SPTBN1 Antibody, HRP conjugated

What is SPTBN1 and what are its key biological functions?

SPTBN1 (spectrin, beta, non-erythrocytic 1) is a cytoskeletal protein with a molecular weight of approximately 275 kDa that functions as the non-erythrocytic form of β-spectrin . It plays crucial roles in multiple biological processes including cytoskeletal organization, protein trafficking, and cell signaling pathways. Recent research has identified SPTBN1 as a significant factor in several pathological conditions, including its tumor-suppressive role in renal clear cell carcinoma (ccRCC), where downregulation correlates with poorer clinical outcomes and increased disease progression . Additionally, SPTBN1 has been implicated in neurodevelopment, as pathogenic variants cause an autosomal dominant neurodevelopmental syndrome . Furthermore, SPTBN1 has been shown to prevent primary osteoporosis by modulating osteoblast function via the TGF-β/Smad3 and STAT1/Cxcl9 pathways .

What applications are SPTBN1 antibodies most commonly used for?

SPTBN1 antibodies are employed across multiple research applications with varying dilution requirements. Western blot (WB) applications typically use dilutions of 1:500-1:1000 for detecting SPTBN1 in tissue lysates from brain, kidney, and lung samples . Immunohistochemistry (IHC) applications use dilutions of 1:50-1:500 and have successfully detected SPTBN1 in human liver cancer tissue, lung tissue, kidney tissue, pancreas cancer tissue, and normal colon tissue . Immunoprecipitation (IP) protocols typically require 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Immunofluorescence (IF) applications have been documented in multiple publications, enabling visualization of SPTBN1's subcellular localization and interactions with binding partners . Additionally, ELISA applications have been validated for quantitative measurement of SPTBN1 in serum, plasma, tissue homogenates, and other biological fluids .

What are the optimal conditions for using SPTBN1 antibodies in Western blot applications?

For optimal Western blot results with SPTBN1 antibodies, researchers should implement specific methodological considerations due to SPTBN1's high molecular weight (275 kDa). Sample preparation should incorporate phosphatase inhibitors to preserve SPTBN1's phosphorylation state, particularly when studying its regulatory mechanisms. Use gradient gels (4-12% or 4-15%) to effectively resolve this large protein, with extended running times to ensure adequate separation. Transfer protocols should employ low-percentage methanol buffers (5-10%) with extended transfer times (overnight at 30V or 2-3 hours at 100V) using PVDF membranes rather than nitrocellulose for superior protein retention. Blocking should utilize 5% BSA rather than milk to prevent non-specific binding. The recommended antibody dilution range is 1:500-1:1000 in TBST with 1% BSA . For HRP-conjugated SPTBN1 antibodies, researchers can proceed directly to detection without secondary antibody incubation, using enhanced chemiluminescence substrates with exposure times adjusted based on expression levels. This approach has been validated for detecting SPTBN1 in mouse brain, kidney, and lung tissues, showing consistent results at the expected 275 kDa molecular weight .

How should researchers optimize SPTBN1 detection in immunohistochemistry?

Successful immunohistochemical detection of SPTBN1 requires careful optimization of several critical parameters. Antigen retrieval represents a crucial first step, with TE buffer at pH 9.0 demonstrating superior epitope exposure compared to citrate buffer (pH 6.0), though the latter may serve as an alternative for certain tissue types . Following retrieval, sections should be treated with peroxidase blocking solution (3% H₂O₂) for 10 minutes to neutralize endogenous peroxidase activity that could otherwise generate false positive signals. Primary antibody dilutions should range from 1:50 (for weakly expressing tissues) to 1:500 (for strongly expressing tissues), with overnight incubation at 4°C yielding optimal signal-to-noise ratios . For HRP-conjugated SPTBN1 antibodies, direct detection is possible following primary antibody incubation, while unconjugated antibodies require appropriate HRP-polymer detection systems. SPTBN1 antibodies have successfully visualized expression patterns in human liver cancer tissue, lung tissue, kidney tissue, pancreas cancer tissue, and normal colon, with differential expression observed between normal and neoplastic tissues . Researchers investigating SPTBN1's role in cancer progression should note that significantly downregulated SPTBN1 expression correlates with advanced clinicopathological characteristics in renal clear cell carcinoma .

What is the sandwich ELISA methodology for quantifying SPTBN1 in biological samples?

The sandwich ELISA methodology for SPTBN1 quantification employs a sequential multi-step protocol for precise detection in complex biological matrices. The procedure begins with antibody-coated 96-well plates containing pre-immobilized SPTBN1-specific capture antibodies . Sample preparation is critical, with serum and plasma typically diluted 1:2 to 1:10 in sample dilution buffer, while tissue homogenates require mechanical disruption followed by centrifugation to obtain clear supernatants. After adding 100 μL of standards (for generating calibration curves) and prepared samples to appropriate wells, plates undergo a 90-minute incubation at 37°C to allow SPTBN1 binding to the capture antibodies . Following washing steps, 100 μL of biotinylated anti-SPTBN1 detection antibody (diluted 1:100 in antibody dilution buffer) is added for a 60-minute incubation, which binds to the captured SPTBN1 molecules . After washing, 100 μL of HRP-Streptavidin conjugate (SABC, diluted 1:100) is added for 30 minutes to bind to the biotinylated antibodies . Following a final washing step, 90 μL of TMB substrate is added, producing a blue color proportional to SPTBN1 concentration, which turns yellow upon addition of acidic stop solution. The optical density is measured at 450 nm, with SPTBN1 concentrations calculated by comparison to the standard curve. This methodology demonstrates high precision with intra-assay CV <8% and inter-assay CV <10% .

How can SPTBN1 antibodies be utilized in investigating its role in cancer progression?

Advanced cancer research applications of SPTBN1 antibodies extend beyond basic protein detection to mechanistic investigations of tumor suppression pathways. Research has demonstrated that SPTBN1 functions as a tumor suppressor in renal clear cell carcinoma (ccRCC), with significant downregulation observed in ccRCC tissues compared to adjacent normal tissues . Multivariate Cox regression analyses have identified SPTBN1 as an independent predictive factor for patient survival (HR = 0.647, 95% CI = 0.489–0.854, P = 0.002) . Researchers investigating SPTBN1's mechanistic role in cancer progression should implement a multi-modal antibody application approach, combining immunohistochemistry for tissue expression patterns with immunoprecipitation to identify novel protein-protein interactions that mediate SPTBN1's tumor-suppressive functions. For functional validation studies, combining SPTBN1 knockdown or overexpression with proliferation assays (CCK-8, colony formation) and cell cycle analysis has effectively demonstrated that SPTBN1 retards G1/S progression and suppresses proliferation both in vitro and in xenograft models . Western blot analysis using SPTBN1 antibodies has revealed its regulatory relationship with GPT2, where SPTBN1 functions as an RNA-binding protein that regulates GPT2 mRNA stability by binding to its 3'-UTR regions . This comprehensive antibody-based approach has established SPTBN1 as a potential therapeutic target and prognostic biomarker in ccRCC.

How can researchers investigate SPTBN1's role in neurodevelopmental disorders?

Investigating SPTBN1's involvement in neurodevelopmental disorders requires specialized antibody applications focusing on neuronal cytoskeletal dynamics and protein interactions. Pathogenic SPTBN1 variants, particularly those affecting the CH domain, can impair neuronal function by altering βII-spectrin binding to or localization of cytoskeletal partners . Researchers can employ immunofluorescence microscopy with SPTBN1 antibodies to visualize protein localization in neuronal cultures, identifying abnormal cytosolic aggregates containing actin and α-spectrin that form with mutant variants . This approach revealed that CH domain variants cause GFP-βIISp aggregation in βII-SpKO cortical neurons, sequestering endogenous αII-spectrin and actin within these aggregates . Co-immunoprecipitation using SPTBN1 antibodies can identify altered protein interaction profiles of wildtype versus mutant SPTBN1, revealing disrupted binding to critical neuronal proteins. For in vivo studies, immunohistochemistry using SPTBN1 antibodies on brain sections from mouse models harboring SPTBN1 variants can visualize altered distribution patterns and potential developmental abnormalities. Western blot analysis comparing wildtype and mutant SPTBN1 expression can detect altered protein stability or post-translational modifications. These methodologies collectively enable researchers to characterize how SPTBN1 mutations contribute to the pathophysiology of neurodevelopmental disorders and potentially identify therapeutic targets for intervention.

What methodologies are effective for studying SPTBN1's role in bone biology and osteoporosis?

Investigating SPTBN1's role in bone biology requires specialized methodologies combining antibody applications with bone-specific analytical techniques. Research has established that SPTBN1 prevents primary osteoporosis by modulating osteoblast function, with its expression significantly downregulated in senile and postmenopausal osteoporosis mouse models . For in vivo studies, micro-CT analysis combined with immunohistochemical staining using SPTBN1 antibodies can correlate protein expression with bone density, trabecular bone parameters, and blood vessel formation . This approach has demonstrated that SPTBN1 silencing leads to decreased bone density, reduced trabecular bone volume, wider trabecular gaps, and decreased blood vessel formation in osteoporosis models . At the molecular level, Western blot analysis using SPTBN1 antibodies can monitor its regulatory effects on osteogenic markers including Runx2, Osterix (Osx), Osteocalcin (Ocn), and vascular endothelial growth factor (VEGF) . In vitro studies with mouse pre-osteoblast MC3T3-E1 cells have shown that SPTBN1 silencing inhibits cell growth and induces apoptosis while increasing expression of TGF-β, Cxcl9, and phosphorylation of STAT1 and Smad3 . Conversely, SPTBN1 overexpression reverses these effects, suggesting therapeutic potential. These methodologies collectively enable comprehensive investigation of SPTBN1's role in bone homeostasis from molecular signaling pathways to macroscopic bone parameters.

What strategies can address non-specific binding in SPTBN1 Western blot applications?

Non-specific binding in SPTBN1 Western blot applications presents significant challenges due to the protein's high molecular weight (275 kDa) and potential cross-reactivity with other spectrin family members. To address these issues, researchers should implement a systematic troubleshooting approach. First, utilize gradient gels (4-12%) to improve separation of high molecular weight proteins, ensuring adequate resolution of SPTBN1 from other large proteins. Implement stringent blocking protocols using 5% BSA in TBST rather than milk, as milk proteins can sometimes introduce artifacts when detecting cytoskeletal components . Optimize antibody dilution through titration experiments, starting with the recommended range of 1:500-1:1000 and adjusting based on signal-to-noise ratio . Include positive controls (mouse brain tissue lysate) and negative controls (tissues known to express minimal SPTBN1) in each experiment to confirm specificity . For problematic samples, consider pre-absorption of the antibody with recombinant SPTBN1 protein to remove any cross-reactive antibodies. If using HRP-conjugated SPTBN1 antibodies, ensure appropriate storage conditions to prevent enzymatic activity loss, and consider adding 0.05% sodium azide to antibody storage buffers for long-term stability (but remove before use, as azide inhibits HRP activity). Additionally, incorporating phosphatase inhibitors in lysis buffers is critical when studying SPTBN1's phosphorylation-dependent interactions and functions.

How can researchers optimize immunoprecipitation protocols for SPTBN1 interaction studies?

Optimizing immunoprecipitation (IP) protocols for SPTBN1 requires careful consideration of several parameters to effectively capture this large cytoskeletal protein and its interaction partners. Begin with cell lysis optimization, using non-denaturing buffers containing 1% NP-40 or 0.5% Triton X-100 supplemented with protease and phosphatase inhibitors to preserve protein-protein interactions. Pre-clearing lysates (2-3 mg total protein) with protein A/G beads for 1 hour at 4°C is essential to reduce non-specific binding . For antibody binding, use 0.5-4.0 μg of SPTBN1 antibody per 1.0-3.0 mg of total protein lysate, with overnight incubation at 4°C under gentle rotation to maintain antibody integrity while maximizing binding . When capturing SPTBN1-antibody complexes, pre-equilibrate protein A/G beads in lysis buffer before adding to the lysate-antibody mixture and incubate for 1-3 hours at 4°C. Washing steps are critical—use progressively stringent washing buffers (from low to moderate salt concentrations) to remove non-specific interactions while preserving genuine binding partners. For specific detection of interaction partners, elute complexes using non-reducing conditions when possible to maintain protein structure. This methodology has been successfully applied to detect SPTBN1 interactions in HEK-293 cells and can be adapted to investigate its role as an RNA-binding protein that regulates GPT2 mRNA stability by binding to its 3'-UTR regions .

What factors affect SPTBN1 antibody performance in sandwich ELISA applications?

Multiple factors can significantly impact SPTBN1 antibody performance in sandwich ELISA applications, requiring careful optimization for accurate quantification. Sample preparation represents a primary concern—serum and plasma samples require appropriate dilution (typically 1:2 to 1:10) in sample dilution buffer to minimize matrix effects, while tissue homogenates should undergo mechanical disruption followed by centrifugation to remove particulates that can interfere with antibody binding . Antibody pair selection is critical, as capture and detection antibodies must recognize distinct, non-overlapping epitopes on SPTBN1 to prevent competition. Incubation times and temperatures substantially affect assay kinetics—standard protocols recommend 90-minute sample incubation at 37°C, followed by 60-minute detection antibody incubation, and 30-minute HRP-Streptavidin conjugate incubation . Washing efficiency between steps is crucial for reducing background signal; insufficient washing results in false positives, while excessive washing can remove specifically bound antibodies, leading to signal reduction. For optimal sensitivity, ensure TMB substrate is freshly prepared and protected from light exposure prior to use. When troubleshooting poor standard curves, verify reagent integrity, particularly HRP-Streptavidin conjugate activity, which can deteriorate with improper storage. Following these optimization strategies enables researchers to achieve high assay precision with intra-assay CV <8% and inter-assay CV <10% .

What methodological approaches can identify novel SPTBN1 binding partners in different cellular contexts?

Identifying novel SPTBN1 binding partners across different cellular contexts requires implementing complementary methodological approaches that leverage antibody specificity while accounting for context-dependent interactions. Immunoprecipitation coupled with mass spectrometry (IP-MS) represents the gold standard approach, using 0.5-4.0 μg of SPTBN1 antibody per 1.0-3.0 mg of total protein lysate to capture SPTBN1 complexes . This methodology has revealed SPTBN1's interactions with cytoskeletal proteins in multiple cell types. For RNA-protein interactions, RNA immunoprecipitation (RIP) using SPTBN1 antibodies has demonstrated that SPTBN1 functions as an RNA-binding protein that can bind to the 3'-UTR regions of target mRNAs like GPT2 . This can be validated using dual-luciferase reporter assays comparing wild-type and mutated 3'-UTR regions of potential target mRNAs . Proximity-dependent biotin identification (BioID) offers complementary insights by fusing SPTBN1 to a biotin ligase that biotinylates proximal proteins, which can then be purified and identified by mass spectrometry. In neuronal contexts, co-immunoprecipitation studies have revealed SPTBN1's interactions with actin and α-spectrin, with CH domain variants causing cytosolic aggregates containing these binding partners . For validation of identified interactions, researchers should implement reciprocal co-immunoprecipitation, proximity ligation assays, and functional studies examining the effects of SPTBN1 knockdown/overexpression on binding partner localization and activity.

How should researchers design experiments to investigate SPTBN1's role in cell signaling pathways?

Designing experiments to elucidate SPTBN1's role in cell signaling pathways requires a comprehensive approach combining genetic manipulation, biochemical analysis, and functional assays. Researchers should begin with SPTBN1 knockdown and overexpression systems in relevant cell types—for osteoblast studies, MC3T3-E1 cells have proven effective in revealing SPTBN1's modulation of TGF-β/Smad3 and STAT1/Cxcl9 signaling pathways . For knockdown validation, Western blot analysis using SPTBN1 antibodies at 1:500-1:1000 dilution can confirm protein reduction . Following genetic manipulation, phosphorylation-specific Western blot analysis should assess activation states of pathway components (e.g., phosphorylated STAT1 and Smad3) . Quantitative RT-PCR analysis of downstream target genes provides complementary data on pathway activity. Functional consequences should be evaluated through context-appropriate assays—in osteoblasts, assessments of proliferation (using CCK-8 assay), apoptosis (flow cytometry), and differentiation (ALP activity, mineralization) . For in vivo validation, conditional knockout models with tissue-specific SPTBN1 deletion enable evaluation of pathway perturbations in physiological contexts. This approach has demonstrated that SPTBN1 silencing significantly increases TGF-β expression and the phosphorylation levels of STAT1 and Smad3 in MC3T3-E1 cells compared to control groups . Integration of these methodologies enables comprehensive characterization of SPTBN1's regulatory roles in diverse signaling networks across different cellular contexts.