CSPG5 Antibody

What is CSPG5 and why is it significant in neuroscience research?

CSPG5, also known as Neuroglycan C, is a 120-150 kDa type I transmembrane glycoprotein belonging to the neuregulin family of proteins. Its significance stems from its expression profile in nervous tissue, particularly in retinal ganglion cells, cerebellar Purkinje cells, and hippocampal neurons . CSPG5 functions as a growth and differentiation factor involved in neuritogenesis, with studies showing that its recombinant ectodomain enhances neurite outgrowth from rat neocortical neurons through phosphatidylinositol 3-kinase and protein kinase C signaling pathways . Additionally, CSPG5 serves as a novel component of midkine receptors, promoting cell attachment and process extension in oligodendroglial precursor-like cells . Its ability to act as a growth factor by directly binding ERbB3 tyrosine kinase and transactivating ErbB2 further emphasizes its importance in neural development and function . Recent investigations have also revealed CSPG5's involvement in pathological conditions such as steroid-induced cataracts, expanding its research significance beyond neuroscience .

What are the structural characteristics of human CSPG5?

Human CSPG5 is synthesized as a 566 amino acid precursor comprising multiple distinct domains: a 30 amino acid signal sequence, a 393 amino acid extracellular domain (ECD), a 21 amino acid transmembrane segment, and a 122 amino acid cytoplasmic region . The ECD contains a chondroitin sulfate (CS) attachment domain (amino acids 34-272), with CS attachment occurring at Ser117 . Additionally, the ECD features one EGF-like domain (amino acids 371-413), two potential sites for N-linked glycosylation, and twelve potential sites for O-linked glycosylation . Human CSPG5 exists in three splice variants: isoform 1 (the long form), isoform 2 (with deletion of amino acids 487-513), and isoform 3 (with an alternative start site at Met139 and the same deletion as isoform 2) . Post-translational modifications include phosphorylation, likely occurring at Ser249, and proteolysis that generates a 75 kDa soluble fragment . When comparing human and mouse CSPG5, there is 84% amino acid identity over residues 31-420 .

How do I select the appropriate CSPG5 antibody for my research?

Selection of the appropriate CSPG5 antibody should be guided by several key considerations related to your experimental design. First, determine the species specificity required—antibodies targeting human CSPG5 (such as AF5685) may not cross-react with mouse/rat CSPG5, while others (like AF5665) are specifically designed for mouse/rat detection . Second, consider the application method: different antibodies perform optimally in specific techniques such as Western blot, immunohistochemistry, or immunofluorescence. For example, the AF5685 antibody has demonstrated efficacy in Western blot analysis of SH-SY5Y human neuroblastoma cell lysates , while the AF5665 antibody has been validated for immunohistochemistry in mouse brain tissue .

Third, evaluate the antibody's target epitope—some antibodies recognize the extracellular domain (Val31-Gln420), while others target internal peptide regions . Fourth, consider the host species and clonality; options include sheep polyclonal (AF5685), goat polyclonal (AF5665), and rabbit polyclonal antibodies . Finally, review the validation data available for each antibody, including detection of specific bands at appropriate molecular weights (120-150 kDa for AF5685, 120 kDa for AF5665) and performance in relevant tissue types. Thorough evaluation of these parameters will ensure selection of an antibody that provides optimal specificity, sensitivity, and reproducibility for your specific research requirements.

What is the expected molecular weight range for CSPG5 detection?

The expected molecular weight range for CSPG5 detection varies based on post-translational modifications, particularly glycosylation status and chondroitin sulfate attachment. In Western blot analyses, CSPG5 typically appears as bands between 120-150 kDa when fully modified . Using the Human Neuroglycan C/CSPG5 Antibody (AF5685) under reducing conditions, specific bands were detected at approximately 120 and 150 kDa in SH-SY5Y human neuroblastoma cell lysates . The presence of two distinct bands likely represents different glycosylation states of the protein. In rat cerebellum tissue lysates, the Mouse/Rat Neuroglycan C/CSPG5 Antibody (AF5665) detected a specific band at approximately 120 kDa .

Interestingly, the CSPG5 Antibody from SAB Biotech reports an SDS-PAGE molecular weight of 60 kDa , which may represent a truncated or differentially processed form of the protein. This variation highlights the importance of understanding the specific isoform and post-translational modifications being targeted by each antibody. Depending on its expression profile, CSPG5 may exist as either a 120 kDa glycoprotein or a 150 kDa chondroitin sulfate proteoglycan . Additionally, proteolytic processing can generate smaller fragments, such as the 75 kDa soluble fragment mentioned in the literature .

How can I optimize Western blot conditions for CSPG5 detection?

Optimizing Western blot conditions for CSPG5 detection requires careful consideration of several technical parameters to account for the protein's complex post-translational modifications. Begin with sample preparation using appropriate lysis buffers that preserve protein integrity while efficiently extracting membrane-associated proteins. Based on published protocols, reducing conditions are recommended for CSPG5 detection . For SH-SY5Y human neuroblastoma cell lysates, Immunoblot Buffer Group 8 was successfully employed with the AF5685 antibody . Similarly, for rat cerebellum tissue, the same buffer group proved effective with the AF5665 antibody .

For gel electrophoresis, select an appropriate percentage acrylamide gel (10% has been reported as effective) to resolve the 120-150 kDa CSPG5 protein bands . During transfer to PVDF membranes, optimize transfer time and voltage to ensure complete transfer of high molecular weight proteins. For primary antibody incubation, a concentration of 1 μg/mL has been validated for both AF5685 and AF5665 antibodies . Following primary antibody incubation, use species-appropriate HRP-conjugated secondary antibodies such as Anti-Sheep IgG Secondary Antibody (HAF016) for AF5685 or Anti-Goat IgG Secondary Antibody (HAF019) for AF5665 .

When troubleshooting weak or absent signals, consider extending primary antibody incubation time or increasing concentration. If background is excessive, optimize blocking conditions or increase washing stringency. For difficult samples, enrichment techniques like immunoprecipitation prior to Western blotting may enhance detection sensitivity. Finally, when analyzing results, remember that CSPG5 typically appears as bands at approximately 120 and 150 kDa, with band intensity variations reflecting different glycosylation states and tissue-specific expression levels .

What are effective immunohistochemistry protocols for CSPG5 detection in brain tissue?

Effective immunohistochemistry protocols for CSPG5 detection in brain tissue must account for the protein's specific localization patterns and preservation of epitope accessibility. Based on validated methods, two primary approaches have demonstrated success: fluorescent immunohistochemistry and chromogenic detection. For fluorescent detection, immersion-fixed frozen sections of mouse brain (particularly cerebellum) have been effectively stained using the Mouse/Rat Neuroglycan C/CSPG5 Antibody (AF5665) at 10 μg/mL with overnight incubation at 4°C . The signal was visualized using NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (NL001), with DAPI counterstaining to provide nuclear context .

For chromogenic detection, the Anti-Goat HRP-DAB Cell & Tissue Staining Kit has been successfully employed with the AF5665 antibody at 15 μg/mL (overnight incubation at 4°C), followed by hematoxylin counterstaining . This approach is particularly valuable for developmental studies, as demonstrated in mouse embryo (15 d.p.c.) tissue sections . When working with human lens samples, a modified protocol has proven effective: extracted anterior capsules are placed flat for 7 days in poly-D-lysine coated 24-well plates, fixed in 4% paraformaldehyde, and blocked using 5% normal donkey serum for 2 hours prior to incubation with anti-CSPG5 (1:500, Abcam) at 4°C .

To optimize staining quality, consider antigen retrieval methods appropriate for fixed tissues. For analysis of immunofluorescence data, specialized software such as Olympus FLUOVIEW Fv10 Asw 4.2 enables quantitative assessment of staining patterns . Additional considerations include careful selection of control tissues (both positive and negative), optimization of antibody concentration through titration experiments, and extended washing steps to minimize background staining in highly autofluorescent tissues like brain.

How does CSPG5 contribute to steroid-induced ocular pathologies?

CSPG5 plays a significant role in steroid-induced ocular pathologies, particularly steroid-induced cataracts (SIC), through its involvement in epithelial-mesenchymal transition (EMT) of lens epithelial cells. Recent research has demonstrated that CSPG5 expression is markedly increased in the lens epithelium of SIC patients compared to controls . This upregulation appears to be mechanistically important, as experimental downregulation of CSPG5 suppresses dexamethasone-induced EMT-related protein expression and cellular motility in human lens epithelial (HLE-B3) cells .

The molecular pathway linking CSPG5 to steroid-induced pathology involves transcription factors EZH2 and B-Myb, which regulate CSPG5 expression in lens epithelial cells . When these transcription factors are disrupted, there is a corresponding downregulation of CSPG5, decreased dexamethasone-induced fibronectin expression, and reduced cell migration in HLE-B3 cells . This provides strong evidence that CSPG5 expression mediates steroid-induced effects on lens epithelial cells, particularly through the modulation of EMT processes.

The connection between CSPG5 and ocular pathologies extends beyond the lens. CSPG5 mRNA and protein expression have been detected in the retina and retinal pigment epithelium in models of Leber congenital amaurosis . Additionally, previous research has shown that nerve growth factor (NGF) remarkably suppresses increased activation of cell migration in lens epithelial cells exposed to high doses of dexamethasone, potentially through pathways involving CSPG5 . These findings collectively emphasize the potential of CSPG5 as a therapeutic target for the prevention and treatment of steroid-induced cataracts and possibly other ocular pathologies.

What signaling pathways involve CSPG5, and how can they be experimentally investigated?

CSPG5 participates in multiple signaling pathways that regulate neural development, cellular differentiation, and pathological processes. One primary pathway involves CSPG5's function as a growth factor through direct binding to ErbB3 tyrosine kinase and subsequent transactivation of ErbB2 . This ErbB receptor signaling cascade can be experimentally investigated using phospho-specific antibodies to detect receptor activation, co-immunoprecipitation to confirm protein-protein interactions, and selective inhibitors of downstream effectors to delineate pathway components.

A second significant pathway involves the enhancement of neurite outgrowth via phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) signaling . Research protocols to investigate this pathway include neurite outgrowth assays with primary neurons or neuroblastoma cell lines treated with recombinant CSPG5 ectodomain, in combination with specific inhibitors of PI3K (e.g., wortmannin, LY294002) or PKC (e.g., staurosporine, Gö6983). Visualization techniques such as immunofluorescence for cytoskeletal markers (β-III-tubulin, F-actin) and automated neurite tracing software can quantify morphological changes.

CSPG5 also functions as a component of midkine receptor complexes, promoting cell attachment and process extension in oligodendroglial precursor-like cells . This pathway can be analyzed through cell adhesion assays, migration studies, and competitive binding experiments with labeled midkine. Additionally, CSPG5's role in steroid-induced epithelial-mesenchymal transition (EMT) involves transcriptional regulation by EZH2 and B-Myb . This pathway can be investigated using chromatin immunoprecipitation (ChIP) to identify transcription factor binding sites, reporter gene assays to quantify promoter activity, and RNA interference to validate functional relationships.

For all these pathways, modern approaches such as phosphoproteomics, CRISPR-Cas9 gene editing, and single-cell transcriptomics can provide systems-level insights into CSPG5's signaling roles under physiological and pathological conditions.

What are the optimal storage and handling conditions for CSPG5 antibodies?

Optimal storage and handling of CSPG5 antibodies requires strict adherence to manufacturer-specified conditions to maintain antibody integrity and performance over time. For most commercial CSPG5 antibodies, including the human-specific AF5685 and mouse/rat-specific AF5665, long-term storage at -20°C to -70°C is recommended, with stability maintained for approximately 12 months from the date of receipt under these conditions . After reconstitution, these antibodies can be stored at 2-8°C under sterile conditions for up to one month, or at -20°C to -70°C for six months .

To minimize antibody degradation, it is critical to avoid repeated freeze-thaw cycles by aliquoting reconstituted antibodies into single-use volumes before freezing . For the CSPG5 Antibody from SAB Biotech, storage at -20°C is specified, with the antibody formulated in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol . The glycerol component serves as a cryoprotectant to maintain antibody stability during freeze-thaw cycles.

When handling antibodies for experimental procedures, maintain sterile technique to prevent microbial contamination. Allow refrigerated antibodies to equilibrate to room temperature before opening to prevent condensation, which can introduce contamination and accelerate degradation. For diluted working solutions, prepare fresh on the day of use whenever possible. If storage of diluted antibody is necessary, keep at 4°C for minimal time periods (typically less than one week) and include appropriate preservatives such as sodium azide (0.02-0.05%) to inhibit microbial growth. Finally, document lot numbers, receipt dates, and preparation details to monitor antibody performance over time and facilitate troubleshooting of experimental variability.

How can I validate the specificity of CSPG5 antibodies for my experimental system?

Validating the specificity of CSPG5 antibodies for your experimental system requires a multi-faceted approach to ensure reliable and reproducible results. Begin with positive and negative control samples: tissues or cell lines with known high expression (e.g., SH-SY5Y neuroblastoma cells, rat cerebellum tissue) versus those with minimal CSPG5 expression . For Western blot validation, confirm detection of bands at the expected molecular weights (120-150 kDa for full-length CSPG5) . The presence of multiple bands may reflect different glycosylation states or proteolytic processing rather than non-specific binding.

For immunohistochemistry validation, compare staining patterns with published CSPG5 localization data. In mouse brain (cerebellum), CSPG5 shows characteristic distribution patterns that can serve as positive controls . Cross-validation using multiple antibodies targeting different epitopes of CSPG5 provides strong evidence of specificity when staining patterns align. For instance, comparing results from the antibody targeting Val31-Gln420 (AF5685) with one recognizing an internal peptide (such as the SAB Biotech antibody) can confirm target specificity .

Genetic approaches offer the most stringent validation: siRNA knockdown of CSPG5 should reduce antibody signal proportionally to knockdown efficiency . Similarly, overexpression systems (transfected cell lines expressing tagged CSPG5 constructs) allow confirmation of antibody recognition through co-localization studies. Peptide competition assays, where pre-incubation of the antibody with its immunizing peptide blocks specific staining, provide another layer of validation. For antibodies used in therapeutic research, validation in disease-relevant contexts is crucial—for example, confirming differential expression in steroid-induced cataract patient samples versus controls, as demonstrated in recent studies .

What are the key considerations for designing CSPG5 functional studies?

Designing robust CSPG5 functional studies requires careful consideration of several experimental parameters to generate reliable and physiologically relevant data. First, select appropriate cellular models that endogenously express CSPG5 or can be manipulated to express it at physiological levels. Neuronal cell lines (SH-SY5Y), primary neurons, lens epithelial cells (HLE-B3), and oligodendroglial precursor cells have all demonstrated CSPG5 expression and are suitable for functional investigations . For in vivo studies, consider both developmental timing and regional specificity, as CSPG5 expression varies across brain regions and developmental stages .

Second, develop appropriate gain- and loss-of-function approaches. For overexpression studies, use expression vectors containing the full-length CSPG5 cDNA or specific domains (e.g., the EGF-like domain or chondroitin sulfate attachment domain) to dissect domain-specific functions . For knockdown studies, siRNA targeting CSPG5 has been successfully employed in HLE-B3 cells . CRISPR-Cas9 gene editing offers an alternative for complete knockout or precise modification of CSPG5 genomic loci.

Third, select functional readouts relevant to CSPG5's known biological roles. For neurite outgrowth studies, measure neurite length, branching complexity, and growth cone morphology in primary neuronal cultures treated with recombinant CSPG5 ectodomain . For EMT studies in lens epithelial cells, monitor expression of EMT markers (fibronectin, vimentin, α-SMA) and cell motility using wound healing or transwell migration assays . For signaling pathway investigations, assess activation of ErbB receptors, PI3K/AKT, and PKC pathways using phospho-specific antibodies .

Finally, include appropriate controls and consider potential compensation by related proteoglycans or EGF-family proteins. Time-course experiments are essential for capturing transient signaling events, while dose-response studies help establish physiological relevance. When interpreting results, consider CSPG5's multiple isoforms and post-translational modifications, which may have distinct functional properties in different cellular contexts.

What techniques are available for analyzing CSPG5 expression in clinical samples?

Analysis of CSPG5 expression in clinical samples requires techniques that account for limited sample availability while providing reliable quantitative or semi-quantitative data. Immunohistochemistry (IHC) offers an excellent approach for formalin-fixed paraffin-embedded (FFPE) tissues, allowing visualization of CSPG5 protein localization while preserving tissue architecture. For lens epithelial samples specifically, a validated protocol involves placing extracted anterior capsules flat for 7 days in poly-D-lysine coated plates, followed by fixation in 4% paraformaldehyde, blocking with 5% normal donkey serum, and incubation with anti-CSPG5 (1:500, Abcam) . Visualization can be achieved using fluorescently-labeled secondary antibodies and analyzed with specialized software such as Olympus FLUOVIEW Fv10 Asw 4.2 .

Western blot analysis provides semi-quantitative assessment of CSPG5 protein levels and can detect specific isoforms or post-translational modifications. For clinical samples, 15-20 μg of protein from tissue lysates separated on 10% polyacrylamide gels has proven effective . After transfer to nitrocellulose membranes and blocking with 5% non-fat dry milk, incubation with anti-CSPG5 antibodies (1:1000, from various suppliers) followed by appropriate secondary antibodies allows detection of specific bands . Protein concentration measurement using BCA protein assay kits ensures equal loading across samples .

Quantitative real-time PCR (qRT-PCR) offers a highly sensitive method for analyzing CSPG5 mRNA expression in minimal amounts of clinical material. This approach has successfully detected CSPG5 transcripts in lens, brain, and retinal tissues . For more comprehensive analysis, RNA-sequencing can provide CSPG5 expression data in the context of global transcriptome changes, while single-cell RNA-seq can reveal cell type-specific expression patterns in heterogeneous clinical samples. Newer technologies like spatial transcriptomics combine the advantages of expression quantification with histological context, particularly valuable for analyzing CSPG5 in complex tissues like brain or eye.

How do human and mouse/rat CSPG5 antibodies differ in specificity and applications?

Human and mouse/rat CSPG5 antibodies exhibit important differences in epitope recognition, species cross-reactivity, and optimized applications that researchers must consider when designing experiments. The Human Neuroglycan C/CSPG5 Antibody (AF5685) specifically targets the Val31-Gln420 region of human CSPG5 (accession # AAQ04776) and has been validated primarily for Western blot applications in human cell lines such as SH-SY5Y neuroblastoma . This antibody detects characteristic bands at approximately 120 and 150 kDa under reducing conditions . In contrast, the Mouse/Rat Neuroglycan C/CSPG5 Antibody (AF5665) targets the Val31-Gln420 region of mouse CSPG5 (accession # AAH55736) and has been validated for multiple applications including Western blot, immunohistochemistry, and immunofluorescence .

While the targeted regions (Val31-Gln420) appear similar, the 84% amino acid identity between human and mouse CSPG5 in this region results in sufficient differences to limit cross-reactivity . The Mouse/Rat antibody has demonstrated particular efficacy in immunohistochemical applications, with validated protocols for both fluorescent and chromogenic detection in mouse brain (cerebellum) and embryonic tissues . Western blot analysis with this antibody typically reveals a single specific band at approximately 120 kDa in rat cerebellum tissue, in contrast to the two bands often observed with the human-specific antibody .

For researchers working with human clinical samples, the rabbit polyclonal antibody from SAB Biotech provides an alternative option, recognizing an internal peptide derived from human CSPG5 . This antibody has been validated specifically for Western blot applications and detects endogenous levels of total CSPG5 protein . When selecting between these antibodies, researchers should consider not only species specificity but also the intended application, target epitope accessibility in their experimental system, and the antibody's validated detection of specific CSPG5 isoforms or post-translational modifications.

What are common challenges in CSPG5 antibody-based experiments and how can they be addressed?

CSPG5 antibody-based experiments present several challenges that researchers must anticipate and address to obtain reliable results. One primary challenge is the variable molecular weight detection due to post-translational modifications, particularly glycosylation and chondroitin sulfate attachment. CSPG5 can appear as either a 120 kDa glycoprotein or a 150 kDa chondroitin sulfate proteoglycan depending on its expression profile . To address this variability, researchers should run appropriate molecular weight markers and positive controls with known CSPG5 expression patterns. Additionally, enzymatic deglycosylation treatments (PNGase F for N-linked glycans or chondroitinase ABC for chondroitin sulfate chains) can help identify the core protein and confirm antibody specificity.

A second challenge involves optimization of extraction conditions for this membrane-associated proteoglycan. Insufficient solubilization may result in poor recovery from tissues or cells. Optimized lysis buffers containing appropriate detergents (such as those in Immunoblot Buffer Group 8, which has been validated for CSPG5 detection) can improve extraction efficiency . For particularly challenging samples, enrichment techniques such as subcellular fractionation or immunoprecipitation may enhance detection sensitivity.

Background staining in immunohistochemistry represents another common challenge, particularly in tissues with high autofluorescence like brain. Extended blocking steps (e.g., 2-hour incubation with 5% normal donkey serum), careful antibody titration, and thorough washing protocols can minimize non-specific binding . For fluorescent detection, inclusion of Sudan Black B in the protocol can reduce tissue autofluorescence.

Reproducibility issues may arise from lot-to-lot antibody variation. Maintaining detailed records of antibody lots, standardizing protocols, and including consistent positive and negative controls in each experiment helps monitor performance over time. Finally, when interpreting results across different experimental systems, consider that CSPG5 expression and processing may vary by cell type, developmental stage, and pathological state. Validation in each new experimental context is essential for confident data interpretation and meaningful cross-study comparisons.

What emerging technologies might enhance CSPG5 research in neurological and ophthalmological disorders?

Emerging technologies offer transformative potential for advancing CSPG5 research in both neurological and ophthalmological disorders. Single-cell RNA sequencing (scRNA-seq) technology allows unprecedented resolution of CSPG5 expression patterns in heterogeneous tissues like brain and retina, potentially revealing cell type-specific expression changes in pathological states that would be masked in bulk tissue analysis. When combined with spatial transcriptomics techniques such as Visium or MERFISH, researchers can map CSPG5 expression within the complex architectural context of neural tissues, providing insights into regional specialization and circuit-specific functions.

CRISPR-Cas9 gene editing represents another revolutionary approach for CSPG5 research. Beyond conventional knockout studies, CRISPR-based technologies now enable precise modification of specific domains (such as the EGF-like domain or chondroitin sulfate attachment sites) to dissect structure-function relationships. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems provide temporally controlled modulation of CSPG5 expression without permanent genetic alterations, valuable for studying developmental processes and acute interventions.

Advanced imaging technologies, including super-resolution microscopy techniques like STORM and PALM, offer nanoscale visualization of CSPG5 localization at synapses and growth cones, potentially revealing previously undetectable protein interactions and trafficking patterns. For in vivo imaging, genetically encoded reporters fused to CSPG5 enable real-time tracking of protein dynamics in living organisms, particularly valuable for understanding its role in neural development and response to injury.

In the therapeutic realm, antibody engineering technologies are producing more specific and functionally active antibodies. Beyond detection tools, engineered antibodies targeting specific CSPG5 epitopes could modulate its function in pathological contexts, such as steroid-induced cataracts where CSPG5 upregulation contributes to disease progression . Additionally, organoid technologies for brain and eye tissues provide physiologically relevant experimental systems that bridge the gap between cell culture and animal models, enabling investigation of CSPG5 function in three-dimensional tissue contexts that better recapitulate human development and disease.