GAS6 Antibody, Biotin conjugated

What are the optimal working dilutions for biotin-conjugated GAS6 antibody across different applications?

Optimal working dilutions vary considerably depending on the specific application and the manufacturer of the biotin-conjugated GAS6 antibody. For Western blotting, concentrations between 0.1-2 μg/mL are typically recommended, with some products specifically suggesting 0.1 μg/mL for optimal results . For immunohistochemistry (IHC) in formalin-fixed tissues, higher concentrations ranging from 5-20 μg/mL are generally required . Immunocytochemistry (ICC) applications similarly require 5-20 μg/mL for optimal staining . For ELISA applications, lower concentrations between 0.05-2 μg/mL are typically sufficient .

It is strongly recommended to perform a titration experiment with your specific sample type and experimental conditions, as the optimal concentration may vary based on protein expression levels, tissue type, fixation method, and detection system. Several manufacturers emphasize that these recommended dilutions should be considered starting points, and each testing system should be individually optimized to obtain reliable results .

What are the recommended storage conditions for maintaining antibody stability and activity?

Biotin-conjugated GAS6 antibodies require specific storage conditions to maintain their activity and stability. Most manufacturers recommend storing the antibody at -20°C for long-term preservation, where it typically remains stable for 12 months after shipment . For reconstituted lyophilized antibodies, stability varies by temperature: at -20°C to -70°C, reconstituted antibodies generally remain stable for 6 months under sterile conditions, while at 2-8°C (refrigerator temperatures), they maintain stability for approximately 1 month .

For frequent use, some biotin-conjugated GAS6 antibodies can be stored at 2-8°C for short periods . Many products are supplied with glycerol (typically 50%) and preservatives like Proclin-300 (0.05%) or sodium azide (0.02%) to enhance stability . To avoid activity loss, it's essential to minimize freeze-thaw cycles by aliquoting the antibody before freezing, although some products specifically note that "aliquoting is unnecessary for -20°C storage" . Always follow the manufacturer's specific recommendations, as formulations may vary between suppliers.

What sample types and validation data exist for biotin-conjugated GAS6 antibodies?

Biotin-conjugated GAS6 antibodies have been validated across multiple sample types. Human samples are the primary validated targets, with specific antibodies showing reactivity in human urine samples, various cell lines including DU 145 and HeLa cells, and tissues such as lung cancer and spleen . Cross-species reactivity has been documented for certain antibodies, with reactivity in mouse and rat samples, particularly in heart tissue .

For human samples, validation data typically shows detection of GAS6 at approximately 75 kDa, consistent with the calculated molecular weight (75 kDa for 678 amino acids) . In Western blot applications, some antibodies exhibit approximately 15% cross-reactivity with recombinant mouse GAS6 . Many antibodies are immunogen-specific, with some developed against specific regions of the human GAS6 protein, such as the Leu136~Phe311 segment or the Asp118-Ala678 region . When selecting an antibody, researchers should review the validation data provided by manufacturers to ensure compatibility with their experimental system and target species.

How can biotin-conjugated GAS6 antibodies be used to investigate neurological diseases and autoimmune processes?

Biotin-conjugated GAS6 antibodies represent valuable tools for investigating neurological and autoimmune diseases due to GAS6's crucial roles in these processes. Research has established that GAS6 and its TAM receptors are expressed in the brain proportionally to synaptogenesis, with GAS6 exerting important protective effects on neurons and oligodendrocytes . For neurological disease research, these antibodies can be employed in immunohistochemistry to visualize GAS6 expression patterns in brain tissue sections, revealing alterations in expression during disease progression.

In multiple sclerosis (MS) research, GAS6 concentration in cerebrospinal fluid (CSF) has been shown to increase during milder relapses, suggesting a protective role . Interestingly, patients with more severe relapses show GAS6 levels comparable to control subjects, potentially indicating a failure of this protective mechanism . Researchers can employ biotin-conjugated GAS6 antibodies in ELISA assays to quantify GAS6 in CSF samples, allowing for correlation with disease severity and progression. A validated ELISA protocol involves coating plates with anti-human-GAS6 primary antibody and detecting the antigen using biotin-conjugated secondary antibody against GAS6 . This methodology allows for sensitive detection of GAS6 in biological fluids and can be modified to investigate various neuroinflammatory conditions.

What role does GAS6 play in cancer progression and how can biotin-conjugated antibodies help study these mechanisms?

GAS6 has emerged as a significant factor in cancer progression, with particular relevance to multiple myeloma (MM). Research has demonstrated that GAS6 is significantly upregulated in bone marrow cells of MM patients, with increased serum levels of soluble GAS6 compared to healthy controls . Biotin-conjugated GAS6 antibodies can be utilized in multiple approaches to investigate these cancer-related mechanisms.

For studying tumor microenvironment interactions, these antibodies can be employed in immunohistochemistry to visualize GAS6 expression patterns in bone marrow biopsies. Additionally, biotin-conjugated antibodies serve as valuable tools in neutralization experiments to block GAS6 activity. Research has shown that GAS6-neutralizing antibodies can reduce the upregulation of IL-6 and ICAM-1 induced by bone marrow stromal cell-conditioned medium in MM cells . This demonstrates how these antibodies can help elucidate paracrine interactions between tumor cells and their microenvironment.

Furthermore, biotin-conjugated antibodies facilitate the development of sensitive ELISAs for quantifying GAS6 in patient samples. By correlating GAS6 levels with clinical parameters and disease progression, researchers can evaluate its potential as a biomarker. These investigations have revealed that GAS6 suppresses apoptosis and enhances proliferation of MM cells through activation of signaling pathways including ERK, Akt, and NF-κB , highlighting the therapeutic potential of targeting GAS6 in cancer treatment.

How do researchers optimize detection protocols when working with both soluble and membrane-bound forms of GAS6?

Optimizing detection protocols for different forms of GAS6 requires careful consideration of sample preparation and antibody selection. For soluble GAS6 detection in serum, plasma, or cell culture supernatants, sandwich ELISA techniques using biotin-conjugated detection antibodies have been validated. One established protocol involves coating plates with anti-human-GAS6 primary antibody, followed by detection with biotinylated anti-human GAS6 antibody and streptavidin-peroxidase conjugate . This method allows for sensitive quantification of soluble GAS6 in biological fluids.

For membrane-bound or intracellular GAS6, immunohistochemistry (IHC) and immunocytochemistry (ICC) require different optimization strategies. When performing IHC, antigen retrieval methods significantly impact detection efficacy. For formalin-fixed tissues, TE buffer at pH 9.0 has been recommended, although citrate buffer at pH 6.0 can serve as an alternative . Appropriate antibody dilutions for IHC applications typically range from 1:50 to 1:500, depending on the specific antibody and tissue type .

Researchers investigating both forms simultaneously should consider dual approaches—combining ELISA for soluble GAS6 quantification with imaging techniques for cellular localization. When interpreting results, it's important to note that different antibodies may preferentially detect specific epitopes that could be differentially accessible in soluble versus membrane-bound forms. Cross-validation using multiple antibodies targeting different epitopes can provide more comprehensive insights into GAS6 biology across its various forms.

What are the most common technical challenges when using biotin-conjugated GAS6 antibodies and how can they be addressed?

When working with biotin-conjugated GAS6 antibodies, researchers frequently encounter several technical challenges that can compromise experimental results. One common issue is background staining in immunohistochemistry and immunocytochemistry applications, which can be addressed through several optimization strategies. Implementing more stringent blocking protocols with 5-10% normal serum matching the species of the secondary antibody helps reduce non-specific binding . Additionally, titrating the primary antibody concentration is essential, as excessive antibody can contribute to background signals.

Another challenge involves potential cross-reactivity between species. Some biotin-conjugated GAS6 antibodies show approximately 15% cross-reactivity with recombinant mouse GAS6 in Western blot applications . Researchers working with multiple species should carefully validate antibody specificity in their specific experimental system. Including appropriate negative controls (samples known to be negative for GAS6) and competitive blocking with recombinant GAS6 can help confirm signal specificity.

For Western blotting applications, obtaining clean, single-band detection can be problematic. This may be addressed by optimizing lysis buffers to ensure complete protein extraction and denaturation, adjusting antibody concentration (typically within the 0.5-2 μg/mL range for GAS6 antibodies) , and fine-tuning blocking and washing steps. For challenging samples, additional optimization of transfer conditions and membrane selection may improve results.

How should researchers properly validate the specificity of biotin-conjugated GAS6 antibodies in their experimental systems?

Thorough validation of biotin-conjugated GAS6 antibodies is essential for generating reliable experimental data. A comprehensive validation approach should include multiple complementary techniques. First, researchers should perform Western blot analysis using positive control samples with known GAS6 expression, such as human urine samples, DU 145 cells, or heart tissue from mice or rats . The observed molecular weight should match the expected 75 kDa for human GAS6 .

Competitive blocking experiments provide additional specificity confirmation. Pre-incubating the antibody with recombinant GAS6 protein (such as human GAS6 Asp118-Ala678) before application to samples should substantially reduce or eliminate specific signals. Similarly, comparing staining patterns between multiple antibodies targeting different GAS6 epitopes can provide confidence in detection specificity.

For researchers utilizing the antibody in novel applications or with untested sample types, it is advisable to include genetic controls where possible. Comparing signal intensity between GAS6-knockdown cells (using siRNA targeting Mer, as demonstrated in multiple myeloma research) and wild-type cells can definitively establish antibody specificity. Additionally, including tissue samples from GAS6 knockout models (when available) as negative controls represents the gold standard for antibody validation.

What considerations are important when designing experiments using biotin-conjugated GAS6 antibodies for multiplex imaging studies?

Designing multiplex imaging studies with biotin-conjugated GAS6 antibodies requires careful planning to ensure compatible detection systems and avoid signal overlap. First, researchers must account for the biotin-streptavidin detection system when selecting other antibodies and fluorophores. Since the biotin-conjugated GAS6 antibody will utilize a streptavidin-coupled detection reagent (typically with fluorophores or enzymes), other detection channels must be spectrally distinct to avoid bleed-through.

Sequential staining protocols often prove more successful than simultaneous approaches, particularly when studying GAS6 alongside its receptors or downstream signaling components. This involves complete detection of the biotin-conjugated GAS6 antibody before introducing additional primary antibodies, helping minimize cross-reactivity between detection systems. When using streptavidin-HRP for colorimetric detection in brightfield imaging, researchers should apply the GAS6 staining last in the sequence to prevent the development reagents from affecting subsequent antibody bindings.

Appropriate controls become especially critical in multiplex studies. These include single-stained controls to establish detection thresholds and identify potential spectral overlap, as well as fluorescence-minus-one (FMO) controls to set accurate gating boundaries for quantitative analysis. For studies examining co-localization between GAS6 and its receptors or downstream targets, super-resolution imaging techniques may be necessary to overcome the diffraction limit of conventional microscopy, providing more definitive evidence of molecular interactions.

How can biotin-conjugated GAS6 antibodies contribute to understanding autoimmune disease mechanisms?

Biotin-conjugated GAS6 antibodies provide valuable tools for investigating autoimmune disease mechanisms, particularly given GAS6's established role in modulating immune responses. Research has demonstrated that the GAS6/TAM receptor system is integrally involved in autoimmunity, with TAM receptor knockout mice exhibiting aberrant lymphocyte proliferation, tissue infiltration, and autoimmune manifestations including vasculitis and lupus-like lesions . Biotin-conjugated GAS6 antibodies can be employed in immunohistochemistry to visualize alterations in GAS6 expression within affected tissues, providing spatial context to expression changes during disease progression.

For quantitative assessment of GAS6 levels in clinical samples, these antibodies enable the development of sensitive ELISA assays. A validated protocol involves coating plates with anti-human-GAS6 primary antibody and detecting the antigen using biotinylated anti-human GAS6 antibody . This approach has successfully quantified GAS6 in cerebrospinal fluid samples from multiple sclerosis patients, revealing increased levels during milder relapses compared to controls . This suggests a protective role for GAS6 in limiting inflammatory demyelination, consistent with its functions in inhibiting macrophage activation and promoting oligodendrocyte survival .

Furthermore, biotin-conjugated GAS6 antibodies can be utilized in neutralization experiments to elucidate the functional consequences of GAS6 activity in disease models. By blocking GAS6 signaling in ex vivo tissue cultures or animal models, researchers can assess downstream effects on inflammatory mediators, cellular infiltration, and tissue damage. These approaches collectively contribute to a more comprehensive understanding of GAS6's role in autoimmune pathogenesis and potential therapeutic targeting strategies.

What methodological approaches can researchers use to study GAS6/TAM receptor interactions using biotin-conjugated antibodies?

Studying GAS6/TAM receptor interactions requires sophisticated methodological approaches where biotin-conjugated GAS6 antibodies play essential roles. Co-immunoprecipitation (Co-IP) experiments represent one powerful technique for investigating these interactions. Researchers can use biotin-conjugated GAS6 antibodies to pull down GAS6 protein complexes from cell lysates, followed by detection of associated TAM receptors (Tyro-3, Axl, and Mer) using receptor-specific antibodies. This approach has helped establish that GAS6/TAM signaling activates downstream pathways including PI3K/Akt, which promotes oligodendrocyte survival during demyelinating events .

Proximity ligation assays (PLA) offer another sophisticated approach for visualizing GAS6/TAM interactions in situ. This technique combines biotin-conjugated GAS6 antibodies with antibodies against TAM receptors, generating fluorescent signals only when the proteins are in close proximity (<40 nm). This enables direct visualization of interaction events within cellular contexts while preserving spatial information. For investigating the dynamics of these interactions, live-cell imaging using biotin-conjugated GAS6 antibodies paired with fluorescently-tagged streptavidin can track receptor engagement and subsequent internalization.

In functional studies, biotin-conjugated GAS6 antibodies have been employed as neutralizing agents to block GAS6/TAM signaling. Research in multiple myeloma has demonstrated that GAS6-neutralizing antibodies can reduce the upregulation of IL-6 and ICAM-1 induced by bone marrow stromal cell-conditioned medium . This approach allows researchers to dissect the specific contributions of GAS6 signaling within complex cellular environments, distinguishing its effects from other signaling pathways.

How are biotin-conjugated GAS6 antibodies being used to investigate novel therapeutic targets in cancer and inflammation?

Biotin-conjugated GAS6 antibodies are increasingly utilized to explore novel therapeutic targets across multiple disease contexts. In cancer research, particularly multiple myeloma, these antibodies have helped uncover the significance of GAS6/TAM signaling in promoting tumor cell survival and proliferation. Studies have demonstrated that GAS6 suppresses apoptosis and enhances proliferation of multiple myeloma cells through activation of ERK, Akt, and NF-κB signaling pathways . By employing biotin-conjugated GAS6 antibodies in neutralization experiments, researchers have established that blocking GAS6 activity can reverse these pro-survival effects, suggesting potential therapeutic applications .

For inflammatory conditions, biotin-conjugated GAS6 antibodies have facilitated investigations into GAS6's role as a modulator of macrophage activation and inflammatory responses. Research has established that GAS6 can inhibit macrophage activation, potentially limiting inflammatory damage in conditions like multiple sclerosis . These antibodies enable quantitative assessment of GAS6 levels in patient samples through ELISA techniques, allowing correlations between GAS6 expression and disease severity. Notably, studies in multiple sclerosis have revealed that CSF GAS6 levels are elevated in patients with milder disease manifestations, suggesting a protective function .

Beyond direct therapeutic targeting, biotin-conjugated GAS6 antibodies support biomarker development efforts. By facilitating sensitive detection of GAS6 in biological fluids, these antibodies help identify patient populations that might benefit from therapies targeting the GAS6/TAM axis. This approach represents a step toward personalized medicine strategies in complex diseases with heterogeneous pathogenic mechanisms.

What are the latest experimental protocols for monitoring GAS6 expression and secretion dynamics in research models?

Recent advancements in monitoring GAS6 expression and secretion dynamics have yielded sophisticated experimental protocols utilizing biotin-conjugated antibodies. For real-time analysis of GAS6 secretion, researchers have developed live-cell immunoassays where biotin-conjugated GAS6 antibodies are applied to cell culture supernatants at defined intervals, followed by detection with fluorescently-labeled streptavidin. This approach enables temporal tracking of GAS6 release in response to experimental manipulations.

For simultaneous assessment of GAS6 expression and signaling outcomes, multiplex imaging approaches have been established. These protocols combine biotin-conjugated GAS6 antibodies with phospho-specific antibodies targeting downstream signaling components (such as phosphorylated Akt or ERK). This methodology has revealed that GAS6 signaling activates multiple pathways in cell-specific contexts, including ERK, Akt, and NF-κB phosphorylation in multiple myeloma cells .

The development of reporter systems represents another frontier in GAS6 research. By engineering cells with fluorescent or luminescent reporters under the control of the GAS6 promoter, researchers can monitor transcriptional regulation in real-time. When combined with biotin-conjugated GAS6 antibodies for protein-level detection, these systems provide comprehensive insights into the relationship between GAS6 transcription, translation, and secretion dynamics. These complementary approaches collectively enhance our understanding of GAS6 biology across diverse experimental contexts.