SAV1 Antibody

What is SAV1 and why is it significant in cellular research?

SAV1 (Salvador Homolog 1) functions as a critical regulator of STK3/MST2 and STK4/MST1 in the Hippo signaling pathway, which plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway consists of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with SAV1, phosphorylate and activate LATS1/2 in complex with MOB1, which subsequently phosphorylates and inactivates the YAP1 oncoprotein and WWTR1/TAZ . SAV1 is particularly significant because it acts as a tumor suppressor, and mutations in SAV1 have been identified in various human cancer cell lines, making it an important target for oncological research . Studies in Drosophila have identified SAV1's role in cell cycle regulation and apoptosis, while embryonic mice lacking Sav1 displayed hyperplastic growth and epithelial differentiation effects, further confirming its importance in developmental and cancer biology .

What are the structural and functional domains of SAV1 protein?

SAV1 is a multi-domain protein characterized by two WW domains, a SARAH domain, and a coiled-coil region. It is ubiquitously expressed in adult tissues . The WW domains are known to facilitate protein-protein interactions with proline-rich regions, which are crucial for SAV1's function in the Hippo pathway. The SARAH domain (Salvador, RASSF, Hippo) mediates interaction with MST1/2 kinases, enabling SAV1 to regulate their activity. Functionally, SAV1 binds to MST1 (mammalian sterile 20-like kinase 1) and promotes MST1-induced apoptosis. Additionally, it has been shown to bind to HAX1 (hematopoietic cell-specific protein 1 (HS1)-associated protein X-1) and attenuate the anti-apoptotic effects of HAX1 . Beyond its role in the Hippo pathway, SAV1 also plays a role in centrosome disjunction by regulating the localization of NEK2 to centrosomes and its ability to phosphorylate CROCC and CEP250 .

What types of SAV1 antibodies are available and which applications are they best suited for?

Several types of SAV1 antibodies are available for research purposes, each optimized for specific applications. The table below summarizes the key characteristics of commercially available SAV1 antibodies:

For Western blotting applications, both polyclonal and monoclonal options are available, with recommended dilutions typically around 1:1000 . For immunoprecipitation studies, rabbit polyclonal antibodies at dilutions of approximately 1:50 are recommended . Researchers conducting immunohistochemistry should consider using antibodies specifically validated for IHC at dilutions of 1:100-1:300 . The choice between polyclonal and monoclonal antibodies should be guided by the specific requirements of the experiment, with monoclonal antibodies offering higher specificity but potentially limited epitope recognition.

How should I optimize Western blotting protocols when using SAV1 antibodies?

When conducting Western blotting with SAV1 antibodies, several optimization steps are crucial for obtaining clear and specific results. First, establish proper protein extraction conditions that preserve SAV1's native structure—typically, RIPA buffer supplemented with protease and phosphatase inhibitors is recommended. For SAV1 detection, load approximately 20-30 μg of total protein lysate per lane. Use 10-12% SDS-PAGE gels for optimal separation, as SAV1 has a molecular weight of approximately 45 kDa .

For transfer, PVDF membranes are generally preferred over nitrocellulose due to their durability and protein binding capacity. Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. When using primary SAV1 antibodies, dilute according to manufacturer specifications, typically 1:1000 for Western blotting , and incubate overnight at 4°C with gentle rocking. After thorough washing with TBST (3-4 times, 5-10 minutes each), apply an appropriate HRP-conjugated secondary antibody and detect using enhanced chemiluminescence.

Importantly, always include positive controls (cell lines known to express SAV1) and negative controls (SAV1 knockout cells if available) to validate antibody specificity. For troubleshooting weak signals, consider longer primary antibody incubation times or signal enhancement systems. Non-specific binding can often be reduced by increasing the concentration of blocking agent or adding 0.1-0.5% Tween-20 to the antibody dilution buffer.

What are the recommended procedures for immunoprecipitation studies using SAV1 antibodies?

For immunoprecipitation (IP) studies involving SAV1, begin with careful cell lysis using a non-denaturing buffer that preserves protein-protein interactions, such as a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and protease/phosphatase inhibitors. After clearing the lysate by centrifugation, pre-clear with Protein A/G beads to reduce non-specific binding. For the IP reaction, use approximately 1-2 mg of total protein and add SAV1 antibody at a 1:50 dilution as recommended for immunoprecipitation applications .

Incubate the antibody-lysate mixture overnight at 4°C with gentle rotation, then add fresh Protein A/G beads and incubate for an additional 2-4 hours. Perform at least 3-5 washes with lysis buffer to remove non-specifically bound proteins. For elution, use either gentle methods (for co-IP studies) such as competitive elution with the immunizing peptide, or more stringent approaches like boiling in SDS sample buffer for subsequent Western blot analysis.

When studying SAV1 interactions with other Hippo pathway components, such as MST1/2 or LATS1/2, consider performing reciprocal IPs to validate interactions and include appropriate controls such as IgG from the same species as the antibody. For detecting weak interactions, chemical crosslinking prior to cell lysis can be employed, though this requires careful optimization to prevent artifactual results.

What are the critical considerations for immunohistochemistry using SAV1 antibodies?

Successful immunohistochemistry (IHC) with SAV1 antibodies requires attention to several key factors. Begin with proper tissue fixation—10% neutral buffered formalin fixed, paraffin-embedded sections are generally suitable, though the optimal fixation method may vary depending on tissue type. Antigen retrieval is critical and should be optimized; typically, heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes works well for SAV1 detection.

For SAV1 antibodies specifically validated for IHC, use the manufacturer-recommended dilution, typically in the range of 1:100-1:300 . Include both positive control tissues (those known to express SAV1) and negative controls (primary antibody omitted) in each staining run. Apply the primary antibody for 1-2 hours at room temperature or overnight at 4°C, followed by an appropriate detection system (such as a polymer-based detection kit).

When analyzing SAV1 expression patterns, note that it may show both cytoplasmic and nuclear localization depending on the cell type and physiological state. Quantification of staining can be performed using established scoring systems such as H-score or Allred score. For dual or multiplex staining to study SAV1 together with other Hippo pathway components, sequential staining protocols with careful antibody stripping between rounds may be necessary to avoid cross-reactivity.

How can I investigate SAV1's role in the Hippo signaling pathway and tumor suppression?

To investigate SAV1's role in the Hippo pathway and its tumor suppression function, a multi-faceted approach is recommended. Begin with expression analysis across normal and cancer tissues/cell lines using validated SAV1 antibodies in Western blotting (1:1000 dilution) or immunohistochemistry (1:100-1:300 dilution) . For mechanistic studies, employ gene knockout or knockdown techniques such as CRISPR-Cas9 or siRNA to ablate SAV1 expression, followed by assessment of downstream effectors like YAP1/TAZ phosphorylation and localization.

Protein-protein interaction studies are crucial for understanding SAV1's function. Use co-immunoprecipitation with SAV1 antibodies (1:50 dilution) to identify binding partners, focusing on known interactors such as MST1/2, LATS1/2, and HAX1. Proximity ligation assays can provide spatial information about these interactions within cells. To examine SAV1's impact on cell cycle regulation and apoptosis, conduct functional assays after SAV1 manipulation, including proliferation assays, cell cycle analysis, and apoptosis assays.

For in vivo studies, consider xenograft models with SAV1-manipulated cancer cells or conditional knockout mouse models. These can reveal SAV1's impact on tumor growth, invasion, and metastasis. Correlative studies examining SAV1 mutations or expression levels in patient samples, coupled with clinical outcome data, can provide translational insights. For a comprehensive understanding, combine these approaches with phosphoproteomics to map the signaling networks affected by SAV1 alterations, focusing on how SAV1 regulation of MST1/2 impacts downstream phosphorylation cascades.

What methods are most effective for studying SAV1 interactions with STK3/MST2 and STK4/MST1?

Studying SAV1 interactions with STK3/MST2 and STK4/MST1 requires a combination of biochemical, cellular, and structural approaches. Co-immunoprecipitation represents a foundational technique—use SAV1 antibodies at the recommended 1:50 dilution for IP applications to pull down the complex, followed by Western blotting with STK3/MST2 or STK4/MST1 antibodies. Perform reciprocal IPs with STK antibodies to confirm the interaction. For detecting endogenous complexes, carefully optimize cell lysis conditions to preserve native interactions.

Proximity-based assays offer in situ detection of these interactions. Techniques such as proximity ligation assay (PLA), fluorescence resonance energy transfer (FRET), or bimolecular fluorescence complementation (BiFC) can confirm protein-protein interactions within living cells and provide spatial information about where these interactions occur. For mapping specific interaction domains, employ truncation or deletion mutants of SAV1, focusing on the SARAH domain known to mediate interaction with MST kinases.

Functional validation is essential to understand the biological significance of these interactions. Assess how disrupting the SAV1-MST interaction (through mutation of key residues or domain deletion) affects MST activation, downstream signaling to LATS kinases, and ultimately YAP/TAZ phosphorylation and localization. Phospho-specific antibodies can be used to monitor MST1/2 activation status in the presence or absence of SAV1. Additionally, in vitro kinase assays with purified components can determine whether SAV1 directly affects MST kinase activity or serves primarily as a scaffold.

How can I analyze SAV1's contribution to centrosome disjunction and cell cycle regulation?

Investigating SAV1's role in centrosome disjunction and cell cycle regulation requires specialized techniques focused on centrosome biology and cell division. Begin with immunofluorescence microscopy using SAV1 antibodies alongside centrosome markers (e.g., γ-tubulin, centrin) to establish SAV1's localization pattern throughout the cell cycle. Time-lapse imaging of fluorescently tagged SAV1 in living cells can provide dynamic information about its recruitment to centrosomes during specific cell cycle phases.

To analyze SAV1's functional impact on centrosome disjunction, manipulate SAV1 levels through knockdown or overexpression and assess centrosome cohesion using measurements of inter-centrosomal distance. Since SAV1 regulates NEK2 localization to centrosomes and its ability to phosphorylate CROCC and CEP250 , examine these downstream events using phospho-specific antibodies or proximity-based assays to detect NEK2-substrate interactions in the presence or absence of SAV1.

For cell cycle regulation studies, synchronize cells at different cell cycle stages and analyze SAV1 expression, phosphorylation status, and interaction partners at each stage. Flow cytometry analysis after SAV1 manipulation can reveal alterations in cell cycle distribution. Connect these observations to the Hippo pathway by simultaneously monitoring YAP/TAZ localization and target gene expression, as these effectors directly influence cell cycle progression and apoptosis.

Biochemical approaches should include co-immunoprecipitation studies using SAV1 antibodies (1:50 dilution) to identify cell cycle-specific interaction partners. Mass spectrometry analysis of SAV1 immunoprecipitates from synchronized cell populations can reveal phase-specific protein complexes and post-translational modifications that regulate SAV1's function in centrosome biology and cell cycle control.

How should I troubleshoot weak or inconsistent signals when using SAV1 antibodies?

When encountering weak or inconsistent signals with SAV1 antibodies, implement a systematic troubleshooting approach. First, verify antibody quality—check expiration dates, storage conditions, and avoid repeated freeze-thaw cycles. Consider testing multiple SAV1 antibodies targeting different epitopes, such as those against N-terminal (AA 1-210), middle (AA 65-92), or C-terminal (AA 300-383) regions of the protein, as availability of epitopes may vary depending on experimental conditions .

For Western blotting, optimize protein extraction—SAV1 is a scaffold protein that forms complexes with other proteins, so extraction buffers containing non-ionic detergents such as NP-40 or Triton X-100 may improve results. Increase protein loading incrementally (20-50 μg) and extend exposure times. If bands appear at unexpected molecular weights, consider the possibility of isoforms, post-translational modifications, or degradation products. The expected molecular weight for SAV1 is approximately 45 kDa .

For immunohistochemistry or immunofluorescence, optimize antigen retrieval methods—test both heat-mediated retrieval with citrate buffer (pH 6.0) and EDTA buffer (pH 9.0). Extend primary antibody incubation times (overnight at 4°C) and test higher antibody concentrations than the recommended 1:100-1:300 range . Signal amplification systems such as tyramide signal amplification may help with low abundance targets.

If SAV1 detection is consistently problematic across multiple antibodies and techniques, consider the possibility that your experimental system has low endogenous expression. Validate with positive control samples known to express SAV1, such as epithelial tissues or certain cancer cell lines. RT-qPCR can confirm SAV1 transcript levels to corroborate protein detection issues.

How can I interpret contradictory results when studying SAV1 across different cell lines or tissue types?

Interpreting contradictory results when studying SAV1 across different experimental systems requires careful consideration of biological and technical variables. First, recognize that SAV1 expression and function can be highly context-dependent. Document baseline expression levels across your cell lines or tissues using Western blotting with recommended antibody dilutions (1:1000) and normalize to appropriate housekeeping proteins. Consider cell type-specific factors—SAV1 functions primarily in epithelial tissues, and its expression or activity may vary in non-epithelial cells.

Cellular microenvironment significantly impacts Hippo pathway regulation. Culture conditions such as cell density, serum levels, substrate stiffness, and cell-cell contacts can dramatically alter SAV1 function and its downstream effects. Standardize these conditions across experiments and explicitly report them when publishing. For tissue samples, factors such as tissue collection methods, preservation techniques, and patient characteristics can influence results.

At the molecular level, investigate whether contradictory findings might be explained by alternative splicing of SAV1, post-translational modifications, or formation of different protein complexes in different cellular contexts. Phosphorylation status of Hippo pathway components often varies across cell types and can alter protein function without changing expression levels. When possible, combine protein expression data with functional readouts such as YAP/TAZ localization or target gene expression to provide a more comprehensive picture.

For reconciling contradictory published findings, consider antibody differences—different studies may use antibodies recognizing distinct epitopes, with varying specificities and sensitivities. When comparing your results to literature, note the exact antibody clones used and their validation status in the experimental systems being compared.

What are the best practices for validating SAV1 antibody specificity?

Thorough validation of SAV1 antibody specificity is crucial for generating reliable research data. Employ a multi-step validation strategy beginning with genetic controls—test the antibody in SAV1 knockout or knockdown models alongside wild-type controls. CRISPR-Cas9 generated knockout cell lines provide the gold standard for specificity validation, while siRNA or shRNA knockdown systems offer accessible alternatives. The antibody signal should be substantially reduced or eliminated in these models.

Peptide competition assays represent another valuable validation approach. Pre-incubate the SAV1 antibody with excess immunizing peptide (if available) before application to your sample. Specific signals should be blocked by this competition, while non-specific signals will persist. For antibodies where the immunizing peptide is not available, recombinant SAV1 protein can serve as an alternative for competition studies.

Orthogonal detection methods provide additional validation. Compare results from antibody-based detection with orthogonal approaches such as mass spectrometry or RNA expression analysis (though the latter only confirms transcript presence, not protein levels or localization). When using multiple SAV1 antibodies targeting different epitopes, concordant results increase confidence in specificity. The table below summarizes commercially available SAV1 antibodies targeting different regions of the protein:

Finally, evaluate antibody performance across multiple applications. An antibody that shows consistent and specific staining patterns across multiple techniques (Western blot, IHC, IF, IP) with expected subcellular localization provides greater confidence in its specificity. Document all validation results thoroughly, including positive and negative controls, for future reference and publication.

How can I investigate SAV1's potential role in regulating the transcriptional activity of ESR1?

Investigating SAV1's role in regulating ESR1 (estrogen receptor alpha) transcriptional activity represents an emerging research area with implications for both Hippo signaling and hormone-responsive cancers. Begin with co-immunoprecipitation studies using SAV1 antibodies at the recommended 1:50 dilution to confirm the physical interaction between SAV1, STK3/MST2, and ESR1 in relevant cell types such as breast cancer cells. Reciprocal IPs with ESR1 antibodies can validate this interaction. For in-cell confirmation, proximity ligation assays can visualize and quantify the interaction in situ.

To assess the functional impact on ESR1 phosphorylation, use phospho-specific antibodies against known ESR1 phosphorylation sites after manipulating SAV1 levels through overexpression or knockdown. Mass spectrometry analysis of immunoprecipitated ESR1 can identify specific phosphorylation sites affected by SAV1 and STK3/MST2. For transcriptional activity assessment, employ reporter gene assays with estrogen response element (ERE)-driven luciferase constructs to measure ESR1 activity in the presence or absence of SAV1 and with or without estrogen stimulation.

Chromatin immunoprecipitation (ChIP) assays can determine whether SAV1 is recruited to ESR1 target gene promoters, either directly or as part of a complex with ESR1 and STK3/MST2. ChIP-seq can provide genome-wide insights into how SAV1 affects ESR1 binding patterns. RNA-seq after SAV1 manipulation can identify the subset of ESR1 target genes specifically regulated by the SAV1-STK3/MST2 axis. For physiological relevance, correlate SAV1 and ESR1 expression/activity in patient samples, particularly in hormone-responsive cancers like breast cancer.

Mechanistically, investigate whether SAV1's effect on ESR1 occurs through the canonical Hippo pathway (via YAP/TAZ) or represents a non-canonical function. Selective inhibitors of Hippo pathway components can help dissect these mechanisms. These multifaceted approaches will provide comprehensive insights into how SAV1 connects Hippo signaling to hormone receptor function.

What are the most promising approaches for studying SAV1 in the context of tissue regeneration and stem cell biology?

Studying SAV1 in tissue regeneration and stem cell biology contexts requires specialized approaches that address the unique aspects of stem cell regulation and tissue homeostasis. Begin with lineage-specific conditional knockout models of SAV1 in regenerative tissues such as liver, intestine, or skin. These models allow temporal control over SAV1 deletion and can reveal its role during homeostasis versus injury-induced regeneration. For stem cell studies, isolate tissue-specific stem cells from these models and assess their self-renewal capacity, differentiation potential, and response to regenerative stimuli.

Organoid culture systems provide valuable platforms for studying SAV1 function in a three-dimensional context that better recapitulates tissue architecture. Generate organoids from wild-type and SAV1-deficient stem cells, then compare growth patterns, differentiation capacity, and response to Hippo pathway modulators. Time-lapse imaging of organoid development can reveal dynamic aspects of SAV1 function during tissue formation. Immunostaining these organoids with SAV1 antibodies at dilutions optimized for immunofluorescence can reveal subcellular localization in different cell populations.

For mechanistic insights, investigate the interplay between SAV1/Hippo signaling and other pathways crucial for stem cell regulation, such as Wnt, Notch, and BMP signaling. Co-immunoprecipitation studies using SAV1 antibodies can identify novel interaction partners in stem cell populations. Single-cell approaches are particularly valuable—single-cell RNA-seq of tissues from SAV1 conditional knockout models can reveal cell type-specific responses to SAV1 loss, while single-cell protein analysis techniques can map signaling network alterations.

Translational aspects can be addressed by examining SAV1 expression patterns during human tissue regeneration and repair processes. Immunohistochemistry with SAV1 antibodies (1:100-1:300 dilution) on tissue sections from patients with various regenerative conditions can provide clinically relevant insights. These comprehensive approaches will advance understanding of how SAV1 and the Hippo pathway regulate the balance between regeneration and tumor suppression.

How can computational and systems biology approaches enhance our understanding of SAV1 function in the Hippo network?

Computational and systems biology approaches offer powerful tools for understanding SAV1's function within the complex Hippo signaling network. Begin with network modeling—integrate experimental data on SAV1 interactions and functional effects into computational models of the Hippo pathway. Ordinary differential equation (ODE) models can simulate the dynamics of SAV1-mediated regulation of MST1/2 and downstream signaling. Parameter sensitivity analysis can identify the most critical nodes and interactions that determine network behavior when SAV1 is perturbed.

Multi-omics data integration provides a comprehensive view of SAV1 function. Combine proteomics data from SAV1 immunoprecipitation studies using antibodies at recommended dilutions with transcriptomics data from SAV1 perturbation experiments and phosphoproteomics to map signaling cascades. Network inference algorithms can identify previously unrecognized connections between SAV1 and other cellular pathways. These integrated networks can reveal how SAV1 connects the Hippo pathway to other cellular processes such as cell cycle regulation, centrosome biology, and apoptosis.

Machine learning approaches can extract patterns from large datasets. Supervised learning algorithms trained on datasets from SAV1 wild-type versus knockout/knockdown experiments can identify gene expression or phosphorylation signatures predictive of SAV1 activity status. These signatures can then be used to infer SAV1 functional status in patient samples or to screen for compounds that mimic or reverse SAV1-dependent effects.

For structural insights, molecular dynamics simulations can model the interactions between SAV1 domains (particularly the SARAH domain) and binding partners like MST1/2. These simulations can predict how mutations affect binding affinity and complex formation, guiding experimental validation with mutant constructs. Virtual screening approaches can identify potential small molecules that might modulate SAV1 interactions or functions, providing starting points for tool compound development to study SAV1 in cellular contexts.

How can SAV1 expression analysis be incorporated into cancer research and potential diagnostic applications?

Incorporating SAV1 expression analysis into cancer research and diagnostic workflows requires careful consideration of methodological approaches and interpretation frameworks. For research applications, begin with comprehensive profiling of SAV1 expression across cancer types and stages using tissue microarrays. Immunohistochemistry with validated SAV1 antibodies at the recommended 1:100-1:300 dilution can provide spatial information about expression patterns and subcellular localization. Develop standardized scoring systems that account for both intensity and percentage of positive cells to ensure consistency across studies.

For potential diagnostic applications, evaluate the prognostic significance of SAV1 expression through retrospective studies correlating expression levels with patient outcomes. Multivariate analysis should control for established prognostic factors to determine whether SAV1 provides independent prognostic information. If promising correlations emerge, prospective validation in independent cohorts is essential before clinical implementation. Consider whether SAV1 analysis alone is sufficient or if it provides greater value as part of a panel including other Hippo pathway components such as YAP/TAZ.

Beyond simple expression analysis, investigate whether specific SAV1 mutations or splice variants have diagnostic or prognostic significance. Next-generation sequencing panels can be designed to include SAV1 alongside other cancer-relevant genes. For functional assessment, develop assays that measure not just SAV1 expression but its activity status, such as its ability to regulate MST1/2 kinases or impact YAP/TAZ nuclear localization.

From a practical implementation perspective, determine the most appropriate specimen types and preservation methods for SAV1 analysis. Compare results from fresh frozen versus formalin-fixed paraffin-embedded tissues to establish concordance. Evaluate whether SAV1 can be reliably detected in liquid biopsy specimens such as circulating tumor cells or cell-free DNA, which would enable less invasive monitoring. These comprehensive approaches will help translate SAV1 research findings into clinically meaningful applications.

What considerations are important when designing experiments to target SAV1 for therapeutic development?

Designing experiments for therapeutic targeting of SAV1 requires multifaceted approaches that address both basic mechanism validation and translational development considerations. First, establish clear rationale for targeting SAV1 by thoroughly characterizing its status (expression, mutation, activity) across cancer types using validated antibodies . Determine whether restoration of SAV1 function (in cases of loss) or inhibition of SAV1 (in contexts where it might promote tumor growth) represents the appropriate strategy for specific cancer types.

For loss-of-function contexts, consider strategies for restoring SAV1 expression or function. This might include identifying compounds that upregulate SAV1 expression through high-throughput screening of small molecule libraries or approaches to re-express SAV1 using viral vectors or mRNA therapeutics. Develop robust cellular assays with clear readouts of SAV1 activity, such as MST1/2 activation, LATS1/2 phosphorylation, or YAP/TAZ nuclear exclusion, that can be adapted to high-throughput screening formats.

For targeting SAV1 protein-protein interactions, detailed structural studies are essential. Focus on the SARAH domain interactions with MST1/2 and employ structural biology techniques (X-ray crystallography, cryo-EM) to identify potential binding pockets for small molecule development. Fragment-based drug discovery or peptide-based approaches may be appropriate for disrupting specific protein-protein interactions. Virtual screening can identify potential hit compounds for experimental validation.

In all therapeutic development efforts, develop appropriate model systems that accurately reflect the biology of SAV1 in human disease. These should include cell line panels with various SAV1 expression levels, patient-derived organoids, and in vivo models such as patient-derived xenografts. For each model, establish clear criteria for therapeutic efficacy, such as tumor growth inhibition, pathway biomarker modulation, or survival extension. These comprehensive experimental approaches will provide the foundation for translating SAV1 biological insights into potential therapeutic strategies.