which should be established based on known synaptic dimensions and validated with co-localization studies using established synaptic markers. Z-stack acquisition and analysis may be necessary to accurately capture the three-dimensional distribution of synaptic CPLX1 immunoreactivity.

Statistical analysis of CPLX1 quantification data requires appropriate consideration of data distribution characteristics. Normality testing should precede selection of parametric or non-parametric statistical approaches. For experiments with multiple variables or repeated measures, appropriate statistical models such as ANOVA with post-hoc tests or mixed-effects models should be employed. Sample size determination through power analysis helps ensure sufficient statistical power to detect biologically meaningful differences in CPLX1 expression. Effect size reporting alongside significance values provides important context for interpreting the biological relevance of observed changes. For complex datasets exploring CPLX1 expression across multiple brain regions or experimental conditions, multivariate analysis approaches may be necessary to identify patterns not evident in univariate analyses. Transparent reporting of all quantification parameters, image processing steps, and statistical methods is essential for reproducibility in CPLX1 expression studies.

What are the key recommendations for optimizing CPLX1 antibody usage in neuroscience research?

Successful implementation of CPLX1 antibody in neuroscience research requires systematic optimization across multiple experimental dimensions. For Western blotting applications, researchers should prioritize sample preparation quality, with fresh or properly preserved neural tissue providing the most reliable results. The recommended dilution range of 1:2000 to 1:10000 should be systematically explored for each specific experimental system, with 1:5000 serving as a reasonable starting point for most applications . Complete protein denaturation and efficient transfer of low molecular weight proteins (using appropriate membrane type and transfer conditions) are critical for accurate detection of the 15-20 kDa CPLX1 protein . Comprehensive controls, including positive controls (brain tissue), negative controls, and loading controls, should be implemented in every experimental run.

For immunohistochemistry and immunofluorescence applications, antigen retrieval methodology represents the most critical variable for successful CPLX1 detection. The specifically recommended TE buffer at pH 9.0 should be used as the first-line approach, with citrate buffer at pH 6.0 serving as an alternative if needed . Antibody dilutions should be titrated within the recommended ranges (1:500-1:2000 for IHC; 1:200-1:800 for IF/ICC) for each specific tissue type and fixation method . Verification of CPLX1 detection patterns should include comparison with published expression patterns and co-localization with known synaptic markers. For all applications, researchers should maintain detailed records of optimization experiments and established protocols to ensure reproducibility within and across research groups.

Cross-validation using orthogonal approaches significantly strengthens research findings. When feasible, researchers should verify key CPLX1 expression findings using multiple detection methods (e.g., Western blotting, immunohistochemistry, and in situ hybridization) and multiple antibodies targeting different CPLX1 epitopes. For interaction studies, co-immunoprecipitation results should be complemented with proximity ligation assays or FRET-based approaches to confirm protein-protein interactions in their native cellular context. Integration of CPLX1 protein studies with mRNA expression analysis provides additional validation and may reveal post-transcriptional regulation mechanisms. By implementing these comprehensive optimization and validation strategies, researchers can maximize the reliability and impact of their CPLX1-focused investigations in neuroscience research.

How can researchers ensure reproducibility in CPLX1 antibody-based experimental findings?

Ensuring reproducibility in CPLX1 antibody-based research requires meticulous attention to methodological details and comprehensive documentation practices. Researchers should maintain detailed records of antibody specifications including catalog number (10246-2-AP), lot number, host species (rabbit), clonality (polyclonal), and immunogen information (CPLX1 fusion protein Ag0387) . These details should be explicitly reported in methods sections of publications to facilitate replication. Antibody validation data, including specificity controls and application-specific optimization experiments, should be systematically documented and made available through supplementary materials or repositories when publishing research findings.

Experimental protocols should be documented with sufficient detail to enable reproduction by other laboratories. This includes specific buffer compositions, incubation times and temperatures, equipment settings, and image acquisition parameters. For quantitative analyses, all data processing steps should be explicitly described, including image processing operations, thresholding methods, region-of-interest selection criteria, and statistical analysis approaches. When possible, automated or semi-automated analysis workflows should be implemented to reduce subjective bias in data interpretation. Research using CPLX1 antibody should adhere to field-specific reporting guidelines, such as those outlined by the ARRIVE guidelines for animal studies or appropriate guidelines for clinical specimen research.