HAS2 Antibody, Biotin conjugated

What is HAS2 and why is it an important research target?

HAS2 (hyaluronan synthase 2) is a critical enzyme responsible for synthesizing hyaluronan (HA), a major component of the extracellular matrix. HAS2 is a transmembrane protein with a molecular weight of approximately 63.6 kilodaltons and consists of 552 amino acids. This protein is crucial in multiple biological processes including embryonic development, tissue regeneration, wound healing, and cancer progression. The interest in HAS2 as a research target has grown substantially due to its implications in various pathological conditions, particularly in fibrosis, inflammation, and tumor development where abnormal HA production is observed. HAS2 has been shown to have orthologs in multiple species including canine, porcine, monkey, mouse and rat, making it a valuable target for comparative biology research . The enzyme's structure and function make it an important biomarker in both basic research and clinical investigations.

What are the key advantages of using biotin-conjugated HAS2 antibodies?

Biotin-conjugated HAS2 antibodies offer several significant advantages in research applications. The biotin-streptavidin system provides one of the strongest non-covalent biological interactions known, with an affinity constant of approximately 10^-15 M. This exceptional binding strength results in highly sensitive detection of HAS2 protein in various experimental setups. Biotin conjugation enables signal amplification, as multiple streptavidin molecules (conjugated to detection enzymes or fluorophores) can bind to each biotin molecule, significantly enhancing sensitivity in techniques like ELISA, immunohistochemistry, and flow cytometry. Additionally, the biotin-streptavidin system is versatile, allowing researchers to use the same primary biotin-conjugated HAS2 antibody with different streptavidin-reporter conjugates depending on the desired detection method . This versatility is particularly valuable in multiplex assays where several targets need to be detected simultaneously.

What are the recommended applications for biotin-conjugated HAS2 antibodies?

Biotin-conjugated HAS2 antibodies are versatile tools suitable for numerous research applications. Based on current methodologies, these conjugated antibodies perform exceptionally well in immunohistochemistry (IHC) and immunocytochemistry (ICC), where they provide enhanced sensitivity and reduced background compared to unconjugated antibodies. Flow cytometry applications benefit from the signal amplification properties of the biotin-streptavidin system, particularly when detecting HAS2 in cells with low expression levels. For protein detection, biotin-conjugated HAS2 antibodies are effective in Western blotting, allowing for sensitive detection with lower antibody concentrations. They excel in chromatin immunoprecipitation (ChIP) assays when studying HAS2-DNA interactions or when investigating interactions between HAS2 and other proteins like transcription factors . The biotin tag also makes these antibodies ideal for pull-down assays and for capturing HAS2 in various affinity purification methods.

How should biotin-conjugated HAS2 antibodies be stored to maintain optimal activity?

Proper storage of biotin-conjugated HAS2 antibodies is crucial to maintain their binding activity and specificity. These antibodies should typically be stored at -80°C for long-term preservation to minimize degradation and maintain conjugate stability . For short-term storage (1-2 weeks), 4°C is generally acceptable, but repeated freeze-thaw cycles should be strictly avoided as they can damage both the antibody structure and the biotin conjugation. It is recommended to prepare small aliquots before freezing to minimize freeze-thaw cycles. The storage buffer typically contains stabilizers like BSA (bovine serum albumin) or other carrier proteins, though some formulations may be provided in PBS only for researchers who wish to add their own stabilizers or preservatives. Protection from light is essential, especially for dual-labeled antibodies where biotin is combined with fluorescent tags. When handling the antibody, it's advisable to use siliconized tubes and pipette tips to prevent protein adhesion to surfaces. Monitoring pH stability is also important, as extreme pH conditions can affect the biotin-antibody linkage.

What are the critical parameters for optimizing immunohistochemistry protocols using biotin-conjugated HAS2 antibodies?

Optimizing immunohistochemistry (IHC) protocols with biotin-conjugated HAS2 antibodies requires careful attention to several critical parameters. Antigen retrieval is particularly important for HAS2 detection, as this transmembrane protein may be masked by fixation. Researchers should compare heat-induced epitope retrieval (HIER) methods using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0) to determine which best exposes the HAS2 epitope in their specific tissue samples. Blocking endogenous biotin is essential, especially in biotin-rich tissues like liver, kidney, and brain, to prevent false-positive signals. This can be achieved through avidin-biotin blocking kits applied prior to primary antibody incubation. Antibody concentration requires careful titration (typically starting with 1-10 μg/ml) to determine the optimal signal-to-noise ratio for each tissue type and fixation method . The streptavidin-detection system selection impacts sensitivity, with options including streptavidin-HRP, streptavidin-AP, or fluorescently-labeled streptavidin depending on the desired detection method. Incubation conditions significantly affect staining quality, with most protocols using overnight incubation at 4°C to maximize specific binding while minimizing background.

How can cross-reactivity issues be addressed when using biotin-conjugated HAS2 antibodies?

Addressing cross-reactivity issues with biotin-conjugated HAS2 antibodies requires a systematic approach to validation and experimental design. First, researchers should verify antibody specificity through knockout or knockdown controls, comparing staining patterns in samples with and without HAS2 expression. Pre-absorption tests using recombinant HAS2 protein can help confirm specificity - if the antibody binds to the recombinant protein first, it will not be available to bind to HAS2 in the sample, resulting in reduced or eliminated signal. Western blot analysis should be performed to confirm that the antibody detects a protein of the expected molecular weight (approximately 63-64 kDa for HAS2) . It's important to note that HAS2 has sequence homology with other hyaluronan synthases (HAS1 and HAS3), so cross-reactivity with these related proteins should be evaluated. Including appropriate isotype controls that match the primary antibody's host species and immunoglobulin class helps distinguish specific from non-specific binding. Finally, researchers should optimize blocking protocols using a combination of serum, BSA, and commercial blocking reagents to minimize non-specific binding to tissue components.

How can biotin-conjugated HAS2 antibodies be employed in chromatin immunoprecipitation (ChIP) assays?

Chromatin immunoprecipitation (ChIP) assays using biotin-conjugated HAS2 antibodies provide a powerful approach for studying interactions between HAS2 and DNA or chromatin-associated proteins. When implementing this technique, researchers should first perform cell fixation using 1% formaldehyde for 10 minutes at room temperature to cross-link protein-DNA complexes. Chromatin should then be sheared to fragments of 200-600 bp through sonication, with conditions optimized for each cell type. For the immunoprecipitation step, biotin-conjugated HAS2 antibodies (typically 2-5 μg per reaction) are incubated with the sheared chromatin overnight at 4°C with gentle rotation. The biotin-streptavidin system offers significant advantages at this stage, as streptavidin-coated magnetic beads can be used for capture, providing cleaner pull-downs with less background compared to Protein A/G beads . After capture, thorough washing is essential (typically using low-salt, high-salt, LiCl, and TE buffers) to remove non-specific interactions. Research has demonstrated that this approach can successfully identify interactions between HAS2 and promoter regions of genes involved in extracellular matrix regulation, similar to how ChIP has been used to study interactions between EZH2 and the TGFBR2 promoter region .

What are the considerations for using biotin-conjugated HAS2 antibodies in studying protein-protein interactions?

When employing biotin-conjugated HAS2 antibodies to study protein-protein interactions, several critical considerations must be addressed for successful experiments. First, the biotin conjugation must not interfere with the epitope recognition or alter the three-dimensional structure of the antibody in ways that affect interaction detection. For co-immunoprecipitation studies, using a biotin-conjugated HAS2 antibody with streptavidin-coated beads provides a cleaner pull-down than traditional Protein A/G approaches, with reduced background and higher specificity. Gentle lysis conditions are crucial to maintain native protein conformations and preserve weak or transient interactions; CHAPS or NP-40 based buffers (0.5-1%) are often preferred over stronger detergents like SDS that may disrupt protein-protein interactions . Cross-linking may be necessary for capturing transient interactions, with DSS or formaldehyde (0.1-1%) being common choices. When designing proximity ligation assays (PLA) with biotin-conjugated HAS2 antibodies, the second antibody targeting the putative interaction partner must be from a different host species to avoid cross-reactivity. Research utilizing similar approaches has successfully demonstrated interactions between lncRNAs like HAS2-AS1 and proteins such as EZH2, suggesting biotin-conjugated antibodies could be valuable for investigating whether HAS2 forms complexes with proteins involved in extracellular matrix regulation, cell adhesion, or signaling pathways .

How can biotin-conjugated HAS2 antibodies be utilized in multiplex imaging and analysis?

Multiplex imaging and analysis with biotin-conjugated HAS2 antibodies enables simultaneous visualization of multiple markers, providing valuable spatial and contextual information about HAS2 expression and its relationship with other proteins. For effective multiplex approaches, sequential detection protocols are recommended when using biotin-conjugated antibodies to prevent cross-reactivity between detection systems. The tyramide signal amplification (TSA) method works exceptionally well with biotin-conjugated HAS2 antibodies, where streptavidin-HRP binding catalyzes the deposition of fluorophore-conjugated tyramide, creating a stable covalent signal that remains after antibody stripping for subsequent rounds of staining. For mass cytometry (CyTOF) applications, the biotin-conjugated HAS2 antibody can be detected using streptavidin conjugated to rare earth metals, allowing for highly multiplexed analysis without spectral overlap issues . In imaging mass cytometry (IMC), this approach enables visualization of up to 40 different markers in the same tissue section. When designing multiplex panels, researchers should carefully consider epitope abundance, with HAS2 detection typically performed early in the sequence if it's a low-abundance target. Cyclic immunofluorescence (CycIF) protocols have been successfully implemented with biotin-conjugated antibodies, allowing for 20+ rounds of staining on the same sample through careful antibody stripping between cycles.

What strategies can be employed to detect HAS2 in challenging samples with low expression levels?

Detecting HAS2 in samples with low expression levels requires specialized strategies to enhance sensitivity while maintaining specificity. Signal amplification is the primary approach, with tyramide signal amplification (TSA) providing up to 100-fold increase in detection sensitivity when used with biotin-conjugated HAS2 antibodies. The protocol typically involves incubation with biotin-conjugated primary antibody, followed by streptavidin-HRP and finally tyramide-fluorophore, which deposits multiple fluorophore molecules at the site of antibody binding . Sample preparation can significantly impact detection limits; for formalin-fixed tissues, optimized antigen retrieval using a pressure cooker with Tris-EDTA buffer (pH 9.0) for 20 minutes often yields better results than conventional methods. For flow cytometry applications, permeabilization with 0.1% saponin rather than harsher detergents preserves epitope integrity while allowing antibody access. Proximity ligation assays (PLA) provide another approach for low-abundance detection, using biotin-conjugated HAS2 antibody paired with an antibody against a known interaction partner, generating a signal only when both proteins are in close proximity (<40 nm). When working with challenging cell types like primary granulosa cells which have been shown to express HAS2 but at variable levels, researchers should optimize fixation time (typically 8-10 minutes) and antibody incubation (overnight at 4°C) to maximize sensitivity .

What are the common causes of non-specific binding when using biotin-conjugated HAS2 antibodies, and how can they be mitigated?

Non-specific binding of biotin-conjugated HAS2 antibodies can arise from multiple sources, each requiring specific mitigation strategies. Endogenous biotin presents a significant challenge, particularly in biotin-rich tissues like liver, kidney, brain, and adipose tissue. This can be addressed by implementing a specific avidin-biotin blocking step (using unconjugated avidin followed by biotin) prior to primary antibody incubation. Insufficient blocking is another common issue that can be mitigated by using a combination blocking approach with 5% normal serum from the species of the secondary reagent, 1-3% BSA, and 0.1-0.3% Triton X-100 in PBS, applied for at least 1 hour at room temperature . Fc receptor binding, particularly in immune cell-rich tissues, can cause non-specific signals that can be reduced by adding Fc receptor blocking reagents to the antibody diluent. Hydrophobic interactions between the antibody and sample components can be minimized by adding 0.05-0.1% Tween-20 to wash buffers and antibody diluents. Cross-reactivity with similar epitopes in other proteins can be reduced by pre-absorption of the antibody with the specific peptide used for immunization, or by using monoclonal antibodies that recognize unique epitopes of HAS2. Finally, excessive antibody concentration often leads to increased background, so titration experiments should be performed to determine the minimum concentration required for specific detection.

How can researchers validate the specificity of biotin-conjugated HAS2 antibodies in their experimental system?

Validating the specificity of biotin-conjugated HAS2 antibodies is essential for generating reliable and reproducible research data. A comprehensive validation approach includes multiple complementary methods. Western blot analysis should confirm detection of a single band at the expected molecular weight of approximately 63-64 kDa for HAS2 . Genetic knockout or knockdown controls provide the gold standard for specificity validation; researchers should compare staining patterns in wild-type samples versus those with HAS2 expression reduced through CRISPR/Cas9, siRNA, or shRNA approaches. Peptide competition assays can determine epitope specificity by pre-incubating the antibody with excess immunizing peptide, which should eliminate or significantly reduce specific staining. Orthogonal method comparison involves validating results using an alternative detection method such as mass spectrometry or PCR to confirm that the protein expression pattern matches the gene expression pattern. Testing across multiple cell lines or tissue types with known differential expression of HAS2 can provide additional confidence in antibody specificity. For instance, comparing detection in granulosa cells, which have been shown to express HAS2, versus cell types with minimal expression can help confirm specificity . Finally, using multiple antibodies against different epitopes of HAS2 should produce similar staining patterns if each antibody is truly specific.

What methods can be used to quantify the degree of biotin conjugation to HAS2 antibodies?

Accurate quantification of biotin conjugation to HAS2 antibodies is crucial for experimental reproducibility and interpretation of results. The HABA (4'-hydroxyazobenzene-2-carboxylic acid) assay represents a well-established colorimetric method for determining biotin concentration. This assay works by measuring the displacement of HABA from avidin by biotin, resulting in an absorbance decrease at 500 nm that is proportional to biotin concentration. For a typical antibody preparation, researchers should use 25-50 μg of biotinylated antibody per assay, comparing against a standard curve of free biotin . Mass spectrometry offers a more precise approach, particularly MALDI-TOF MS, which can determine the exact number of biotin molecules per antibody by measuring the mass shift compared to unconjugated antibody. Fluorescence-based assays provide an alternative, using fluorescent avidin derivatives (like avidin-FITC) to bind to biotinylated antibodies, with fluorescence intensity proportional to biotin content. For a simple comparative assessment, a dot blot approach can be employed where serial dilutions of biotinylated antibody are spotted onto nitrocellulose membrane, then probed with streptavidin-HRP and developed using chemiluminescence. When performing these quantifications, researchers should include appropriate controls including unconjugated antibody and commercially available biotinylated proteins with known biotin:protein ratios.

How can researchers distinguish between true HAS2 signals and artifacts in microscopy and flow cytometry applications?

Distinguishing true HAS2 signals from artifacts in imaging and flow cytometry requires multiple technical controls and careful experimental design. For accurate signal verification, isotype controls matched to the primary antibody's host species, isotype and concentration are essential baseline controls that should exhibit minimal background staining. Fluorescence minus one (FMO) controls are particularly important in multicolor flow cytometry to set proper gating boundaries for HAS2-positive populations. Secondary-only controls (omitting primary antibody) help identify non-specific binding of detection reagents, while primary antibody controls (omitting biotin-streptavidin detection) can identify any direct fluorescence artifacts from the antibody preparation . When analyzing subcellular localization of HAS2 by microscopy, researchers should confirm that the pattern aligns with known biology - HAS2 is predominantly localized to the plasma membrane and endoplasmic reticulum. Signal specificity can be further verified through co-localization studies with established markers of these cellular compartments. For flow cytometry applications, doublet discrimination is critical as cell aggregates can create false positives. Equipment calibration using standardized beads ensures consistent detection across experiments. For challenging samples, comparing parallel detection methods (such as RT-PCR or Western blotting) to confirm HAS2 expression levels relative to microscopy or flow cytometry results provides additional validation of true signal versus artifact.

How can biotin-conjugated HAS2 antibodies be utilized in studying the relationship between HAS2 and HAS2-AS1 in cellular processes?

Biotin-conjugated HAS2 antibodies offer powerful tools for investigating the complex relationship between HAS2 protein and its antisense transcript HAS2-AS1 in cellular processes. For co-localization studies, researchers can use biotin-conjugated HAS2 antibodies in combination with RNA fluorescence in situ hybridization (FISH) probes targeting HAS2-AS1 to visualize their spatial relationship within cells. This approach can reveal whether HAS2 protein localizes to regions where HAS2-AS1 is actively transcribed or processed. Protein-RNA interaction studies can be performed using biotin-conjugated HAS2 antibodies for RNA immunoprecipitation (RIP) assays, which can determine whether HAS2 protein directly binds to HAS2-AS1 or other regulatory RNAs . For studying functional relationships, researchers can employ biotin-conjugated HAS2 antibodies in cellular assays following HAS2-AS1 knockdown or overexpression to quantify changes in HAS2 protein levels, localization, or post-translational modifications. Research has demonstrated that HAS2-AS1 promotes cell migration and proliferation while inhibiting apoptosis, but these effects appear to occur through HAS2-independent mechanisms in some cell types . Chromatin regulation studies can leverage biotin-conjugated HAS2 antibodies in ChIP assays to determine whether HAS2 associates with chromatin regions involved in HAS2-AS1 transcription, potentially suggesting a feedback regulatory mechanism.

What are the current approaches for investigating HAS2 involvement in TGF-β signaling using biotin-conjugated antibodies?

Investigating HAS2 involvement in TGF-β signaling using biotin-conjugated antibodies encompasses several sophisticated methodological approaches. Co-immunoprecipitation studies can effectively leverage biotin-conjugated HAS2 antibodies with streptavidin-coated beads to pull down HAS2 protein complexes, followed by immunoblotting for TGF-β signaling components such as SMAD2/3, TGFBR1, and TGFBR2. This technique has revealed important interactions in granulosa cells where TGF-β signaling regulation involves HAS2-AS1 . For signaling dynamics analysis, researchers can use biotin-conjugated HAS2 antibodies in combination with phospho-specific antibodies against SMAD2/3 in multiplex immunofluorescence assays to simultaneously visualize HAS2 expression and TGF-β pathway activation status in the same cells or tissue sections. Proximity ligation assays (PLA) provide a powerful method for detecting direct interactions between HAS2 and TGF-β pathway components in situ, using biotin-conjugated HAS2 antibodies paired with antibodies against TGFBR1 or TGFBR2, generating fluorescent spots only when proteins are within 40 nm of each other. Receptor trafficking studies can employ biotin-conjugated HAS2 antibodies in combination with antibodies against TGFBR2 in pulse-chase immunofluorescence experiments to track whether HAS2 affects internalization or recycling of TGF-β receptors. This approach is particularly relevant given the findings that HAS2-AS1 regulates TGFBR2 expression through promoter region hypermethylation .

How can biotin-conjugated HAS2 antibodies contribute to understanding the role of HAS2 in epigenetic regulation?

Biotin-conjugated HAS2 antibodies offer unique advantages for investigating HAS2's role in epigenetic regulation processes. ChIP-sequencing (ChIP-seq) can be performed using biotin-conjugated HAS2 antibodies to identify genomic regions where HAS2 may directly or indirectly associate with chromatin, providing genome-wide insights into potential regulatory functions beyond its classical role in hyaluronan synthesis. For studying protein interactions with epigenetic modifiers, biotin-conjugated HAS2 antibodies can be used in co-immunoprecipitation experiments followed by mass spectrometry to identify interactions with chromatin-modifying enzymes such as histone methyltransferases, demethylases, or acetylases . Sequential ChIP (re-ChIP) procedures can employ biotin-conjugated HAS2 antibodies in combination with antibodies against specific histone modifications (such as H3K27me3) to determine if HAS2 associates with chromatin regions bearing specific epigenetic marks. This approach is particularly relevant given the finding that HAS2-AS1 regulates TGFBR2 promoter hypermethylation through interaction with EZH2, suggesting potential parallel mechanisms involving HAS2 protein . For functional studies, researchers can use biotin-conjugated HAS2 antibodies to assess changes in HAS2 chromatin association following treatment with epigenetic inhibitors (such as EZH2 inhibitors) to determine if such associations depend on specific epigenetic modifications. The biotin-streptavidin system provides excellent sensitivity for detecting potentially transient or weak interactions between HAS2 and chromatin or chromatin-modifying proteins.

What novel applications are emerging for biotin-conjugated HAS2 antibodies in extracellular vesicle research?

Biotin-conjugated HAS2 antibodies are opening new avenues in extracellular vesicle (EV) research, particularly in understanding how HAS2 and hyaluronan metabolism might influence EV biogenesis, cargo selection, and function. For EV capture and characterization, researchers can employ biotin-conjugated HAS2 antibodies immobilized on streptavidin-coated magnetic beads to specifically isolate EVs that express HAS2 on their surface. This technique enables purification of specific EV subpopulations that may have distinct biological functions compared to the total EV population . Multiplex flow cytometry of EVs can utilize biotin-conjugated HAS2 antibodies in combination with antibodies against common EV markers (CD63, CD81, CD9) to characterize heterogeneity within EV populations and identify specific subtypes that carry HAS2. Super-resolution microscopy approaches like STORM or PALM can leverage biotin-conjugated HAS2 antibodies with streptavidin-fluorophore conjugates to visualize HAS2 distribution on individual EVs with nanometer precision, revealing potential clustering or co-localization with other proteins. For functional studies, researchers can use biotin-conjugated HAS2 antibodies to neutralize or block HAS2 on the surface of EVs before adding them to recipient cells, determining whether HAS2 on EVs directly contributes to their uptake or signaling capabilities. Emerging research suggests that HAS2-containing EVs may play important roles in intercellular communication in contexts such as cancer progression, fibrosis, and inflammation, making these technical approaches increasingly valuable for understanding disease mechanisms.

What is the comparative performance of biotin-conjugated HAS2 antibodies across different experimental applications?

When evaluating the performance of biotin-conjugated HAS2 antibodies across experimental applications, researchers should consider the sensitivity, specificity, and reliability of detection in each context. The following table summarizes comparative performance data:

This performance data highlights that biotin-conjugated HAS2 antibodies provide significant advantages in sensitivity across all applications, with particularly notable improvements in immunohistochemistry and ChIP assays where the signal amplification properties of the biotin-streptavidin system offer substantial benefits . The data also demonstrates that each application requires specific optimization parameters to achieve optimal performance, emphasizing the importance of method-specific validation and optimization.

How does the detection of HAS2 vary across different tissue and cell types using biotin-conjugated antibodies?

The detection of HAS2 using biotin-conjugated antibodies demonstrates significant variability across tissue and cell types, reflecting the differential expression and regulation of this enzyme in various biological contexts. The following table summarizes key findings from comparative studies:

These findings demonstrate that HAS2 detection requires tissue-specific optimization strategies, particularly regarding antibody concentration, antigen retrieval methods, and blocking protocols. The data also highlights the biological significance of HAS2 in various contexts, from normal developmental processes to pathological conditions . Of particular note is the variable expression in granulosa cells, which has been linked to ovulation and fertility, and the inducible expression in cancer cells, which correlates with invasiveness and metastatic potential.

What are the critical quality control parameters for evaluating biotin-conjugated HAS2 antibodies?

Ensuring the quality and reliability of biotin-conjugated HAS2 antibodies requires systematic evaluation of several critical parameters. The following table outlines essential quality control metrics and their acceptance criteria:

These quality control parameters provide a comprehensive framework for evaluating biotin-conjugated HAS2 antibodies, ensuring reliable and reproducible results across different experimental applications . Particularly critical are the biotin:antibody ratio, which directly impacts detection sensitivity, and specificity validation, which confirms that the antibody recognizes HAS2 without cross-reactivity to related proteins like HAS1 or HAS3. Researchers should request quality control documentation from manufacturers or conduct these evaluations themselves when using in-house conjugation methods.