CPLX2 Antibody

What is CPLX2 and what is its primary function?

CPLX2 (Complexin-2, also known as Complexin II or Synaphin-1) is a cytoplasmic protein of approximately 15 kDa that interacts with the SNARE complex to regulate vesicle fusion. It exhibits dual functionality in vesicle exocytosis processes. CPLX2 negatively regulates the formation of synaptic vesicle clustering at the active zone to the presynaptic membrane in postmitotic neurons. Simultaneously, it positively regulates a late step in exocytosis of various cytoplasmic vesicles, including synaptic vesicles and other secretory vesicles . This dual regulatory mechanism makes CPLX2 a critical component in controlled neurotransmitter release. Recent research has also uncovered CPLX2's involvement in mast cell exocytosis, suggesting broader functions beyond neuronal systems .

In which cell types is CPLX2 expressed?

CPLX2 shows a selective expression pattern across different cell types. It is predominantly expressed in neurons throughout the central nervous system, with documented expression in the dorsolateral prefrontal cortex (DLPFC) and superior temporal cortex (STC) . Interestingly, recent research has revealed that CPLX2 is also expressed in B lymphocytes but not in T lymphocytes, making this distinction important for immunological studies . When designing experiments, SH-SY5Y neuroblastoma cells serve as a reliable positive control for CPLX2 expression in neurological studies . The differential expression pattern of CPLX2 across immune cells provides valuable internal controls when studying its function in the immune system, with B cells serving as positive controls and T cells as negative controls.

What applications are CPLX2 antibodies validated for?

CPLX2 antibodies have been validated for multiple research applications, allowing comprehensive investigation of this protein across different experimental contexts:

• Western blot (WB): For detecting CPLX2 protein in lysates, with an expected band size of 15 kDa

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For localizing CPLX2 in tissue samples, particularly brain tissue

• Immunocytochemistry (ICC): For subcellular localization studies in cultured cells

• Flow cytometry (intracellular): For quantifying CPLX2 expression in cell populations

Different antibody formats may have specific application strengths. For example, rabbit recombinant monoclonal CPLX2 antibodies have been specifically validated for immunocytochemistry and flow cytometry applications , while rabbit polyclonal antibodies may be particularly effective for Western blot and immunohistochemistry applications . When selecting an antibody, researchers should carefully review the validation data for their specific application of interest.

What are appropriate controls for CPLX2 antibody validation?

Rigorous validation of CPLX2 antibodies requires multiple layers of controls to ensure specificity and reliability. For positive controls, recombinant human CPLX2 protein provides a definitive standard for Western blot applications . SH-SY5Y neuroblastoma cells, which express CPLX2 endogenously, serve as excellent cellular positive controls for immunocytochemistry and flow cytometry applications . Brain tissue sections, particularly from cerebral cortex and hippocampus, offer tissue-level positive controls for immunohistochemistry.

For negative controls, CPLX2 knockout tissues or cells provide the gold standard for antibody specificity validation . The differential expression between B cells (positive) and T cells (negative) offers a valuable internal control system for immunological studies . Additional controls should include primary antibody omission, isotype controls, and blocking peptide competition to distinguish specific from non-specific signals.

When validating antibodies against post-translational modifications, appropriate stimulation or inhibition of pathways known to modulate those modifications is essential, as gene expression knockdown alone would not indicate modification-specific binding . This comprehensive approach to controls ensures reliable interpretation of CPLX2 antibody-generated results.

How should samples be prepared for optimal CPLX2 detection?

Sample preparation critically influences CPLX2 detection quality. For immunocytochemistry applications, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization in PBS has been validated for optimal results . Blocking with 10% serum prior to antibody incubation reduces background signal. For maximal sensitivity, overnight incubation of the primary antibody at 4°C is recommended rather than shorter incubations at room temperature .

For Western blot applications, standard protein extraction methods using RIPA or NP-40 based lysis buffers with protease inhibitors are suitable. Given CPLX2's relatively small size (15 kDa), using appropriate percentage acrylamide gels (12-15%) will provide optimal resolution. For immunohistochemistry on paraffin-embedded sections, heat-induced epitope retrieval may be necessary to unmask epitopes after formalin fixation, with published protocols using concentrations around 20 μg/ml for human brain tissue .

For flow cytometry applications, thorough cell permeabilization is essential since CPLX2 is an intracellular protein. Following manufacturer-recommended fixation and permeabilization protocols, with attention to buffer composition and incubation times, will ensure consistent results across experiments.

How does CPLX2 expression differ between neuronal and immune cells?

CPLX2 exhibits distinct expression patterns and functions between neuronal and immune systems. In neurons, CPLX2 is highly expressed in presynaptic terminals where it regulates neurotransmitter release through SNARE complex interactions . It plays a critical role in synaptic plasticity, with CPLX2-deficient mice showing decreased post-tetanic potentiation and impaired long-term potentiation (LTP) induction .

In the immune system, CPLX2 expression is selectively present in B lymphocytes but notably absent in T lymphocytes . This distinctive expression pattern contrasts with other complexins and SNARE proteins, which show similar expression levels across both lymphocyte types. This selective expression correlates with CPLX2's functional role in regulating antibody secretion from B cells. The differential expression suggests tissue-specific adaptations of CPLX2 function: in neurons, it primarily regulates neurotransmitter release, while in B cells, it modulates antibody secretion processes.

When investigating CPLX2 across different systems, researchers should account for these tissue-specific expression patterns by using appropriate positive and negative controls. The selective expression in B cells but not T cells provides a valuable internal control system for immunological studies, allowing researchers to validate antibody specificity within the same experimental preparation.

How do CPLX2 knockout models affect immunoglobulin secretion?

CPLX2 knockout (KO) mouse models have revealed CPLX2's unexpected role in regulating immunoglobulin secretion. Studies show that CPLX2 KO mice exhibit significantly elevated serum levels of IgM compared to wild-type (WT) mice, while other immunoglobulin isotypes (IgG, IgA, IgE) remain at similar levels between genotypes . This selective effect on IgM is particularly notable because IgM, especially natural IgM antibodies, is constitutively secreted and plays important roles in preventing infection and maintaining homeostasis.

At the cellular level, splenic antibody-secreting cells (ASCs) from CPLX2 KO mice demonstrate enhanced spontaneous secretion of both IgM and IgG1 . This effect appears most prominent in splenic B cells rather than bone marrow or peritoneal cavity B cells, suggesting CPLX2 primarily regulates antibody secretion in specific B cell subpopulations, likely B-1 and marginal zone (MZ) B cells, which are major sources of natural antibodies .

These findings establish CPLX2 as a negative regulator of constitutive antibody secretion, particularly for natural IgM from splenic ASCs. The mechanism likely involves CPLX2's interaction with the SNARE complex in regulating vesicle fusion during antibody release. When designing experiments with CPLX2 KO models, researchers should measure multiple immunoglobulin isotypes and examine the effect across different lymphoid tissues to comprehensively capture CPLX2's regulatory impact on humoral immunity.

What are the differences between monoclonal and polyclonal CPLX2 antibodies for research applications?

Monoclonal and polyclonal CPLX2 antibodies offer distinct advantages for different research applications, with selection criteria depending on experimental goals:

Rabbit recombinant monoclonal CPLX2 antibodies have been successfully used for immunocytochemistry on 4% PFA fixed, 0.1% Triton X-100 permeabilized SH-SY5Y cells and for intracellular flow cytometry . Rabbit polyclonal CPLX2 antibodies have demonstrated effectiveness for Western blotting of recombinant CPLX2 protein and rat spinal cord lysate, as well as immunohistochemistry on formalin-fixed, paraffin-embedded human brain tissue .

When selecting between monoclonal and polyclonal CPLX2 antibodies, researchers should consider their specific experimental requirements, balancing the trade-offs between specificity, sensitivity, and application suitability.

How can researchers effectively study CPLX2's interaction with the SNARE complex?

Investigating CPLX2's interaction with the SNARE complex requires specialized approaches that preserve protein-protein interactions while providing meaningful functional insights. Co-immunoprecipitation (Co-IP) represents a foundational approach, using CPLX2 antibodies to pull down CPLX2 and its associated SNARE proteins (syntaxin, SNAP-25, synaptobrevin/VAMP). This technique requires gentle lysis conditions (e.g., NP-40 or digitonin-based buffers) to maintain native protein interactions .

For in situ analysis of these interactions, proximity ligation assays (PLA) offer high specificity and sensitivity by combining CPLX2 antibodies with antibodies against SNARE components. This technique not only confirms interactions but also enables quantification of interaction frequency in different cellular compartments or under various experimental conditions. Immunofluorescence colocalization studies using high-resolution microscopy (confocal, STED, or SIM) provide spatial information about CPLX2-SNARE interactions, though they require careful controls and quantification methods.

When designing these experiments, researchers should select antibodies raised against different species for multi-label experiments and include appropriate negative controls for each technique. Validation of antibody specificity in CPLX2 knockout/knockdown systems is essential to ensure reliable interpretation of results. To connect molecular interactions with function, these protein interaction studies should be integrated with functional readouts such as neurotransmitter release assays or antibody secretion measurements, potentially using CPLX2 mutants that disrupt specific SNARE interactions.

What methodologies exist for studying CPLX2 in B cell function and antibody secretion?

Recent discoveries about CPLX2's role in B cell function have necessitated specialized methodologies to investigate this previously unrecognized aspect of CPLX2 biology. For expression analysis, intracellular flow cytometry can quantify CPLX2 levels in different B cell subsets, while RT-qPCR and Western blotting provide complementary information on mRNA and protein expression in sorted B cell populations (B-1, marginal zone, follicular B cells) .

Antibody secretion assays represent the functional core of CPLX2 studies in B cells. ELISA measurement of spontaneous antibody secretion from cultured splenic cells, peritoneal cavity cells, or bone marrow cells allows quantification of multiple immunoglobulin isotypes (IgM, IgG, IgA, IgE) and IgG subclasses. This approach has revealed that CPLX2 knockout leads to enhanced spontaneous secretion of IgM and IgG1 specifically from splenic antibody-secreting cells . ELISPOT assays provide complementary information by enumerating antibody-secreting cells in different tissues.

Genetic approaches using CPLX2 knockout mouse models serve as powerful tools for studying systemic effects on humoral immunity. These can be complemented with B cell-specific CPLX2 knockout using Cre-lox systems or CRISPR/Cas9-mediated CPLX2 deletion in B cell lines for cell-type specific analysis. For mechanistic studies, live-cell imaging with fluorescent vesicle markers can visualize secretory vesicle dynamics, while total internal reflection fluorescence (TIRF) microscopy enables monitoring of vesicle fusion events at the plasma membrane.

How does post-translational modification affect CPLX2 function and detection?

Post-translational modifications (PTMs) of CPLX2 can significantly impact both its functional properties and experimental detection. CPLX2 can undergo various modifications, with phosphorylation being particularly important for regulating its interaction with the SNARE complex. Phosphorylation by protein kinase A (PKA) has been reported to modulate CPLX2 function, potentially altering its binding affinity for SNARE proteins and switching between inhibitory and facilitatory roles in exocytosis.

These modifications present important technical considerations for antibody-based detection. PTMs can alter epitope accessibility or antibody binding sites, potentially reducing detection efficiency with certain antibodies. Some antibodies may preferentially detect specific modified or unmodified forms of CPLX2, leading to incomplete representation of the total CPLX2 population in a sample. For comprehensive CPLX2 detection, researchers should use antibodies targeting different epitopes to ensure detection of various modified forms .

When studying CPLX2 phosphorylation specifically, inclusion of phosphatase inhibitors in lysis buffers is essential. For detailed mapping of modification sites, mass spectrometry approaches can identify specific PTM locations. Researchers should also be aware that recombinant CPLX2 proteins used as positive controls may lack important PTMs present in vivo, potentially affecting their behavior in comparison to native CPLX2. Understanding these modification-related considerations is particularly important when studying CPLX2 in dynamic processes like neurotransmission or B cell activation.

What experimental approaches are recommended for investigating CPLX2's role in neurological conditions?

Investigating CPLX2's potential involvement in neurological conditions requires integrated approaches spanning molecular, cellular, and systems levels. Expression studies in relevant patient samples offer a starting point, analyzing CPLX2 mRNA and protein levels in post-mortem brain tissue from patients with neurological disorders. These studies should focus on regions like the dorsolateral prefrontal cortex and superior temporal cortex, where CPLX2 expression alterations have been observed in schizophrenia patients .

Functional analyses using electrophysiological recordings provide critical insights into how CPLX2 affects synaptic transmission. CPLX2-deficient mice show decreased post-tetanic potentiation and impaired long-term potentiation (LTP) induction, particularly when subjected to environmental stress like maternal deprivation . This suggests examining both basal synaptic properties and various forms of synaptic plasticity in CPLX2-modified systems.

Genetic approaches using CPLX2 knockout or knockdown in neuronal models can be combined with environmental stressors to investigate gene-environment interactions. CPLX2 has been linked to working memory-related neural activity, suggesting cognitive testing focusing on working memory tasks in animal models . When designing such experiments, researchers should consider both pre- and post-synaptic effects of CPLX2 manipulation and examine multiple brain regions and cell types to capture the full spectrum of CPLX2's neurological functions.

What are the best practices for validating CPLX2 antibody specificity?

Rigorous validation of CPLX2 antibody specificity is essential for reliable research outcomes. A comprehensive validation approach combines multiple complementary strategies. Genetic validation using tissues or cells from CPLX2 knockout models provides the gold standard negative control for antibody specificity testing . The absence of signal in these samples strongly supports antibody specificity. Comparative analysis across multiple antibodies targeting different CPLX2 epitopes offers another level of validation; concordant results from diverse antibodies increase confidence in specificity.

Biological validation leverages known expression patterns, such as CPLX2's presence in B cells but absence in T cells . This differential expression provides a natural internal control system for evaluating antibody specificity within the same experimental preparation. Technical controls, including primary antibody omission, isotype controls, and pre-adsorption with recombinant CPLX2 protein, help distinguish specific from non-specific signals.

Application-specific validation is also crucial. For Western blot, confirming the expected 15 kDa band size and using recombinant CPLX2 protein as a positive control establishes specificity . For immunostaining, demonstration of the expected subcellular localization pattern provides additional validation. For flow cytometry, using appropriate gating strategies and comparing staining in positive and negative cell populations ensures reliable detection . This multi-layered validation approach maximizes confidence in the specificity and reliability of CPLX2 antibody-based experiments.