SPTBN1 Antibody, FITC conjugated

What is SPTBN1 and what biological functions does it serve in normal and pathological conditions?

SPTBN1 (spectrin, beta, non-erythrocytic 1) is a non-erythrocytic form of β-spectrin, also known as β-fodrin. It belongs to the superfamily of F-actin cross-linking proteins with a calculated molecular weight of 275 kDa . SPTBN1 plays crucial roles in multiple biological processes and disease states.

Research has demonstrated that SPTBN1 suppresses tumor progression through multiple mechanisms, including regulation of Wnt signaling through its control of Kallistatin (a Wnt inhibitor), modulation of E-cadherin expression, and influencing stem cell phenotypes in liver tissue . Beyond liver cancer, SPTBN1 has been implicated in attenuating rheumatoid arthritis synovial cell proliferation, invasion, migration, and inflammatory responses by binding to PIK3R2 and suppressing the PI3K/AKT signaling pathway .

What is the difference between unconjugated SPTBN1 antibodies and FITC-conjugated versions?

Unconjugated SPTBN1 antibodies, such as the 19722-1-AP product, are supplied without any fluorescent or enzymatic labels attached to the antibody molecule . These require secondary antibody detection systems in applications like Western blotting, immunohistochemistry, and immunofluorescence.

FITC (fluorescein isothiocyanate)-conjugated SPTBN1 antibodies, in contrast, have the fluorescent dye FITC directly attached to the antibody molecule. This direct conjugation offers several methodological advantages:

• It eliminates the need for secondary antibodies in fluorescence-based applications

• Reduces background by decreasing the number of steps in staining protocols

• Enables direct visualization in applications such as flow cytometry, immunofluorescence microscopy, and fluorescence-activated cell sorting (FACS)

• Allows for multiplexing with other antibodies conjugated to different fluorophores

FITC-conjugated antibodies emit green fluorescence at approximately 525 nm when excited with light at 490 nm, making them compatible with standard fluorescence microscopy filter sets and flow cytometry instrumentation.

What are the recommended applications and optimal dilutions for SPTBN1 antibodies?

Based on available research data, SPTBN1 antibodies have been validated for multiple applications with specific recommended dilution ranges for optimal results:

For FITC-conjugated SPTBN1 antibodies specifically, dilutions may need to be optimized depending on the fluorescence intensity of the conjugate and the application. Generally, fluorophore-conjugated antibodies may require different dilutions than their unconjugated counterparts due to potential effects of conjugation on antibody binding efficiency and fluorescence yield.

Note that these recommendations serve as starting points, and each experimental system requires titration for optimal results .

In which tissues and cell types has SPTBN1 been successfully detected?

SPTBN1 has been successfully detected in multiple tissues and cell types using appropriate antibodies:

Research has demonstrated reactivity with human, mouse, and rat samples, making SPTBN1 antibodies versatile tools for comparative studies across species . The broad tissue distribution reflects SPTBN1's importance in maintaining cellular architecture and signaling in multiple organ systems.

In cancer research specifically, SPTBN1 expression has been studied in hepatocellular carcinoma, gastric cancer, and lung cancer tissues, where significant reductions in expression are frequently observed .

How can FITC-conjugated SPTBN1 antibodies be optimized for flow cytometry and cell sorting applications?

When optimizing FITC-conjugated SPTBN1 antibodies for flow cytometry and cell sorting, researchers should consider several methodological approaches:

• Titration optimization: Perform a dilution series (typically 0.1-10 μg/ml) to determine the optimal antibody concentration that maximizes specific signal while minimizing background. Plot the signal-to-noise ratio against antibody concentration to identify the optimal point.

• Permeabilization protocol selection: Since SPTBN1 is primarily an intracellular protein, effective permeabilization is crucial. Compare different permeabilization methods:

  • Saponin (0.1-0.5%) for gentle permeabilization

  • Triton X-100 (0.1-0.5%) for more thorough permeabilization

  • Methanol-based fixation/permeabilization for certain epitopes

• Blocking optimization: Test different blocking reagents (BSA, normal serum, commercial blocking solutions) to minimize non-specific binding.

• Compensation controls: Create single-color controls for accurate compensation when multiplexing with other fluorophores.

• Gating strategy development: Design gating strategies incorporating forward/side scatter profiles and viability dyes before examining SPTBN1 expression.

Research has shown that FACS analysis can successfully identify and quantify EpCAM-positive cells in SPTBN1+/− mouse livers compared to wild-type controls, demonstrating the utility of flow cytometry in SPTBN1-related research . In these studies, the number of EpCAM-positive cells doubled in SPTBN1+/− mouse liver compared to wild-type controls, highlighting the relationship between SPTBN1 expression and stem cell marker expression.

What mechanisms underlie SPTBN1's role in suppressing cancer progression, and how can antibody-based techniques help elucidate these pathways?

SPTBN1 suppresses cancer progression through multiple interconnected mechanisms that can be effectively studied using antibody-based techniques:

• Wnt signaling regulation: SPTBN1 regulates the Wnt inhibitor Kallistatin, influencing β-catenin phosphorylation and nuclear localization . FITC-conjugated SPTBN1 antibodies can be used in conjunction with antibodies against β-catenin to perform dual immunofluorescence studies that visualize the subcellular localization of these proteins in cancer cells.

• E-cadherin/EMT modulation: Loss of SPTBN1 decreases E-cadherin expression while increasing vimentin, promoting epithelial-to-mesenchymal transition (EMT) . Multiplex immunofluorescence with FITC-conjugated SPTBN1 antibodies alongside E-cadherin and vimentin markers can help visualize this relationship.

• Stem cell trait regulation: SPTBN1+/− mice have increased EpCAM-positive liver cells, suggesting a role in regulating stem cell populations . Flow cytometry using FITC-conjugated SPTBN1 antibodies combined with stem cell markers can quantify this relationship in various experimental models.

• PI3K/AKT pathway inhibition: In rheumatoid arthritis research, SPTBN1 has been shown to bind PIK3R2, inhibiting the PI3K/AKT signaling pathway . Co-immunoprecipitation studies using SPTBN1 antibodies can help identify additional binding partners involved in this signaling cascade.

Research has demonstrated that HCC cells with decreased SPTBN1 show reduced Kallistatin expression, decreased β-catenin phosphorylation, and increased β-catenin nuclear localization . Antibody-based techniques have been instrumental in establishing that restoration of Kallistatin expression reverses the observed Wnt activation, providing mechanistic insights into SPTBN1's tumor-suppressive functions.

What approaches can be used to validate the specificity of FITC-conjugated SPTBN1 antibodies in complex experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For FITC-conjugated SPTBN1 antibodies, consider these methodological approaches:

• Genetic knockdown/knockout controls: Use SPTBN1 siRNA knockdown or CRISPR/Cas9 knockout cells alongside wild-type cells to confirm signal specificity. Loss of signal in knockdown/knockout samples provides strong evidence of antibody specificity.

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide before staining to block specific binding sites. Comparison of blocked versus unblocked antibody staining can reveal non-specific binding.

• Isotype controls: Use FITC-conjugated isotype-matched immunoglobulins that have no relevant specificity to establish background fluorescence levels.

• Cross-validation with multiple antibodies: Compare staining patterns between different antibody clones targeting distinct SPTBN1 epitopes.

• Correlation with orthogonal methods: Compare protein detection by FITC-conjugated antibodies with mRNA expression data from RT-qPCR or RNA-seq to ensure concordance between protein and transcript levels.

• Western blot confirmation: Verify that the antibody detects a single band of appropriate molecular weight (275 kDa for SPTBN1) in the same samples used for fluorescence applications.

In published research, SPTBN1 expression has been validated using multiple techniques. For example, studies have shown consistent results between mRNA and protein levels of SPTBN1 in mouse liver tissues, with SPTBN1+/− mice showing approximately half the expression level compared to wild-type controls .

How can FITC-conjugated SPTBN1 antibodies be employed to study the intersection of SPTBN1 with Wnt signaling in cancer research?

FITC-conjugated SPTBN1 antibodies offer powerful tools for investigating the relationship between SPTBN1 and Wnt signaling in cancer contexts:

• Co-localization studies: Perform dual immunofluorescence with FITC-conjugated SPTBN1 antibodies and red-fluorescent antibodies against Wnt pathway components (e.g., β-catenin, LRP6, Wnt3a). This allows visualization of potential spatial relationships between SPTBN1 and these proteins within the same cells.

• Pathway activation dynamics: Use time-course experiments with Wnt stimulation to track changes in SPTBN1 localization and expression levels using FITC-conjugated antibodies, potentially revealing dynamic regulatory relationships.

• Sorted cell population analysis: Use FACS with FITC-conjugated SPTBN1 antibodies to isolate cells with varying SPTBN1 expression levels, then analyze these populations for differential expression of Wnt pathway components.

• 3D culture systems: Employ FITC-conjugated SPTBN1 antibodies in 3D organoid cultures that better recapitulate tumor environments to visualize SPTBN1 and Wnt pathway markers in more physiologically relevant contexts.

Research has established that SPTBN1+/− mouse livers exhibit significantly increased mRNA levels of several Wnt-related genes, including LRP6, Wnt3a, and Wnt10a, which play critical roles in HCC pathogenesis . Furthermore, the positive correlation between SPTBN1 and Kallistatin expression in human HCC tissues suggests a mechanistic relationship that can be further explored using fluorescence-based techniques.

What are common pitfalls when working with FITC-conjugated antibodies, and how can they be addressed in SPTBN1 research?

When working with FITC-conjugated SPTBN1 antibodies, researchers may encounter several technical challenges that require specific troubleshooting approaches:

• Photobleaching: FITC is relatively prone to photobleaching compared to other fluorophores.

  • Solution: Use anti-fade mounting media, minimize exposure to light during preparation, and consider using newer generation fluorophores like Alexa Fluor 488 for critical applications requiring extended imaging.

• pH sensitivity: FITC fluorescence is sensitive to pH changes, with optimal emission at pH 8.0.

  • Solution: Maintain consistent buffer pH during staining and imaging procedures. Consider using buffered mounting media specifically formulated for fluorescence preservation.

• Autofluorescence: Tissues like liver (where SPTBN1 is commonly studied) often exhibit significant green autofluorescence.

  • Solution: Include unstained controls for each tissue type, use spectral imaging systems when available, and consider Sudan Black B treatment (0.1-0.3%) to reduce autofluorescence.

• Fixation artifacts: Overfixation can mask SPTBN1 epitopes and alter subcellular localization.

  • Solution: Compare multiple fixation methods (paraformaldehyde, methanol, acetone) and durations to optimize protocol for your specific application.

• Specificity confirmation: Ensuring signals represent true SPTBN1 distribution rather than non-specific binding.

  • Solution: Always include appropriate controls including isotype controls, peptide competition controls, and when possible, SPTBN1 knockdown/knockout samples.

Researchers studying SPTBN1 in liver tissues should be particularly attentive to autofluorescence issues, as studies have shown significant autofluorescence in this tissue type that can confound interpretation of green fluorescent signals.

How can researchers quantitatively analyze SPTBN1 expression patterns using fluorescence-based techniques?

Quantitative analysis of SPTBN1 expression using FITC-conjugated antibodies requires rigorous methodological approaches:

• Flow cytometry quantification:

  • Use standardized protocols with consistent voltage settings

  • Include fluorescence calibration beads to convert arbitrary units to molecules of equivalent fluorochrome (MEF)

  • Report median fluorescence intensity (MFI) and percent positive cells

  • Consider using stimulation index (ratio of sample MFI to control MFI) for comparisons across experiments

• Image-based quantification:

  • Acquire images with identical exposure settings for all samples

  • Perform background subtraction using adjacent negative areas

  • Use thresholding algorithms to define positive staining

  • Quantify parameters such as:

    • Mean fluorescence intensity per cell

    • Integrated density (product of area and mean intensity)

    • Nuclear/cytoplasmic ratio of signal intensity

• Subcellular distribution analysis:

  • Use nuclear counterstains to define cellular compartments

  • Employ specialized software (ImageJ, CellProfiler) to create masks of cellular compartments

  • Calculate the percentage of SPTBN1 signal in each compartment

• Statistical considerations:

  • Use appropriate statistical tests based on data distribution

  • Report both biological and technical replicates

  • Consider normalization to housekeeping proteins for Western blot quantification

Published research has successfully used flow cytometry to quantify EpCAM-positive cells in SPTBN1+/− and wild-type mouse liver, demonstrating that quantitative approaches can reveal significant biological differences in SPTBN1-related research .

What considerations should be made when designing dual or multiple immunofluorescence protocols involving FITC-conjugated SPTBN1 antibodies?

Designing effective multiple immunofluorescence protocols with FITC-conjugated SPTBN1 antibodies requires careful planning:

• Fluorophore selection and spectral separation:

  • Pair FITC (green) with fluorophores having minimal spectral overlap like Cy3 (red) or Cy5 (far-red)

  • If using confocal microscopy, optimize detection channels to minimize bleed-through

  • Consider sequential scanning approaches for closely overlapping fluorophores

• Antibody compatibility:

  • Ensure primary antibodies are raised in different host species to avoid cross-reactivity

  • When using antibodies from the same species, consider direct conjugation or specialized blocking steps

  • Test each antibody individually before combining to ensure performance isn't compromised

• Optimization of fixation and antigen retrieval:

  • Different antigens may require different fixation methods

  • For SPTBN1, antigen retrieval with TE buffer pH 9.0 is recommended, but this must be compatible with other target proteins

  • Consider a compromise protocol that adequately preserves all antigens of interest

• Controls for multiple immunostaining:

  • Include single-stained controls for each fluorophore

  • Use isotype controls for each primary antibody

  • Include absorption controls where feasible

• Order of application:

  • For directly conjugated antibodies, consider sequential application to avoid steric hindrance

  • When one antigen is significantly less abundant, apply that antibody first

Research applications might include dual staining for SPTBN1 and β-catenin to study their relationship in Wnt signaling, or SPTBN1 with E-cadherin or vimentin to investigate EMT processes in cancer contexts .

How can SPTBN1 antibodies be utilized to develop prognostic biomarkers in cancer research?

SPTBN1 shows significant potential as a prognostic biomarker in multiple cancer types, particularly hepatocellular carcinoma. Research indicates that decreased SPTBN1 expression correlates with decreased relapse-free survival in HCC patients . FITC-conjugated SPTBN1 antibodies can facilitate translational research in several ways:

• Tissue microarray analysis:

  • Develop standardized immunofluorescence protocols for SPTBN1 detection in tissue microarrays

  • Establish quantitative scoring systems based on staining intensity and distribution

  • Correlate SPTBN1 expression patterns with clinical outcomes and other prognostic factors

• Circulating tumor cell (CTC) detection:

  • Use FITC-conjugated SPTBN1 antibodies in multiparameter flow cytometry panels to characterize CTCs

  • Investigate whether SPTBN1 expression in CTCs correlates with disease progression or treatment response

• Liquid biopsy approaches:

  • Develop assays to detect SPTBN1 protein in extracellular vesicles using FITC-conjugated antibodies

  • Correlate SPTBN1 levels with disease status and clinical outcomes

• Multimarker prognostic panels:

  • Combine SPTBN1 with other markers (e.g., E-cadherin, Kallistatin) to develop comprehensive prognostic signatures

  • Use machine learning approaches to identify optimal marker combinations

Clinical relevance is supported by data showing that SPTBN1 gene expression is positively and significantly correlated with E-cadherin and Kallistatin gene expression in both HCV-induced and HBV-induced HCC. Furthermore, decreased levels of SPTBN1 and SERPINA4 (the gene encoding Kallistatin) genes are significantly correlated with decreased relapse-free survival (p<0.001 and p=0.0193 respectively) .

What are the methodological considerations for studying SPTBN1's role in rheumatoid arthritis pathogenesis using fluorescence-based techniques?

Recent research has revealed SPTBN1's role in attenuating rheumatoid arthritis synovial cell proliferation, invasion, migration, and inflammatory response by binding to PIK3R2 . When studying this role using fluorescence-based techniques, researchers should consider:

• Sample preparation from synovial tissue:

  • Fresh synovial biopsies require gentle processing to maintain cell viability

  • Optimize digestion protocols (e.g., collagenase concentration, incubation time) to isolate fibroblast-like synoviocytes (FLS) while preserving SPTBN1 epitopes

  • Consider cryopreservation methods that maintain antigen integrity

• Co-visualization of SPTBN1 and interacting partners:

  • Design dual immunofluorescence protocols to visualize SPTBN1 and PIK3R2 co-localization

  • Include markers for specific cell populations (CD68 for macrophages, CD3 for T cells)

  • Quantify co-localization coefficients (e.g., Pearson's correlation coefficient, Manders' overlap coefficient)

• Functional assays with fluorescence readouts:

  • Use FITC-conjugated SPTBN1 antibodies in combination with functional assays that measure:

    • Cell proliferation (EdU incorporation)

    • Apoptosis (TUNEL assay)

    • Migration (wound healing with live cell imaging)

    • Invasion (fluorescence-based transwell assays)

• Inflammatory response assessment:

  • Combine SPTBN1 detection with fluorescence-based cytokine assays

  • Consider multiplex systems that allow simultaneous detection of multiple inflammatory mediators

Research has shown that SPTBN1 overexpression represses the proliferation, migration, invasion, and inflammation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) while promoting apoptosis . These effects were mediated through interaction with PIK3R2 and suppression of the PI3K/AKT signaling pathway, providing a potential therapeutic target for RA treatment.

How can researchers design experiments to investigate the relationship between SPTBN1 and the PI3K/AKT signaling pathway?

The interaction between SPTBN1 and the PI3K/AKT pathway, particularly through PIK3R2 binding, represents a critical area for investigation in both cancer and rheumatoid arthritis research . FITC-conjugated SPTBN1 antibodies can be employed in several experimental approaches:

• Proximity ligation assays (PLA):

  • Combine FITC-conjugated SPTBN1 antibodies with antibodies against PI3K/AKT pathway components

  • PLA technology generates fluorescent signals only when proteins are in close proximity (<40nm)

  • Quantify interaction events per cell and analyze their subcellular distribution

• FRET (Förster Resonance Energy Transfer) analysis:

  • Design FRET pairs using FITC-conjugated SPTBN1 antibodies and red-shifted fluorophore-conjugated antibodies against pathway components

  • Measure energy transfer efficiency as an indicator of protein-protein interactions

  • Perform acceptor photobleaching to confirm FRET signals

• Live cell imaging with pathway activation:

  • Use cell-permeable FITC-conjugated SPTBN1 antibody fragments

  • Stimulate cells with growth factors to activate PI3K/AKT pathway

  • Track dynamic changes in protein localization and interaction

• Fluorescence-based signaling readouts:

  • Combine SPTBN1 detection with phospho-specific antibodies against AKT, PI3K

  • Quantify changes in phosphorylation status following SPTBN1 manipulation

  • Use ratiometric approaches to normalize signals

Research has demonstrated that PIK3R2 interference increases the levels of phosphorylated PI3K and AKT in SPTBN1-overexpressing cells, suggesting that SPTBN1 represses the PI3K/AKT signaling pathway via interaction with PIK3R2 . These findings highlight the importance of studying the spatial and temporal dynamics of these interactions using advanced fluorescence techniques.

What emerging techniques might enhance the utility of FITC-conjugated SPTBN1 antibodies in complex experimental systems?

As technology advances, several emerging techniques show promise for enhancing SPTBN1 research using fluorescence-based approaches:

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED can overcome the diffraction limit

  • Enable visualization of SPTBN1 distribution at nanoscale resolution (20-50 nm)

  • Allow precise localization relative to cytoskeletal components and signaling platforms

• Expansion microscopy:

  • Physical expansion of specimens using hydrogel technology

  • Can achieve 4-10x physical expansion, effectively improving resolution

  • Particularly valuable for densely packed cellular structures where SPTBN1 may function

• Lattice light-sheet microscopy:

  • Enables rapid 3D imaging with minimal photobleaching

  • Ideal for tracking dynamic SPTBN1 interactions in living cells

  • Can reveal previously undetectable transient interactions

• Mass cytometry (CyTOF) with conjugated antibodies:

  • Metal-tagged rather than fluorophore-tagged antibodies

  • Eliminates spectral overlap issues in highly multiplexed assays

  • Enables simultaneous detection of 40+ markers including SPTBN1 and related pathway components

• Spatial transcriptomics integration:

  • Combine FITC-conjugated SPTBN1 antibody staining with spatial transcriptomics

  • Correlate protein expression with local transcriptional programs

  • Reveal tissue microenvironments where SPTBN1 function is particularly important

These advanced techniques could yield deeper insights into SPTBN1's role in cancer progression, Wnt signaling regulation, and inflammatory responses in rheumatoid arthritis, potentially identifying new therapeutic targets or diagnostic approaches.

How might single-cell analysis approaches using FITC-conjugated SPTBN1 antibodies advance our understanding of heterogeneous disease states?

Single-cell analysis represents a powerful frontier for SPTBN1 research, particularly in heterogeneous diseases like cancer and rheumatoid arthritis:

• Single-cell proteomics:

  • Use FITC-conjugated SPTBN1 antibodies in flow cytometry-based single-cell western blot systems

  • Identify rare cell populations with distinct SPTBN1 expression patterns

  • Correlate SPTBN1 levels with other protein markers at single-cell resolution

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

  • Combine FITC-conjugated SPTBN1 antibody detection with single-cell RNA sequencing

  • Correlate SPTBN1 protein levels with transcriptional programs in individual cells

  • Identify cellular states where SPTBN1 regulation may be particularly important

• Microfluidic approaches:

  • Isolate single cells based on SPTBN1 expression using fluorescence-activated cell sorting

  • Perform single-cell secretome analysis to correlate SPTBN1 levels with inflammatory mediator production

  • Study clonal growth properties of cells with varying SPTBN1 expression

• Spatial single-cell analysis:

  • Use FITC-conjugated SPTBN1 antibodies in multiplexed ion beam imaging (MIBI) or imaging mass cytometry

  • Preserve spatial context while achieving single-cell resolution

  • Map SPTBN1 expression in relation to tissue architecture and cellular neighborhoods

Research has already demonstrated heterogeneity in SPTBN1 expression within tissues, such as the increased number of EpCAM-positive cells in SPTBN1+/− mouse livers compared to wild-type controls . Single-cell approaches could further dissect this heterogeneity and its functional consequences in disease progression and treatment response.

What are the considerations for developing therapeutic strategies targeting the SPTBN1 pathway based on insights from antibody-based research?

Research using SPTBN1 antibodies has revealed several potential therapeutic targets and strategies:

• Wnt pathway modulation:

  • SPTBN1 regulates Wnt signaling through Kallistatin

  • Therapeutic strategies could include:

    • Kallistatin augmentation in SPTBN1-deficient tumors

    • Small molecule inhibitors of β-catenin nuclear translocation

    • Targeted delivery of SPTBN1 expression vectors to tumor cells

• PI3K/AKT pathway targeting:

  • SPTBN1 represses PI3K/AKT signaling via PIK3R2 binding

  • Potential approaches include:

    • PI3K inhibitors that mimic SPTBN1's regulatory effect

    • Peptide mimetics that recapitulate SPTBN1-PIK3R2 binding interface

    • Combined targeting of multiple nodes in the pathway

• EMT reversal strategies:

  • Loss of SPTBN1 promotes EMT through decreased E-cadherin and increased vimentin

  • Therapeutic concepts could include:

    • E-cadherin stabilization approaches

    • Vimentin targeting in SPTBN1-deficient tumors

    • Epigenetic modifiers to restore epithelial phenotype

• Biomarker-guided therapy:

  • SPTBN1 and Kallistatin expression correlate with survival in HCC

  • Clinical applications might include:

    • Stratification of patients for therapeutic selection

    • Monitoring of SPTBN1 pathway activation during treatment

    • Combination therapies targeting multiple aspects of SPTBN1 deficiency

Translational research could utilize FITC-conjugated SPTBN1 antibodies to monitor target engagement and pathway modulation in preclinical models and potentially in clinical samples. The development of companion diagnostics based on SPTBN1 and related biomarkers could ultimately guide personalized therapeutic strategies in both cancer and inflammatory diseases.