CSTB Antibody
Description
Introduction to CSTB Antibody
CSTB Antibodies are monoclonal or polyclonal immunoglobulins raised against epitopes of the CSTB protein. CSTB itself is a 12 kDa intracellular thiol protease inhibitor that regulates cathepsins (e.g., cathepsin B) to prevent uncontrolled proteolysis . These antibodies enable researchers to:
-
Visualize CSTB localization in tissues or cells via immunohistochemistry (IHC) or immunofluorescence (IF).
-
Quantify CSTB expression using Western blotting or ELISA.
Key Applications in Research
CSTB Antibodies are pivotal in elucidating CSTB's functions and pathophysiological roles:
Types and Specificity
CSTB Antibodies vary in clonality, epitope targeting, and species reactivity:
Specificity Notes:
-
Monoclonal antibodies (e.g., CPTC-CSTB-1) often target full-length CSTB and show minimal cross-reactivity with homologs like CSTA .
-
Polyclonal antibodies (e.g., Cusabio CSB-PA00224A0Rb) may recognize broader epitopes but require validation for cross-reactivity .
Research Findings and Mechanisms
CSTB Antibodies have advanced understanding of CSTB's roles in disease:
Neurological Disorders
-
Epilepsy (EPM1A): Mutant CSTB proteins fail to localize to lysosomes, leading to cathepsin-induced neuronal damage .
-
Neuroprotection: CSTB interacts with SOD-1 to mitigate oxidative stress in the cerebellum and hippocampus .
Oncology
-
Gastric Cancer: CSTB downregulation correlates with enhanced PI3K/Akt/mTOR signaling, promoting cell proliferation and migration .
-
Bladder Cancer: CSTB serves as a urinary biomarker for recurrence and progression .
Amyloid-β Pathology
-
Alzheimer’s Disease: CSTB duplication in mice increases CSTB protein but does not alter amyloid-β plaque deposition, suggesting indirect effects on cathepsin activity .
References
Product Specs
Q&A
What is Cystatin B and what is its biological significance in research contexts?
Cystatin B (CSTB) is an intracellular thiol proteinase inhibitor that functions as a tight binding reversible inhibitor of cathepsins L, H, and B. Also known as CST6, STFB, CPI-B, liver thiol proteinase inhibitor, or Stefin-B, CSTB plays a critical role in maintaining proteolytic balance within cells. The inhibition of cathepsins by CSTB is physiologically significant because dysregulation of cathepsin activity has been implicated in various pathological conditions, including cancer progression and neurodegenerative disorders . Understanding CSTB's role is essential for researchers studying protease regulation and associated disease mechanisms.
What research applications are CSTB antibodies optimized for?
CSTB antibodies are suitable for multiple laboratory applications:
| Application | Typical Dilution | Notes |
|---|---|---|
| Western Blotting (WB) | 1:1000 or 2 μg/mL | Detects bands at approximately 11-12 kDa |
| Immunohistochemistry (IHC-P) | 1:100 | For paraffin-embedded tissue sections |
| Immunocytochemistry (ICC/IF) | Varies by antibody | For cellular localization studies |
| Immunoprecipitation (IP) | Varies by antibody | For isolating CSTB protein complexes |
| Direct ELISA | Varies by antibody | For quantitative measurement |
Different antibodies may show varying efficiencies across these applications. For example, rabbit polyclonal antibody ab236646 is reported suitable for IP, WB, IHC-P, and ICC/IF with human samples , while the mouse monoclonal antibody MAB1408 has been validated for Western blots and direct ELISAs with less than 5% cross-reactivity to related cystatins .
What molecular weight should researchers expect when detecting CSTB by Western blot?
Researchers should expect to observe CSTB at approximately 11-12 kDa in Western blot applications. While the predicted band size based on amino acid sequence is 11 kDa , the protein typically appears at approximately 12 kDa in experimental settings . This slight discrepancy between predicted and observed molecular weight may be attributed to post-translational modifications or the specific properties of the protein when separated by SDS-PAGE. When validating a new CSTB antibody, confirming this expected molecular weight is an important specificity control.
How can researchers validate the specificity of CSTB antibodies in experimental systems?
Multiple approaches should be employed to validate CSTB antibody specificity:
-
Knockout validation: Using CSTB knockout cell lines provides the most stringent control. Western blot analysis of HEK293T parental cells versus Cystatin B knockout HEK293T cells demonstrates this approach, with specific bands observed at approximately 12 kDa in parental cells but absent in knockout lines .
-
Cross-reactivity testing: Evaluating potential cross-reactivity with related proteins is essential. Some validated antibodies show less than 5% cross-reactivity with related proteins like recombinant human Cystatins A and S .
-
Species specificity validation: When working with models expressing both human and mouse CSTB, testing antibody specificity across species is crucial. Human euploid and trisomic fibroblast homogenates can serve as controls for anti-mouse CSTB antibodies, while negligible signal should be observed with anti-human CSTB antibodies in samples from mice not expressing human CSTB .
-
siRNA knockdown: Reducing CSTB expression via siRNA should result in corresponding signal reduction when probed with specific antibodies, providing functional validation .
-
Multiple detection methods: Consistent results across different applications (Western blot, IHC, ICC/IF) strengthen confidence in antibody specificity.
What controls are essential when using CSTB antibodies in immunohistochemistry?
Rigorous controls are necessary for reliable immunohistochemical analysis of CSTB:
-
Negative controls: Use non-immune rabbit serum (1:200 dilution) in phosphate-buffered saline instead of primary antibody to assess non-specific binding .
-
Positive controls: Include tissues with known CSTB expression patterns.
-
Antibody dilution optimization: Titrate antibody concentrations; successful staining has been achieved at 1:100 dilution for paraffin-embedded tissues .
-
Blinded assessment: Immunostaining should be evaluated by at least two independent investigators blinded to clinical characteristics and outcomes to prevent bias .
-
Comprehensive scoring system: Implement a quantitative scoring method that incorporates both:
How should samples be prepared for optimal CSTB detection in different applications?
Sample preparation varies by application technique:
For Western blot analysis:
-
Lyse tissues in SDS buffer supplemented with 1% phenylmethanesulfonyl fluoride and 1% phosphatase inhibitor
-
Normalize protein loading with β-actin
-
Detect signals using ECL detection systems (e.g., BeyoECL Moon Kit)
For immunohistochemistry:
-
Properly de-paraffinize tissue sections
-
Use standardized detection systems (e.g., Mouse/Rabbit Specific HRP/DAB Detection Kit)
-
Examine multiple representative fields (minimum 12) to account for heterogeneity
For functional studies with CSTB knockdown:
-
Optimize siRNA transfection conditions (1:400 dilution of transfection reagent has been effective)
-
Confirm knockdown efficiency via Western blot before conducting functional assays
How can CSTB antibodies be used to investigate the relationship between CSTB and cathepsin activity?
Researchers can employ several strategies using CSTB antibodies to explore this regulatory relationship:
-
Neutralization assays: CSTB antibodies can block CSTB's inhibitory effect on cathepsins, allowing measurement of the resulting change in protease activity. For example, papain (a cysteine protease similar to cathepsins) activity can be measured in the presence of recombinant human CSTB that has been preincubated with increasing concentrations of CSTB antibody. The typical neutralization dose (ND50) has been reported as 5.4 μg/mL .
-
Knockdown studies with activity measurement: After siRNA-mediated CSTB reduction, cathepsin B activity can be quantified using substrate cleavage assays (e.g., with Ac-RR-AFC substrate). Importantly, this approach has revealed context-dependent effects—reduction in CSTB abundance increases cathepsin B activity in disomic (normal) human fibroblasts but not in trisomy 21 fibroblasts, despite similar post-knockdown CSTB levels .
-
Complementary genetic approaches: Studies using mice with reduced Cstb gene copy number (+/- Cstb) in both normal and Tc1 (carrying human chromosome 21) backgrounds have demonstrated that reducing Cstb from two to one copies, or three to two copies, reduces protein abundance in cortical tissues .
These methodologies highlight the complex, context-dependent relationship between CSTB levels and cathepsin activity that varies with genetic background.
What is the significance of CSTB as a potential biomarker in cancer research?
CSTB has emerged as a promising biomarker candidate in cancer research, particularly for cervical cancer:
-
Differential expression: High CSTB mRNA and protein expression has been documented in cervical cancer tissues compared to paired adjacent non-cancerous tissue, as confirmed by multiple methodologies including tissue microarray analysis .
-
Correlation with disease progression: Elevated CSTB expression has been associated with pathological progression in cervical cancer, suggesting potential prognostic value .
-
Multi-omics validation: The biomarker potential of CSTB has been validated through:
-
Therapeutic implications: Research suggests CSTB may aid in developing intervention strategies, positioning it as not only a diagnostic/prognostic marker but potentially a therapeutic target .
How should researchers interpret context-dependent effects when studying CSTB in different genetic backgrounds?
The interpretation of CSTB function requires careful consideration of genetic context, as demonstrated by several findings:
-
Differential effects in normal versus trisomy backgrounds: Reducing CSTB abundance increases cathepsin B activity in disomic (normal) human fibroblasts but not in trisomy 21 fibroblasts . This suggests that the trisomy 21 genetic background contains additional factors that modify the CSTB-cathepsin relationship.
-
Quantitative consistency: When interpreting results, researchers should confirm that experimental manipulations (e.g., siRNA knockdown) achieve comparable CSTB reduction across different genetic backgrounds to ensure valid comparisons .
-
Species-specific effects: When working with both human and mouse CSTB in model systems, researchers must distinguish between species-specific proteins using appropriate antibodies. Studies have used human CSTB-specific antibodies that show negligible signal in samples from mice not expressing the human protein .
-
Integrated analysis approaches: To fully understand CSTB's role in different contexts, researchers should integrate:
-
Protein abundance measurements
-
Enzymatic activity assays
-
Gene expression analysis
-
Pathway enrichment analysis
-
These considerations highlight the importance of comprehensive experimental design when studying CSTB across different disease models or genetic backgrounds.
What are the major technical challenges in quantifying CSTB expression in tissue samples?
Researchers face several technical challenges when quantifying CSTB expression:
How can functional enrichment analysis enhance CSTB research in disease contexts?
Functional enrichment analysis offers valuable insights into CSTB's broader biological context:
-
Co-expression network analysis: Tools like LinkedOmics can predict genes positively and negatively correlated with CSTB expression in disease contexts (e.g., cervical cancer data from TCGA), visualized through heatmaps .
-
Gene-gene functional interactions: Platforms such as GenMANIA prediction can analyze functional interactions between CSTB and its top correlated genes, revealing potential mechanistic relationships .
-
Pathway enrichment: KEGG pathway analysis of CSTB-associated genes can identify biological processes potentially influenced by CSTB dysregulation in disease states .
-
GO term enrichment: Gene Ontology analysis helps classify CSTB-associated genes into functional categories, providing insights into biological processes, molecular functions, and cellular components relevant to CSTB's role .
-
Integration with clinical data: Correlating these bioinformatic findings with clinical parameters enhances the translational relevance of CSTB research.
This multi-layered analytical approach helps contextualize experimental findings and generates hypotheses for future research directions.
What is the relevance of CSTB in coronavirus research?
Recent research has identified connections between CSTB, cathepsins, and coronavirus infections:
Cathepsin B, which is inhibited by CSTB, has been identified as a SARS-CoV-2-related target for research into the mechanisms of the Novel Coronavirus causing the COVID-19 pandemic . While the search results don't provide extensive details on this relationship, this connection suggests that modulating the CSTB-cathepsin B axis could potentially influence viral entry or replication mechanisms.
Researchers investigating this area should consider:
-
How CSTB expression patterns might affect cathepsin activity in tissues susceptible to SARS-CoV-2 infection
-
Whether CSTB levels correlate with COVID-19 severity or susceptibility
-
The potential for targeting CSTB-cathepsin interactions as a therapeutic approach
This emerging area represents a promising direction for researchers with expertise in CSTB biology.
Product Science Overview
Cystatin B, also known as Stefin B, is a protein encoded by the CSTB gene in humans. It belongs to the cystatin superfamily, which comprises proteins that function as cysteine protease inhibitors. These inhibitors play a crucial role in regulating proteolytic activities within cells, thereby maintaining cellular homeostasis .
Cystatin B is an intracellular thiol proteinase inhibitor that tightly binds and reversibly inhibits cathepsins L, H, and B . These cathepsins are lysosomal proteases involved in various cellular processes, including protein degradation, antigen presentation, and apoptosis. By inhibiting these proteases, Cystatin B helps prevent excessive proteolysis, which can lead to cellular damage and disease.
Mutations in the CSTB gene are associated with a rare genetic disorder known as progressive myoclonic epilepsy type 1A (EPM1A), also referred to as Unverricht-Lundborg disease . This condition is characterized by myoclonic seizures, ataxia, and cognitive decline. The exact mechanism by which CSTB mutations lead to EPM1A is not fully understood, but it is believed that the loss of Cystatin B’s inhibitory function results in increased protease activity, leading to neuronal damage and neurodegeneration.
Mouse anti-human Cystatin B antibodies are monoclonal antibodies specifically designed to target and bind to human Cystatin B. These antibodies are commonly used in various research applications, including immunohistochemistry (IHC), immunocytochemistry (ICC), Western blotting (WB), and flow cytometry . They are valuable tools for studying the expression, localization, and function of Cystatin B in different biological contexts.
- Immunohistochemistry (IHC): Mouse anti-human Cystatin B antibodies are used to detect Cystatin B in tissue sections. This technique helps researchers study the distribution and localization of Cystatin B in various tissues and understand its role in different physiological and pathological conditions .
- Immunocytochemistry (ICC): These antibodies are also used to visualize Cystatin B in cultured cells. ICC allows researchers to examine the subcellular localization of Cystatin B and investigate its interactions with other cellular components .
- Western Blotting (WB): In WB, mouse anti-human Cystatin B antibodies are used to detect Cystatin B in protein extracts from cells or tissues. This technique helps quantify Cystatin B levels and assess its expression under different experimental conditions .
- Flow Cytometry: Flow cytometry with mouse anti-human Cystatin B antibodies enables the analysis of Cystatin B expression in individual cells within a mixed population. This application is particularly useful for studying cell-specific expression patterns and identifying cell types that express Cystatin B .
