What is HS3ST3B1 and what biological processes does it influence?

HS3ST3B1 (Heparan Sulfate Glucosamine 3-O-Sulfotransferase 3B1) is a key enzyme involved in the sulfation of heparan sulfate proteoglycans, particularly at the 3-O-position of glucosamine residues. This enzyme participates in the final modification steps of heparan sulfate chains, creating mature sugar structures. HS3ST3B1 plays crucial roles in various biological processes including cell signaling, cell adhesion, and extracellular matrix organization. Studies have shown that dysregulation of HS3ST3B1 is associated with developmental disorders, cancer progression, and inflammatory diseases, highlighting its significance in physiological and pathological conditions . In particular, research has demonstrated that HS3ST3B1 can positively contribute to acute myeloid leukemia progression through induction of proangiogenic factor VEGF expression and release .

What are the key differences between the various forms of HS3ST3B1 antibodies available?

Various forms of HS3ST3B1 antibodies differ primarily in their host species, clonality, conjugation, and target epitopes. The most common antibodies are raised in rabbits and are polyclonal in nature. Available conjugates include unconjugated, biotin-conjugated, FITC-conjugated, and HRP-conjugated forms, each optimized for specific applications. The biotin-conjugated variant (e.g., ABIN7155015) specifically targets amino acids 54-390 of the human HS3ST3B1 protein and is particularly suited for ELISA applications . Other variants may target different regions, such as the N-terminal region (e.g., ABIN2782700), which affects their specificity and application suitability . These differences should be considered when selecting an antibody for specific experimental applications, as they can significantly impact sensitivity and specificity.

What species reactivity can be expected from HS3ST3B1 antibodies?

The species reactivity of HS3ST3B1 antibodies varies depending on the specific product. Based on the search results, the biotin-conjugated HS3ST3B1 antibody (ABIN7155015) is specifically reactive to human samples . Some unconjugated variants show broader reactivity profiles. For instance, ABIN2782700 demonstrates predicted reactivity with multiple species including human (100%), cow (92%), dog (93%), guinea pig (86%), horse (93%), pig (93%), rabbit (87%), and rat (93%) . This cross-reactivity information is crucial for researchers working with animal models, as it determines whether a particular antibody can be effectively used in their experimental system. When planning experiments with non-human samples, researchers should verify the cross-reactivity data or consider performing validation tests to confirm antibody recognition in their specific model organism.

How should biotin-conjugated HS3ST3B1 antibodies be optimally stored and handled to maintain functionality?

The biotin-conjugated HS3ST3B1 antibody should be stored at -20°C or -80°C upon receipt to maintain its functionality and prevent degradation. Repeated freeze-thaw cycles should be avoided as they can damage the antibody structure and compromise its performance . For short-term storage during ongoing experiments, keeping the antibody at 4°C is recommended, but this should not exceed one week. When handling the antibody, use sterile techniques and avoid contamination. The antibody is typically supplied in a buffer containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) , which help maintain its integrity. Prior to use, allow the antibody to equilibrate to room temperature and gently mix by inverting the tube rather than vortexing, which can damage the protein structure. Aliquoting the antibody upon initial thawing is highly recommended to minimize freeze-thaw cycles if multiple experiments are planned over time.

What are the recommended dilution ratios and incubation conditions for ELISA using biotin-conjugated HS3ST3B1 antibody?

For ELISA applications using biotin-conjugated HS3ST3B1 antibody, the recommended dilution ranges typically fall between 1:100 to 1:500, though this may vary slightly depending on the specific experimental conditions and sensitivity requirements . Optimal incubation conditions generally involve applying the diluted antibody to wells pre-coated with the antigen or capture antibody and incubating for 1-2 hours at room temperature or overnight at 4°C. Following this, thorough washing with PBS-T (PBS containing 0.05-0.1% Tween-20) is necessary to remove unbound antibody. Since the antibody is biotin-conjugated, a streptavidin-HRP conjugate (typically at 1:1000 to 1:5000 dilution) should be used as the detection system, followed by incubation with an appropriate substrate. For precise applications, researchers should perform a titration experiment using various antibody dilutions (e.g., 1:100, 1:200, 1:500, 1:1000) to determine the optimal concentration that provides maximum specific signal with minimal background.

What positive controls are recommended when validating an HS3ST3B1 antibody for the first time?

When validating an HS3ST3B1 antibody for the first time, several positive controls should be considered to ensure specificity and functionality. Cell lysates from cell lines known to express HS3ST3B1 provide excellent positive controls . Based on expression data, human acute myeloid leukemia (AML) cell lines would serve as appropriate positive controls due to their documented expression of HS3ST3B1 . Additionally, tissue samples from salivary glands, particularly from myoepithelial cells and intercalated ducts where HS3ST3B1 has been shown to be expressed, can serve as positive controls for immunohistochemistry . Recombinant human HS3ST3B1 protein can be used as a definitive positive control for ELISA and Western blotting. For cellular distribution studies, myoepithelial cells and developing epithelial cells from salivary glands are recommended, as these have been shown to express HS3ST3B1 during development and in adult tissues . Proper validation should include both positive and negative controls (cells or tissues not expressing the target) to confirm specificity.

What sample preparation protocols optimize detection of HS3ST3B1 in different tissue types?

Sample preparation protocols for optimal detection of HS3ST3B1 vary depending on the tissue type and application. For Western blotting, cells or tissues should be lysed in a buffer containing protease inhibitors to prevent degradation of the target protein. RIPA buffer (containing 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with a protease inhibitor cocktail is recommended. For immunohistochemistry of tissues expressing HS3ST3B1 such as salivary glands, 10% neutral buffered formalin fixation followed by paraffin embedding is standard, with antigen retrieval using citrate buffer (pH 6.0) prior to antibody incubation . For studies involving epithelial tissues, which have demonstrated significant HS3ST3B1 expression, careful isolation of the epithelial component from surrounding mesenchyme may be necessary, particularly in developmental studies . In all cases, freshly prepared samples yield better results than archived specimens, and optimization of fixation time is critical to balance between preserving tissue morphology and maintaining antigen accessibility.

How can non-specific binding be minimized when using biotin-conjugated HS3ST3B1 antibodies?

Non-specific binding with biotin-conjugated HS3ST3B1 antibodies can be minimized through several strategies. First, implement a thorough blocking step using 3-5% BSA or 5-10% normal serum from the species in which the secondary reagent was raised. For biotin-conjugated antibodies specifically, it's crucial to block endogenous biotin by pre-incubating samples with an avidin/biotin blocking kit, particularly important for tissues with high endogenous biotin such as liver, kidney, and brain. Optimize antibody dilution by performing titration experiments; over-concentrated antibody solutions frequently lead to increased background. Including 0.1-0.3% Triton X-100 or Tween-20 in blocking and antibody dilution buffers helps reduce hydrophobic interactions. For problematic samples, pre-absorption of the antibody with non-specific proteins from the same species as the sample can significantly reduce cross-reactivity. Additionally, reducing incubation temperature from room temperature to 4°C may decrease non-specific interactions, though this requires extending incubation time. Finally, increase washing steps (at least 3-5 washes of 5 minutes each) between all incubations using buffers containing 0.05-0.1% Tween-20.

What are common sources of false positive results with HS3ST3B1 antibodies and how can they be identified?

Common sources of false positive results with HS3ST3B1 antibodies include cross-reactivity with structurally similar sulfotransferases, endogenous biotin or peroxidase activity, and non-specific binding to extracellular matrix components. To identify and mitigate these issues, several approaches are recommended. First, include appropriate negative controls such as isotype controls and samples from HS3ST3B1 knockout models where available . Second, use competitive binding assays with recombinant HS3ST3B1 protein to confirm specificity; true positive signals should be blocked by pre-incubation with the target protein. Third, when using biotin-conjugated antibodies, include a biotin blocking step to prevent detection of endogenous biotin. Fourth, cross-validate results using different detection methods (e.g., comparing ELISA results with Western blot or IHC). Fifth, sequence comparison analysis can help identify potential cross-reactive proteins; antibodies targeting highly conserved regions may react with related sulfotransferases. Finally, correlation of staining patterns with known expression profiles of HS3ST3B1 in tissues can help verify results; discrepancies may indicate false positives.

What are the optimal antigen retrieval methods for detecting HS3ST3B1 in formalin-fixed paraffin-embedded tissues?

For optimal detection of HS3ST3B1 in formalin-fixed paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval (HIER) methods are generally most effective. Citrate buffer (10 mM, pH 6.0) provides good results for most applications involving HS3ST3B1 detection. The protocol should include heating the deparaffinized sections in the buffer at 95-100°C for 20-30 minutes, either using a microwave, pressure cooker, or water bath, followed by cooling at room temperature for at least 20 minutes. For tissues with high extracellular matrix content, such as salivary glands where HS3ST3B1 is known to be expressed , enzymatic retrieval using proteinase K (10-20 μg/mL for 10-15 minutes at 37°C) as a complementary or alternative approach may improve antibody accessibility to the target. In some cases, a dual retrieval method combining both HIER and enzymatic treatment yields superior results. Optimization is essential, as excessive antigen retrieval can damage tissue morphology while insufficient retrieval leads to weak staining. Testing multiple retrieval conditions side by side on serial sections is recommended to determine the optimal protocol for specific tissue types.

How can signal amplification be achieved when working with low abundance HS3ST3B1 samples?

When working with low abundance HS3ST3B1 samples, several signal amplification strategies can be employed to enhance detection sensitivity. For biotin-conjugated antibodies specifically, utilizing streptavidin-based amplification systems can significantly boost signal intensity. The biotin-streptavidin system offers natural amplification due to streptavidin's four biotin-binding sites. For additional enhancement, employ tyramide signal amplification (TSA), which can increase sensitivity by 10-100 fold; this technique uses HRP to catalyze the deposition of additional biotin or fluorophore-labeled tyramide molecules near the antibody binding site. Another effective approach is to use polymer-based detection systems that incorporate multiple HRP or alkaline phosphatase molecules per antibody binding event. For ELISA applications, extending substrate incubation time allows for greater chromogen development, though this should be balanced against increased background. Sandwich ELISA configurations, where a capture antibody first immobilizes the target before detection with the biotin-conjugated HS3ST3B1 antibody, can significantly improve sensitivity. Additionally, sample concentration techniques through immunoprecipitation prior to analysis can effectively increase the target protein concentration above detection thresholds.

How do HS3ST3B1 expression patterns correlate with developmental stages and pathological conditions?

HS3ST3B1 expression demonstrates specific temporal and spatial patterns throughout development and in various pathological conditions. During embryonic development, particularly in salivary glands, HS3ST3B1 is highly expressed in developing myoepithelial cells, endbud cells, and basal duct cells at E16 (embryonic day 16), the onset of cell differentiation. It is also expressed in Krt19+ differentiated duct cells . Postnatally (P1), HS3ST3B1 continues to be expressed in myoepithelial cells, and is also detected in mitotic cells, Krt19+ duct cells, and Bpifa2+ proacinar cells . In adult tissues, HS3ST3B1 expression is maintained in myoepithelial cells and intercalated ducts . In pathological contexts, HS3ST3B1 shows significant involvement in cancer progression, particularly in acute myeloid leukemia (AML) where it promotes angiogenesis and proliferation through induction of VEGF expression . Studies have demonstrated that HS3ST3B1 positively contributes to AML progression both in vitro and in vivo, with these activities associated with proangiogenic factor VEGF expression and shedding . Research also indicates that loss of HS3ST3B1 enzymes alters heparan sulfate structure, which can reduce epithelial morphogenesis and adult salivary gland function , suggesting its importance in maintaining normal tissue architecture and function.

What is known about the interaction between HS3ST3B1 and VEGF signaling in cancer progression?

Research has revealed a significant relationship between HS3ST3B1 and VEGF signaling in cancer progression, particularly in acute myeloid leukemia (AML). HS3ST3B1 has been shown to positively contribute to AML progression both in vitro and in vivo, with these activities directly associated with an induction of proangiogenic factor VEGF expression and shedding . The 3-O-sulfation of heparan sulfate by HS3ST3B1 facilitates VEGF release from the cell surface or extracellular matrix. This mechanism can be disrupted by treatment with heparanase inhibitors such as suramin, which prevents VEGF secretion and subsequently blocks VEGF-induced activation of downstream signaling pathways including ERK and AKT . Additionally, the effects of HS3ST3B1 on activation of these pathways can be blocked by VEGFR inhibitors like axitinib, further supporting the relationship between heparan sulfate 3-O-sulfation and VEGF-activated signaling cascades . These findings establish VEGF as an important functional target of HS3ST3B1 and provide insights into the mechanism by which HS3ST3B1 contributes to cancer progression, suggesting potential therapeutic approaches targeting this pathway in cancers where angiogenesis plays a crucial role.

What is the current understanding of compensatory mechanisms between different HS3ST isoforms when HS3ST3B1 is disrupted?

Current research provides insights into compensatory mechanisms between different HS3ST isoforms when HS3ST3B1 is disrupted. Gene expression analysis of isolated epithelial cultures from HS3ST3B1 knockout models has revealed a trend of increased expression of other HS3ST isoforms, particularly HS3ST1 and HS3ST3A1, suggesting these isoforms may attempt to compensate for the loss of HS3ST3B1 function . This compensatory upregulation is more readily detectable when analyzing isolated epithelium rather than whole tissue samples, indicating tissue-specific regulation. Interestingly, there appears to be a regulatory relationship between HS3ST isoforms, as knockout of HS3ST3A1 leads to reduced expression of HS3ST3B1, suggesting HS3ST3A1 may regulate HS3ST3B1 expression . Despite these compensatory mechanisms, functional studies demonstrate that these adaptations are insufficient to fully restore normal tissue function and morphogenesis, as evidenced by reduced branching morphogenesis in HS3ST3B1 knockout salivary glands . The compensatory mechanisms do not extend to all sulfotransferase families, as expression of HS2ST1 and HS6ST1 remains unchanged in HS3ST3B1 knockout models . This selective compensation among HS3ST family members suggests specific functional overlaps within this subfamily of enzymes, while maintaining distinct roles that cannot be fully compensated when disrupted.

How should researchers quantitatively analyze HS3ST3B1 expression data from ELISA assays?

Quantitative analysis of HS3ST3B1 expression data from ELISA assays requires a systematic approach to ensure accuracy and reliability. First, establish a standard curve using purified recombinant HS3ST3B1 protein at known concentrations (typically ranging from 0-1000 pg/mL in a serial dilution), plotting optical density against concentration. The standard curve should be fitted using appropriate regression models; a four-parameter logistic curve fit is often optimal for most ELISA data. Calculate sample concentrations by interpolating their optical density values against this standard curve, ensuring they fall within the linear range of detection. For comparative studies, normalize HS3ST3B1 protein levels to total protein concentration in each sample, determined by methods such as Bradford or BCA assays. Statistical analysis should include assessment of technical replicates (coefficient of variation should be <15%) and biological replicates (minimum n=3). When comparing HS3ST3B1 levels across different conditions or tissues, employ appropriate statistical tests (t-test for two groups, ANOVA for multiple groups) with post-hoc corrections for multiple comparisons. For time-course studies or developmental analyses, consider repeated measures ANOVA or mixed models to account for temporal correlations. Report data with appropriate measures of central tendency and dispersion (mean ± standard deviation or median with interquartile range), and include p-values and confidence intervals for statistical comparisons.

What control experiments are necessary to validate HS3ST3B1 antibody specificity in different experimental systems?

To thoroughly validate HS3ST3B1 antibody specificity across different experimental systems, several critical control experiments are necessary. First, perform peptide competition assays where the antibody is pre-incubated with excess recombinant HS3ST3B1 protein or immunizing peptide before application to samples; this should substantially reduce or eliminate specific signals. Second, utilize genetic models such as HS3ST3B1 knockout tissues or cells as negative controls and compare against wild-type samples; true specific antibodies will show absence of signal in knockout samples. Third, employ siRNA or shRNA knockdown of HS3ST3B1 in relevant cell lines, confirming reduction of both mRNA (by qPCR) and protein levels detected by the antibody. Fourth, compare staining patterns across multiple antibodies targeting different epitopes of HS3ST3B1; concordant patterns suggest specificity. Fifth, verify tissue or cellular localization patterns against published expression data, such as the documented expression of HS3ST3B1 in myoepithelial cells and intercalated ducts in adult salivary glands . Sixth, perform Western blot analysis to confirm detection of a protein of the expected molecular weight (approximately 43 kDa for human HS3ST3B1). Finally, conduct cross-reactivity tests against closely related family members (other HS3ST isoforms) to confirm the antibody does not recognize these related proteins.

How can researchers differentiate between direct and indirect effects of HS3ST3B1 modulation in functional studies?

Differentiating between direct and indirect effects of HS3ST3B1 modulation in functional studies requires sophisticated experimental approaches. First, employ acute manipulation systems such as inducible expression or degradation systems (e.g., doxycycline-inducible expression or auxin-inducible degradation) to observe immediate effects of HS3ST3B1 modulation before secondary adaptations occur. Second, utilize structure-function analysis by comparing wild-type HS3ST3B1 with catalytically inactive mutants (site-directed mutagenesis of critical residues in the sulfotransferase domain) to determine whether enzymatic activity is required for observed phenotypes. Third, perform domain deletion/mutation studies to identify specific protein interaction regions that mediate particular functions. Fourth, implement rescue experiments where specific downstream pathways are independently activated or inhibited following HS3ST3B1 modulation; if phenotypes are reversed, those pathways likely mediate the effects. Fifth, conduct temporal analysis of molecular changes following HS3ST3B1 modulation, as direct effects typically manifest more rapidly than indirect consequences. Sixth, use proximity labeling methods (BioID or APEX) to identify direct interaction partners of HS3ST3B1. Finally, employ specific inhibitors targeting the interaction between HS3ST3B1 and potential effector molecules, such as VEGF signaling pathway inhibitors like axitinib , to determine if blocking specific downstream pathways prevents the effects of HS3ST3B1 modulation.

What analytical approaches can identify potential compensatory mechanisms when HS3ST3B1 is knocked out or inhibited?

Identifying potential compensatory mechanisms when HS3ST3B1 is knocked out or inhibited requires a multi-faceted analytical approach. First, conduct comprehensive transcriptomic analysis (RNA-seq or microarray) comparing wild-type and HS3ST3B1-deficient samples to identify upregulated genes, particularly other sulfotransferases such as HS3ST1 and HS3ST3A1, which have shown trends of increased expression in HS3ST3B1 knockout models . Second, perform time-course analysis of gene expression changes following HS3ST3B1 disruption to distinguish immediate responses from adaptive mechanisms, focusing particularly on isolated epithelial tissues where compensatory expression changes are more readily detectable . Third, employ proteomics approaches (mass spectrometry) to identify changes in protein abundance and post-translational modifications, including alterations in heparan sulfate structure that might compensate for HS3ST3B1 loss. Fourth, analyze heparan sulfate disaccharide composition using specialized mass spectrometry or HPLC approaches to determine if alternative sulfation patterns emerge following HS3ST3B1 knockout. Fifth, conduct enzyme activity assays for related sulfotransferases to determine if increased enzymatic activity occurs independently of expression changes. Sixth, use double or triple knockout models (e.g., HS3ST3B1/HS3ST3A1 double knockout) to reveal functional redundancy and compensation; more severe phenotypes in multiple knockouts compared to single knockouts would suggest compensatory mechanisms. Finally, employ computational network analysis to identify altered signaling pathways and regulatory networks that might represent systemic compensatory responses to HS3ST3B1 deficiency.

What is the sensitivity range of biotin-conjugated HS3ST3B1 antibody in ELISA applications?

The sensitivity range of biotin-conjugated HS3ST3B1 antibody in ELISA applications typically spans from 10-20 pg/mL at the lower detection limit to approximately 2000 pg/mL at the upper quantification limit, though this can vary depending on the specific experimental conditions and detection systems employed. When used at the recommended dilution range of 1:100 to 1:500 , and coupled with a high-sensitivity streptavidin-HRP detection system, the antibody demonstrates optimal performance within this range. The standard curve typically shows linearity between approximately 50-1000 pg/mL, with some deviation at very low and very high concentrations. For maximum sensitivity, extended substrate incubation times (up to 30 minutes) and optimized washing procedures are recommended. The intra-assay coefficient of variation (CV) should be less than 10% and the inter-assay CV less than 15% across this range for reliable quantification. Researchers should note that sample matrix effects can influence sensitivity, and appropriate dilution of complex biological samples in assay buffer may be necessary to minimize interference. For applications requiring detection of extremely low abundance HS3ST3B1, signal amplification techniques such as tyramide signal amplification can further enhance sensitivity into the sub-picogram range.

What is the amino acid sequence of the immunogen used to generate the biotin-conjugated HS3ST3B1 antibody?

The biotin-conjugated HS3ST3B1 antibody (ABIN7155015) was generated using a recombinant human Heparan sulfate glucosamine 3-O-sulfotransferase 3B1 protein fragment corresponding to amino acids 54-390 as the immunogen . This extensive region represents a significant portion of the full-length protein, which typically consists of approximately 390 amino acids. While the exact sequence used in ABIN7155015 is not fully detailed in the provided search results, related antibodies targeting the N-terminal region of HS3ST3B1 have used peptide sequences including "AMLCVWLYMF LYSCAGSCAA APGLLLLGSG SRAAHDPPAL ATAPDGTPPR" . The selection of amino acids 54-390 as the immunogenic region is strategically significant as it encompasses most of the functional domains of the protein, including the catalytic sulfotransferase domain, while excluding the signal peptide and transmembrane regions that might interfere with antibody generation or specificity. This extended immunogen likely contributes to the antibody's effectiveness in recognizing the native protein in various applications, while the biotin conjugation provides additional versatility for detection systems utilizing streptavidin conjugates.

What Buffer Composition is optimal for maintaining biotin-conjugated HS3ST3B1 antibody stability?

The optimal buffer composition for maintaining biotin-conjugated HS3ST3B1 antibody stability combines several key components to preserve both protein integrity and biotin conjugation. The standard storage buffer includes 50% glycerol, which prevents freezing at -20°C and reduces protein denaturation during freeze-thaw cycles. A neutral buffering system of 0.01M PBS at pH 7.4 maintains optimal pH for antibody stability. Addition of 0.03% Proclin 300 as a preservative prevents microbial growth without damaging the antibody structure, unlike higher concentrations of sodium azide which can interfere with biotin-streptavidin interactions . For working solutions, dilution in PBS containing 1-2% BSA or other carrier proteins helps prevent antibody adsorption to surfaces and maintains stability. Antioxidants such as 0.01% thimerosal or 5mM EDTA can be added to prevent oxidative damage to both the antibody and the biotin moiety. Avoid buffers containing primary amines (e.g., Tris) when preparing working dilutions, as these can compete with the NHS-ester reaction used in biotin conjugation. For long-term storage beyond 6 months, addition of stabilizing proteins such as 0.1-1% BSA can provide further protection. The antibody should be stored in aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing significantly reduces both antibody activity and the integrity of the biotin conjugation.