HS3ST3B1 Antibody, HRP conjugated

What is HS3ST3B1 and what biological functions does it serve?

HS3ST3B1 (Heparan Sulfate Glucosamine 3-O-Sulfotransferase 3B1) is an enzyme responsible for generating 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. These modified epitopes are rare but highly sulfated structures that play crucial roles in developmental processes. Research has shown that 3-O-sulfated HS stabilizes the FGF10/FGFR2b complex, promoting MAPK signaling and expansion of progenitor cells within developing structures such as salivary gland endbuds . Additionally, 3-O-sulfated HS serves as a binding preference for multiple receptors and ligands including FGFR1 ectodomain, FGF1, cyclophilin, stabilin, neuropilin, FGF10-FGFR2b complex, Tau, BMP4, and FGF8 . These interactions suggest that 3-O-sulfated HS epitopes modulate growth factor functions across various biological contexts.

What are the specifications of commercially available HS3ST3B1 antibodies conjugated to HRP?

The HRP-conjugated rabbit polyclonal anti-HS3ST3B1 antibody targets the amino acid region 54-390 of human HS3ST3B1 . This antibody is designed for research applications with specific reactivity to human samples . Alternative forms of HS3ST3B1 antibodies include unconjugated versions and those with different conjugates such as FITC and biotin . When selecting an antibody for your research, consider the specific amino acid regions targeted, as some antibodies target the N-terminal region while others target specific amino acid ranges (e.g., AA 38-87 or AA 54-390) . The polyclonal nature of these antibodies provides broad epitope recognition but may introduce batch-to-batch variation that should be considered in experimental design.

What sample types and applications are validated for HS3ST3B1 antibody, HRP conjugated?

The HRP-conjugated HS3ST3B1 antibody has been validated for human samples . While specific applications for the HRP-conjugated variant focus on ELISA , related unconjugated HS3ST3B1 antibodies have been validated for Western Blotting (WB) using cell lysates as positive controls . For optimal results, it is recommended to determine working dilutions experimentally for your specific application and sample type . The antibody has demonstrated reactivity with human samples, though predicted cross-reactivity exists with various species including cow (92%), dog (93%), guinea pig (86%), horse (93%), pig (93%), rabbit (87%), and rat (93%) . This cross-reactivity profile should be considered when designing experiments with non-human tissue samples.

How should I design experiments to study HS3ST3B1 expression patterns using HRP-conjugated antibodies?

For studying HS3ST3B1 expression patterns, design a multi-method approach combining immunohistochemistry, Western blotting, and qPCR for comprehensive validation. When using HRP-conjugated antibodies for immunohistochemistry, implement a tiered protocol starting with fixation optimization (test both PFA and acetone fixation), antigen retrieval assessment (citrate buffer, pH 6.0 is recommended), and antibody concentration gradients (typically 1:100 to 1:1000). Include parallel experiments with unconjugated primary antibodies and separate HRP-conjugated secondary antibodies as a procedural control to validate direct conjugate performance . Based on single-cell RNA-seq data from salivary gland development, HS3ST3B1 shows differential expression in endbud epithelial cells and myoepithelial cells across developmental stages . This expression pattern can guide your experimental tissue selection, with E13-E16 embryonic tissues and adult tissues being particularly relevant for studying developmental expression patterns.

What controls should I include when using HRP-conjugated HS3ST3B1 antibody in my experiments?

A comprehensive control strategy for HRP-conjugated HS3ST3B1 antibody experiments should include multiple elements to ensure reliable results. First, incorporate both positive and negative tissue controls—use tissues known to express HS3ST3B1 (such as embryonic kidney ureteric bud tips or salivary gland endbuds) as positive controls , and include tissue from Hs3st3b1 knockout models as negative controls where available . Second, include isotype controls using non-specific rabbit IgG at matching concentrations to assess non-specific binding. Third, implement absorption controls by pre-incubating the antibody with excess target peptide (the immunogen used for antibody production) to confirm binding specificity. Fourth, cross-validate with alternative detection methods such as unconjugated primary antibody plus HRP-secondary antibody to verify that direct HRP conjugation doesn't interfere with epitope recognition. Finally, include Western blot validation alongside immunohistochemistry to confirm antibody specificity to the target protein size.

What is the best methodology for quantifying HS3ST3B1 enzymatic activity in conjunction with immunodetection?

Quantifying HS3ST3B1 enzymatic activity alongside immunodetection requires a complementary approach that correlates protein expression with functional activity. Begin with immunoprecipitation using the unconjugated HS3ST3B1 antibody to isolate the enzyme from tissue or cell lysates. Then conduct an in vitro sulfotransferase assay using purified heparan sulfate substrate, [35S]PAPS (3'-phosphoadenosine 5'-phosphosulfate) as the sulfate donor, and measure incorporation of radiolabeled sulfate through scintillation counting. Alternatively, implement a non-radioactive assay using a colorimetric detection of released PAP (3'-phosphoadenosine 5'-phosphate). The enzymatic activity should be normalized to the amount of immunoprecipitated protein as quantified by Western blot using the HRP-conjugated antibody. For in situ activity correlation, perform sequential tissue sections analysis with one section for immunohistochemistry using the HRP-conjugated antibody and adjacent sections for detection of 3-O-sulfated HS epitopes using specialized phage display antibodies such as HS4C3V . This approach provides spatial correlation between enzyme expression and functional output.

How do Hs3st3a1 and Hs3st3b1 enzymes compensate for each other in knockout models and what implications does this have for antibody-based detection?

Studies of knockout models reveal complex compensatory mechanisms between Hs3st3a1 and Hs3st3b1 enzymes. Analysis of isolated epithelial cultures from Hs3st3b1 knockout embryos showed a trend toward increased Hs3st1 and Hs3st3a1 expression, suggesting these isoforms may compensate for Hs3st3b1 loss . Interestingly, in Hs3st3a1 knockout epithelia, Hs3st3b1 expression was reduced, indicating potential regulatory relationships between these enzymes . This interplay complicates antibody-based detection approaches, as changes in one isoform may affect expression of others. When designing antibody-based experiments with knockout models, researchers should implement multiplexed detection of all relevant Hs3st isoforms, particularly when analyzing developmental processes. Additionally, single-cell RNA-seq data from various developmental stages shows that Hs3st3b1 is generally more highly expressed than Hs3st3a1, with both enzymes predominantly expressed in endbud and myoepithelial cells . This differential expression pattern suggests tissue-specific compensatory mechanisms that should be considered when interpreting antibody staining patterns in different developmental contexts.

What methodological approaches can reveal the relationship between HS3ST3B1 activity and FGF signaling pathways?

To investigate the relationship between HS3ST3B1 activity and FGF signaling, implement a comprehensive approach combining biochemical, cellular, and developmental techniques. First, establish protein-protein interaction studies using co-immunoprecipitation with the HS3ST3B1 antibody to isolate protein complexes, followed by immunoblotting for FGF10 and FGFR2b components. Second, conduct bead-based binding assays where heparan sulfate modified by recombinant HS3ST3B1 is conjugated to beads and used to assess FGF10-FGFR2b complex formation and stability. Third, implement developmental organ culture assays using submandibular glands (SMGs) isolated from wild-type and Hs3st3b1 knockout embryos, treated with FGF10, and assessed for branching morphogenesis . In these cultures, quantify downstream FGFR signaling by measuring phosphorylation of ERK1/2 and expression of transcriptional targets like Etv5 . Finally, use live cell imaging with fluorescently-tagged FGF10 to track receptor binding and internalization rates in cells with normal or reduced HS3ST3B1 expression. Knockout models show that deletion of Hs3st3b1 reduces branching morphogenesis in ex vivo culture of embryonic salivary glands, confirming its importance in FGF10-dependent morphogenesis .

How can the HRP-conjugated HS3ST3B1 antibody be used to investigate developmental timing of HS3ST3B1 expression?

To investigate developmental timing of HS3ST3B1 expression using HRP-conjugated antibodies, implement a chronological tissue sampling approach combined with multiplexed detection systems. Collect tissue samples across critical developmental timepoints, guided by the single-cell RNA sequencing data that demonstrates dynamic expression of Hs3st3b1 from embryonic day 12 (E12) through adulthood . Process tissues for immunohistochemistry using the HRP-conjugated antibody with 3,3'-diaminobenzidine (DAB) detection, and perform parallel immunofluorescence with unconjugated antibodies on adjacent sections to facilitate co-localization studies. To maximize information yield, implement multiplexed immunofluorescence combining HS3ST3B1 detection with markers for specific cell populations identified in scRNA-seq studies: KRT5 for basal cells, KRT19 for differentiated duct cells, ACTA2 for myoepithelial cells, and KIT for progenitor cells . For developmental timing analysis, establish a semi-quantitative scoring system for staining intensity across these timepoints, and correlate protein expression patterns with the transcript expression data from scRNA-seq. This approach will reveal potential post-transcriptional regulation mechanisms if protein and transcript patterns diverge.

How should I optimize immunohistochemistry protocols using HRP-conjugated HS3ST3B1 antibody?

Optimizing immunohistochemistry with HRP-conjugated HS3ST3B1 antibody requires systematic parameter adjustment across multiple dimensions. Begin with a fixation comparison, testing both 4% paraformaldehyde (12-24 hours) and acetone fixation (10 minutes) on parallel tissue sections to determine optimal epitope preservation. Next, evaluate multiple antigen retrieval methods including heat-induced epitope retrieval with citrate buffer (pH 6.0), EDTA buffer (pH 9.0), and enzymatic retrieval with proteinase K (10 μg/mL, 10 minutes at 37°C). For blocking, test a gradient of bovine serum albumin concentrations (1-5%) combined with normal serum from a species distinct from the antibody host. The antibody dilution optimization should follow a geometric series (1:100, 1:200, 1:400, 1:800, 1:1600) to determine optimal signal-to-noise ratio. When working with the HRP conjugate, implement hydrogen peroxide blocking (0.3% H₂O₂ in methanol for 30 minutes) before antibody application to quench endogenous peroxidase activity. For visualization, compare DAB development times (30 seconds to 10 minutes) and consider signal amplification using tyramide signal amplification for low-abundance targets. Validate specificity using tissues from Hs3st3b1 knockout models as negative controls .

What are the major sources of background and non-specific staining when using HRP-conjugated antibodies, and how can they be mitigated?

Major sources of background with HRP-conjugated antibodies include endogenous peroxidase activity, non-specific antibody binding, tissue autofluorescence, and cross-reactivity issues. To address endogenous peroxidase activity, implement dual blocking with 0.3% hydrogen peroxide in methanol for 30 minutes, followed by 3% hydrogen peroxide in water for 10 minutes. For non-specific binding, use a comprehensive blocking solution containing 5% BSA, 5% normal serum from the species unrelated to primary antibody host, 0.3% Triton X-100, and 0.05% Tween-20 in PBS for 2 hours at room temperature. Tissue autofluorescence can be mitigated using Sudan Black B treatment (0.1% in 70% ethanol) for 20 minutes after antibody incubation when performing bright-field microscopy. To address potential cross-reactivity (given the antibody's predicted reactivity with multiple species including cow, dog, guinea pig, horse, pig, rabbit, and rat ), pre-absorb the antibody with tissue homogenates from non-target species when working with multi-species samples. Additionally, optimize incubation conditions by comparing overnight incubation at 4°C with 2-hour incubation at room temperature, and test multiple washing regimens varying in duration, buffer composition, and detergent concentration.

What is the best approach for multiplexing HS3ST3B1 antibody with other markers in the same tissue section?

For effective multiplexing of HRP-conjugated HS3ST3B1 antibody with other markers, implement a sequential detection approach with appropriate enzymatic inactivation steps. Begin with detection of the HRP-conjugated HS3ST3B1 antibody using DAB substrate, followed by thorough washing and heat treatment (80°C in citrate buffer, pH 6.0 for 10 minutes) to denature and inactivate the first set of immunoreagents. Subsequently apply the second primary antibody, which should be from a different host species than the HS3ST3B1 antibody, followed by an AP-conjugated secondary antibody and visualization with Fast Red or Vector Blue substrate to achieve color distinction. For fluorescent multiplexing, use tyramide signal amplification (TSA) for the HRP-conjugated antibody, which allows for subsequent heat-mediated antibody stripping while preserving the covalently bound fluorophore signal. This enables sequential application of multiple antibodies to the same section. Based on single-cell RNA-seq data, strategic marker combinations for studying HS3ST3B1 in salivary gland development would include ACTA2 for myoepithelial cells, KRT19 for duct cells, and BPIFA2 for proacinar cells, as these populations show differential expression of Hs3st3a1 and Hs3st3b1 across developmental stages .

How should I interpret discrepancies between immunohistochemistry results and RNA expression data for HS3ST3B1?

When confronting discrepancies between HS3ST3B1 immunohistochemistry results and RNA expression data, implement a systematic analysis framework to identify the source of incongruence. First, verify antibody specificity through parallel testing in knockout models where available . Second, consider post-transcriptional regulation mechanisms—RNA expression may not directly correlate with protein levels due to differential translation efficiency or protein stability. Third, evaluate temporal dynamics by examining multiple timepoints, as the scRNA-seq data reveals stage-specific expression patterns of Hs3st3b1 during development . Fourth, assess cell type heterogeneity through parallel section analysis with cell type-specific markers corresponding to populations identified in scRNA-seq (endbud, myoepithelial, ductal cells). Fifth, consider technical variables including sensitivity differences between antibody detection and RNA quantification methods. Sixth, examine potential compensatory mechanisms, as knockout studies have revealed complex interplay between different Hs3st isoforms . Finally, validate your findings through alternative methods such as RNAscope to visualize transcripts in tissue context, or Western blotting to quantify protein levels across developmental stages.

What are the most robust methods to validate specificity of the HS3ST3B1 antibody in experimental systems?

Validating HS3ST3B1 antibody specificity requires a multi-modal approach incorporating genetic, biochemical, and analytical methods. The gold standard is testing the antibody in Hs3st3b1 knockout tissues, which should show absence of specific staining . When knockouts are unavailable, implement siRNA knockdown in appropriate cell lines, followed by Western blotting to confirm specificity. Peptide competition assays provide another validation strategy—pre-incubate the antibody with excess immunogen peptide before application to tissues or Western blots; specific staining should be abolished. For cross-validation, compare detection patterns across multiple antibodies targeting different epitopes of HS3ST3B1. Orthogonal validation methods include correlation of antibody staining with mRNA detection by in situ hybridization or RNAscope in the same tissue regions. Mass spectrometry analysis of immunoprecipitated proteins can confirm antibody capture of the intended target. Additionally, validate the antibody across multiple applications (IHC, Western blot, IP) to ensure consistent target recognition. The antibody specifications indicate it was validated on Western Blot using cell lysate as positive control , providing a baseline for expected performance.

How can I quantitatively assess the co-localization of HS3ST3B1 with 3-O-sulfated heparan sulfate epitopes?

To quantitatively assess co-localization between HS3ST3B1 and its enzymatic product (3-O-sulfated heparan sulfate epitopes), implement a dual labeling approach followed by rigorous image analysis. First, perform sequential immunostaining on adjacent tissue sections: one section with the HRP-conjugated HS3ST3B1 antibody and the adjacent section with the HS4C3V phage display antibody that specifically recognizes 3-O-sulfated HS epitopes . Alternatively, for direct co-localization, use fluorescent multiplexing with the unconjugated HS3ST3B1 primary antibody paired with a fluorescent secondary antibody, combined with fluorescently-labeled HS4C3V antibody. Acquire high-resolution confocal z-stack images and process them using specialized co-localization analysis software. Quantitative assessment should include multiple metrics: Pearson's correlation coefficient (values > 0.5 indicating significant co-localization), Mander's overlap coefficient to determine the fraction of each signal overlapping with the other, and intensity correlation analysis to assess whether the intensities of both signals vary together. For spatial pattern analysis, implement distance mapping to measure the proximity of HS3ST3B1-positive cells to 3-O-sulfated HS deposits. Research has shown that in Hs3st3b1 knockout mice, there is reduced 3-O-sulfated HS in the basement membrane , providing a valuable negative control for validating detection specificity.

What methodological approaches can distinguish between different Hs3st isoforms (Hs3st1, Hs3st3a1, Hs3st3b1, Hs3st6) when using antibody-based detection?

Distinguishing between highly homologous Hs3st isoforms requires a sophisticated approach combining isoform-specific antibodies with complementary techniques. First, perform epitope mapping to identify unique regions for each isoform, focusing on the N-terminal domains which show greater sequence divergence than the conserved catalytic domains. Design a panel of blocking experiments where tissues are pre-incubated with recombinant proteins of each isoform before antibody application to identify cross-reactivity. Implement Western blotting with recombinant proteins of all isoforms to assess antibody cross-reactivity quantitatively. For tissue detection, use sequential sections stained with isoform-specific antibodies and analyze expression patterns in reference to scRNA-seq data showing that Hs3st1 is broadly expressed across cell types, while Hs3st3a1 and Hs3st3b1 show more restricted expression in endbud and myoepithelial cells, and Hs3st6 is expressed in specific duct populations . The developmental expression patterns provide a natural specificity control, as Hs3st3a1 and Hs3st3b1 show increased expression in specific cell types like myoepithelial cells at P1 and adult stages . For ultimate specificity validation, use tissues from the respective knockout models, testing each antibody against all available Hs3st knockouts to confirm absence of cross-reactivity.