STARD13 Antibody, Biotin conjugated

What is STARD13 and why is it significant in research?

STARD13 (StAR-related lipid transfer domain containing 13), also known as DLC2 (Deleted in liver cancer 2), functions as a GTPase-activating protein primarily for RhoA and possibly Cdc42. It plays crucial roles in regulating cytoskeletal reorganization, cell proliferation, and cell motility pathways. Most significantly, STARD13 acts as a tumor suppressor in hepatocellular carcinoma cells, making it an important research target in oncology . The protein contains a START domain that facilitates lipid transfer functions, positioning it at the intersection of membrane dynamics and signal transduction. For researchers, STARD13's involvement in cancer suppression mechanisms makes antibodies against this protein valuable tools for studying tumor progression and potential therapeutic interventions.

What are the key characteristics of commercially available STARD13 antibodies?

Most commercially available STARD13 antibodies are rabbit polyclonal antibodies that target specific epitopes within the human STARD13 protein. These antibodies typically have a molecular weight recognition of approximately 125 kDa, which corresponds to the full-length STARD13 protein . For instance, the STARD13 antibody catalog #21325-1-AP shows reactivity with human and mouse samples, while NBP2-85837 is designed specifically for human samples . The immunogens used to generate these antibodies vary, with some derived from recombinant protein fragments and others from synthetic peptides targeting specific regions of the STARD13 protein . Understanding these characteristics is essential for selecting the appropriate antibody for your experimental system.

What applications are STARD13 antibodies validated for?

STARD13 antibodies have been validated for multiple research applications, primarily Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence/Immunocytochemistry (IF/ICC). The recommended dilutions vary by application: for Western Blot, dilutions typically range from 1:500-1:2000; for IF/ICC applications, dilutions of 1:50-1:500 are common . Published studies have successfully employed these antibodies in knockout/knockdown validation experiments, further confirming their specificity . When using STARD13 antibodies for Western Blot, researchers have successfully detected the protein in various human cell lines including MCF-7, HeLa, RT4, U251 MG, and PANC1, as well as in human tissue lysates from liver, tonsil, and pancreas . This broad validation across multiple techniques and sample types provides researchers with confidence in antibody performance across diverse experimental contexts.

How should STARD13 antibodies be stored and handled to maintain optimal activity?

STARD13 antibodies should be stored at -20°C for long-term stability. Most formulations contain preservatives such as sodium azide (typically 0.02-0.09%) and stabilizers like glycerol (often 50%) to maintain antibody integrity . The storage buffer generally consists of PBS with specific pH (typically 7.3-7.4) to ensure optimal protein stability . For antibodies stored as liquids, it's recommended to aliquot upon receipt to avoid repeated freeze-thaw cycles that can degrade antibody quality. Most manufacturers indicate that properly stored antibodies remain stable for at least one year after shipment . When working with biotin-conjugated antibodies specifically, similar storage conditions apply, but special attention should be paid to protecting the conjugate from light exposure during handling to prevent photobleaching of the biotin molecule.

How can STARD13 antibodies be utilized to investigate its tumor suppressor function in cancer research?

STARD13 has been identified as a significant tumor suppressor, particularly in hepatocellular carcinoma and papillary thyroid carcinoma . To investigate this function, researchers can employ multiple antibody-based approaches. First, immunohistochemistry using STARD13 antibodies on tissue microarrays comparing normal versus cancerous tissues can reveal expression pattern changes associated with malignant transformation. Western blot analysis across cancer progression models can quantify STARD13 downregulation during carcinogenesis. For mechanistic studies, researchers should combine STARD13 immunoprecipitation with mass spectrometry to identify novel interaction partners in the tumor suppression pathway. Additionally, chromatin immunoprecipitation (ChIP) experiments using transcription factor antibodies can elucidate how STARD13 expression is regulated at the genomic level. Recent publications have demonstrated that loss of STARD13 contributes to aggressive phenotype transformation in papillary thyroid carcinoma, suggesting that quantifying STARD13 levels may serve as a prognostic biomarker .

What methodological considerations should be addressed when studying STARD13's interaction with RhoA and Cdc42?

Studying STARD13's interactions with RhoA and Cdc42 requires careful experimental design due to the dynamic nature of these GTPase interactions. Researchers should employ co-immunoprecipitation assays using STARD13 antibodies under both basal and stimulated conditions to capture physiologically relevant interactions. It's critical to use lysis buffers that preserve GTPase-protein interactions (typically containing low concentrations of non-ionic detergents). To distinguish between active and inactive GTPase binding, researchers should perform pull-down assays with GTP-γS or GDP-loaded RhoA/Cdc42 alongside STARD13 immunoprecipitation. For live-cell imaging of these interactions, consider using proximity ligation assays (PLA) with STARD13 antibodies and RhoA/Cdc42 antibodies to visualize interaction events in situ. When analyzing these interactions by Western blot, remember that the GAP activity of STARD13 may alter the GTP/GDP-bound state of these GTPases, potentially affecting their migration patterns and antibody recognition .

How can biotin-conjugated STARD13 antibodies enhance detection sensitivity in complex tissue samples?

Biotin-conjugated antibodies offer significant advantages for detecting low-abundance proteins like STARD13 in complex tissue samples. The biotin-streptavidin system provides signal amplification through the high-affinity binding (Kd ≈ 10^-15 M) of streptavidin to biotin, enabling detection of proteins expressed at low levels. For optimal results in immunohistochemistry of tissues with high endogenous biotin (like liver or kidney), researchers should implement an avidin/biotin blocking step prior to primary antibody incubation. When working with formalin-fixed paraffin-embedded samples, antigen retrieval methods should be optimized specifically for STARD13 detection, as the protein's conformation may be particularly sensitive to fixation artifacts. For multiplex imaging, biotin-conjugated STARD13 antibodies can be visualized using fluorescently labeled streptavidin conjugates with distinct emission spectra, allowing simultaneous detection of multiple targets without cross-reactivity issues common with secondary antibodies .

What approaches can be used to validate STARD13 antibody specificity in the context of biotin conjugation?

Validating the specificity of biotin-conjugated STARD13 antibodies requires multiple complementary approaches. First, perform parallel Western blots comparing the biotin-conjugated antibody against a well-validated unconjugated STARD13 antibody to confirm identical banding patterns at the expected 125 kDa molecular weight . Incorporate STARD13 knockdown/knockout controls to demonstrate signal reduction or elimination with decreased target expression. For cell and tissue applications, pre-absorption controls using the recombinant STARD13 protein immunogen should substantially reduce or eliminate specific staining. Additionally, conduct peptide competition assays using the synthetic peptide immunogen (particularly for antibodies like NBP2-85837 generated against peptide sequences) to confirm epitope-specific binding . When validating biotin-conjugated antibodies specifically, include controls for potential streptavidin binding to endogenous biotin by performing detection protocols with streptavidin reagents alone, without the primary biotin-conjugated antibody.

What is the mechanism behind biotin conjugation and how does it affect antibody performance?

Biotin conjugation involves the covalent attachment of biotin molecules to antibodies, typically through N-hydroxysuccinimide (NHS) ester chemistry targeting primary amines on lysine residues or the N-terminus of the antibody. This conjugation creates a detection system leveraging the extraordinary binding affinity between biotin and avidin/streptavidin (Kd ≈ 10^-15 M), one of the strongest non-covalent interactions in nature. The conjugation ratio (number of biotin molecules per antibody) significantly impacts performance—too few biotin molecules reduce detection sensitivity, while excessive conjugation can impair antibody binding by sterically hindering the antigen-binding site. Most commercial biotin-conjugated antibodies are optimized with 3-8 biotin molecules per antibody to balance these considerations . The biotin conjugation process may slightly alter antibody dynamics, potentially affecting binding kinetics, so optimization of incubation conditions and dilution factors is necessary when transitioning from unconjugated to biotin-conjugated formats of the same antibody.

How do storage conditions specifically affect biotin-conjugated STARD13 antibodies?

Biotin-conjugated antibodies require specific storage considerations to maintain optimal activity. The recommended storage temperature is -20°C, similar to unconjugated antibodies, but with additional precautions . Storage buffers for biotin-conjugated antibodies typically contain glycerol (approximately 50%) to prevent freeze-thaw damage, stabilizing proteins like BSA (1%), and preservatives such as Proclin300 (0.03%) rather than higher concentrations of sodium azide, as azide can interfere with some downstream applications involving peroxidase enzymes . Light exposure should be minimized during handling, as biotin conjugates can be susceptible to photobleaching. Storage stability data indicates that properly stored biotin-conjugated antibodies maintain their activity for approximately 12 months, though aliquoting upon receipt is strongly recommended to avoid repeated freeze-thaw cycles that can accelerate degradation of both the antibody and the biotin conjugate .

What detection systems work optimally with biotin-conjugated STARD13 antibodies?

For biotin-conjugated STARD13 antibodies, several detection systems offer superior sensitivity and specificity. The streptavidin-horseradish peroxidase (HRP) system is most commonly used for Western blotting and immunohistochemistry, offering amplified signal through the enzymatic conversion of substrates like DAB or enhanced chemiluminescence reagents. For fluorescent detection, streptavidin conjugated to fluorophores such as Alexa Fluor 488, 555, or 647 provides excellent signal-to-noise ratios and photostability for immunofluorescence applications. When designing multiplex experiments, researchers should consider that the biotin-streptavidin interaction is essentially irreversible under physiological conditions, meaning streptavidin-based detection reagents bind permanently and cannot be stripped for reprobing . For Western blot applications specifically, titration experiments starting at 1:500 dilution are recommended to determine optimal signal-to-background ratios for biotin-conjugated STARD13 antibodies, as excessive antibody can lead to higher background due to non-specific biotin interactions.

How should I design a Western blot protocol optimized for STARD13 detection?

For optimal STARD13 detection by Western blot, begin with careful sample preparation. Since STARD13 is a large protein (125 kDa) with both cytoplasmic and nuclear localization, use a lysis buffer containing both ionic and non-ionic detergents (e.g., RIPA buffer with 0.1% SDS) to ensure complete extraction . When preparing your gel, select 8% polyacrylamide to provide better resolution in the 125 kDa range. Transfer conditions should be optimized for large proteins—use a wet transfer system with 10% methanol for at least 2 hours at constant amperage (300-400 mA) or overnight at lower current. For primary antibody incubation, dilute STARD13 antibodies between 1:500-1:2000 in 5% BSA rather than milk, as the latter may contain phosphatases that affect phosphorylated epitopes . Include positive control lysates from cell lines known to express STARD13, such as MCF-7, HeLa, or PANC1 cells . The following table summarizes optimal Western blot conditions based on published protocols:

What are the common issues encountered when using STARD13 antibodies and how can they be resolved?

Several challenges may arise when working with STARD13 antibodies. One common issue is weak or absent signal, which can be addressed by increasing antibody concentration, extending incubation time, or implementing signal enhancement techniques like biotin-streptavidin amplification. For high background problems, optimize blocking conditions (try 3-5% BSA instead of milk for phospho-sensitive epitopes) and increase wash duration and frequency. If multiple bands appear on Western blots, verify sample preparation (add protease inhibitors to prevent degradation) and confirm antibody specificity through knockout/knockdown controls . For tissue immunohistochemistry, antigen retrieval optimization is critical—test both citrate (pH 6.0) and EDTA (pH 9.0) buffers at different durations to determine optimal conditions for STARD13 epitope exposure. When using biotin-conjugated antibodies specifically, endogenous biotin in tissues can cause high background; implement an avidin/biotin blocking step prior to primary antibody incubation to minimize this issue .

How can I determine the optimal working concentration for biotin-conjugated STARD13 antibodies in different applications?

Determining the optimal working concentration for biotin-conjugated STARD13 antibodies requires systematic titration across applications. For Western blotting, prepare a dilution series (1:250, 1:500, 1:1000, 1:2000) using a positive control lysate known to express STARD13 . Evaluate signal-to-noise ratio at each concentration to identify the dilution producing specific bands at 125 kDa with minimal background. For immunofluorescence, start with a broader range (1:50 to 1:500) as recommended for unconjugated STARD13 antibodies, then narrow down based on signal specificity and intensity . In all optimization experiments, include appropriate negative controls (secondary detection reagents alone, isotype controls) to distinguish specific signal from background. The table below provides a starting framework for antibody titration experiments:

What considerations are important when analyzing STARD13 in cancer progression models?

When analyzing STARD13 in cancer progression models, researchers must account for several critical factors. First, as a tumor suppressor, STARD13 expression typically decreases during malignant transformation, but this pattern may vary by cancer type and stage . Longitudinal studies examining STARD13 across disease progression should include multiple time points or stages to capture transition dynamics. When performing immunohistochemical analyses, implement quantitative scoring methods (H-score or Allred) rather than qualitative assessments to detect subtle changes in expression levels. The subcellular localization of STARD13 may shift during cancer progression—cytoplasmic-to-nuclear translocation or membrane association changes may indicate functional alterations beyond simple expression differences. Published research indicates that STARD13 loss contributes to aggressive phenotype transformation in papillary thyroid carcinoma, suggesting that quantitative thresholds of expression may correlate with clinical outcomes . For mechanistic insights, always correlate STARD13 expression with known downstream effectors, particularly RhoA activity status, as the tumor suppressive function of STARD13 operates largely through its GAP activity on this GTPase .

How can researchers properly validate findings from STARD13 antibody-based experiments?

Comprehensive validation of STARD13 antibody-based findings requires a multi-method approach. First, confirm key results using at least two antibodies targeting different epitopes of STARD13 to rule out epitope-specific artifacts . For expression studies, complement protein-level detection with mRNA quantification via qRT-PCR or RNA-seq. Functional validation through genetic manipulation is essential—both knockdown (siRNA/shRNA) and overexpression experiments should demonstrate the expected reciprocal effects on STARD13-dependent phenotypes. For biotin-conjugated antibody studies specifically, parallel experiments with unconjugated versions should yield concordant results, accounting for sensitivity differences. When studying protein interactions, confirm co-immunoprecipitation results with proximity ligation assays or FRET/BRET approaches for in vivo validation. Finally, for clinical correlations, ensure statistical power through appropriate sample sizes and employ multivariate analyses to control for confounding factors. The table below summarizes a comprehensive validation strategy:

What emerging methodologies could enhance STARD13 detection and functional analysis?

Several cutting-edge methodologies hold promise for advancing STARD13 research beyond conventional antibody-based approaches. Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling fused to STARD13 could map its protein interaction network in living cells with temporal resolution. CRISPR-based endogenous tagging of STARD13 with split fluorescent proteins or enzymatic tags would enable tracking of native STARD13 without overexpression artifacts. Single-molecule imaging techniques using biotin-conjugated antibodies with quantum dots could reveal the dynamics of STARD13's interactions with RhoA/Cdc42 at unprecedented resolution. For tissue-level analysis, multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) using metal-conjugated STARD13 antibodies could simultaneously visualize dozens of markers in the STARD13 signaling network. These advanced techniques would complement traditional antibody applications while providing deeper insights into STARD13's spatial and temporal regulation in normal and pathological contexts . Implementation of these emerging technologies requires careful validation against established antibody-based methods to ensure continuity with the existing knowledge base.

How might biotin-conjugated STARD13 antibodies contribute to therapeutic development targeting RhoA pathways?

Biotin-conjugated STARD13 antibodies could accelerate therapeutic development targeting RhoA pathways through several innovative applications. In drug discovery pipelines, these antibodies can enable high-throughput screening assays to identify compounds that modulate STARD13-RhoA interactions or enhance STARD13 expression in cancer models where it is downregulated. The biotin-streptavidin system's high sensitivity could improve detection of STARD13 restoration after drug treatment, even at low expression levels . For targeted drug delivery approaches, understanding STARD13's tissue distribution using biotin-conjugated antibodies could help identify cancer types most likely to respond to RhoA pathway inhibitors. In patient stratification, immunohistochemical analysis of STARD13 levels using biotin-amplified detection systems might predict responsiveness to cytoskeletal-targeting therapies. Additionally, biotin-conjugated antibodies could facilitate the development of proximity-based screening systems to identify peptides or small molecules that specifically disrupt the interaction between STARD13 and its negative regulators, potentially restoring tumor suppressor function in cancers with reduced but not absent STARD13 expression .

What are the implications of STARD13's role in pancreatic cancer metastasis for diagnostic applications?

Recent research highlighting the role of STARD13 in pancreatic cancer metastasis opens significant avenues for diagnostic applications. Studies have shown that exosomal microRNA-125b-5p influences metastatic phenotypes in pancreatic cancer cells by targeting STARD13, suggesting potential for liquid biopsy applications . For developing such diagnostics, researchers should focus on quantitative immunoassays using biotin-conjugated STARD13 antibodies to detect subtle changes in protein levels that may indicate early metastatic potential. Since STARD13 functions as a tumor suppressor, decreased expression relative to matched normal tissue may serve as a prognostic biomarker . Immunohistochemical protocols using biotin-streptavidin amplification systems could improve detection sensitivity in small biopsy specimens. To develop clinically relevant cutoff values, researchers should analyze STARD13 expression across large patient cohorts with known outcomes, correlating expression patterns with survival data and metastatic progression. The incorporation of STARD13 into multi-marker panels alongside established pancreatic cancer biomarkers could enhance diagnostic accuracy compared to single-marker approaches. Advanced image analysis algorithms applied to STARD13 immunohistochemistry could extract subtle features like subcellular localization changes that may have prognostic significance beyond simple expression level measurements.

What key considerations should researchers keep in mind when selecting and using STARD13 antibodies?

When selecting and using STARD13 antibodies, researchers should prioritize several critical factors to ensure experimental success. First, match the antibody to your specific application—while some STARD13 antibodies perform well across multiple applications, others may be optimized for specific techniques like Western blot or immunohistochemistry . Consider the species reactivity carefully; most commercial STARD13 antibodies react with human samples, but cross-reactivity with mouse or rat samples varies between products . For biotin-conjugated antibodies specifically, evaluate whether your experimental system contains endogenous biotin that might interfere with detection, particularly in tissues like liver, kidney, and brain. Always include appropriate positive controls (cell lines with known STARD13 expression like MCF-7, HeLa, or PANC1) and negative controls (knockdown samples or isotype controls) to validate specificity . Finally, consider the target region of the antibody—those recognizing different epitopes may yield different results depending on potential post-translational modifications or protein interactions that might mask specific regions of STARD13 in your experimental context.