SLC9A3R2 Antibody

What is SLC9A3R2 and why is it an important research target?

SLC9A3R2, also known as Na(+)/H(+) exchange regulatory cofactor NHE-RF2, NHERF-2, or SIP-1, is a 37.414 kDa protein involved in the regulation of membrane protein trafficking and ion transport systems . This protein functions as a scaffold protein that connects membrane proteins with cytoskeletal components and signaling molecules, making it an important research target in cellular physiology, ion transport mechanisms, and potential disease associations. The protein has several alternative names including E3KARP, NHE3RF2, NHERF2, OCTS2, SIP-1, SIP1, and TKA-1, which reflect its various discovered functions and interactions across different research contexts . Understanding SLC9A3R2 is crucial for researchers investigating cellular signaling pathways, membrane protein regulation, and associated pathologies.

What are the common applications for SLC9A3R2 antibodies in research?

SLC9A3R2 antibodies are utilized across multiple experimental applications in research settings. Based on validated protocols, these antibodies have demonstrated effectiveness in Western blot (WB), immunohistochemistry (IHC) on paraffin-embedded tissues, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) . Each application requires specific optimization parameters, with Western blot typically employing 0.25-0.5 μg/ml concentrations for human and mouse samples, while IHC applications generally use 2-5 μg/ml concentrations across human, mouse, and rat tissues . Flow cytometry applications typically utilize 1-3 μg per 10^6 cells for optimal staining. The versatility of these antibodies across multiple applications makes them valuable tools for researchers investigating SLC9A3R2 expression, localization, interactions, and functions in various experimental contexts.

What species reactivity can be expected with SLC9A3R2 antibodies?

Commercial SLC9A3R2 antibodies, such as the Boster Bio Anti-NHERF-2/SIP-1/SLC9A3R2 Antibody (A05624-2), demonstrate validated cross-reactivity with human, mouse, and rat samples . This cross-species reactivity is particularly valuable for comparative studies and translational research. Validation images confirm detection of SLC9A3R2 in tissues from all three species, including human cancer cell lines (MCF-7, T-47D, PC-3), human tissues (thyroid cancer, colorectal adenocarcinoma, lung cancer, laryngeal squamous cell carcinoma), mouse tissues (kidney, lung), and rat tissues (kidney, skin, lung) . Researchers should note that while the antibody has been validated for these three species, experimental conditions may need species-specific optimization to achieve optimal signal-to-noise ratios when comparing expression patterns across different organisms.

How should SLC9A3R2 antibodies be validated for research applications?

Following current best practices in antibody validation, researchers should implement multiple validation approaches when working with SLC9A3R2 antibodies. According to recent guidelines, effective antibody validation employs five key strategies: orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis . For SLC9A3R2 specifically, researchers should compare antibody-based detection with orthogonal methods that measure mRNA expression, validate specificity using SLC9A3R2 knockdown/knockout models, confirm detection of recombinant SLC9A3R2, compare results with alternative antibodies targeting different epitopes, and when possible, verify detection through mass spectrometry . This multi-pillar approach is particularly important for scaffold proteins like SLC9A3R2 that may have multiple interaction partners and exist in different conformational states depending on cellular context.

What are the optimal conditions for Western blot detection of SLC9A3R2?

For optimal Western blot detection of SLC9A3R2, researchers should follow validated protocols that address sample preparation, electrophoresis conditions, and detection parameters. Based on validated protocols, SDS-PAGE should be performed on 5-20% gradient gels at 70V (stacking) followed by 90V (resolving) for 2-3 hours . Sample loading should include approximately 30 μg of protein per lane under reducing conditions. After electrophoresis, proteins should be transferred to nitrocellulose membranes at 150 mA for 50-90 minutes . Blocking should be performed with 5% non-fat milk in TBS for 1.5 hours at room temperature, followed by overnight incubation with the primary anti-SLC9A3R2 antibody at 0.5 μg/mL at 4°C . After washing with TBS-0.1% Tween (3 times, 5 minutes each), membranes should be probed with goat anti-rabbit IgG-HRP secondary antibody at 1:5000 dilution for 1.5 hours at room temperature . While the expected molecular weight for SLC9A3R2 is 37 kDa, researchers should note that the observed band typically appears at approximately 45 kDa, likely due to post-translational modifications.

What controls are essential when working with SLC9A3R2 antibodies?

When designing experiments with SLC9A3R2 antibodies, researchers should incorporate multiple controls to ensure reliable and interpretable results. Positive controls should include tissues or cell lines with known SLC9A3R2 expression, such as MCF-7, T-47D, or PC-3 cells for Western blot applications, and various human cancer tissues (thyroid, colorectal, lung, laryngeal) as well as mouse and rat tissues (kidney, lung, skin) for IHC applications . For flow cytometry, PC-3 cells serve as effective positive controls . Negative controls should include samples where SLC9A3R2 is knocked down or knocked out, as well as technical controls where primary antibody is omitted or replaced with non-specific IgG of the same species and concentration. For IHC applications, tissue sections should be blocked with 10% goat serum prior to antibody incubation, and heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended for optimal epitope exposure . Additional methodology controls might include detection of recombinant SLC9A3R2 protein as a standard reference.

How can researchers differentiate between SLC9A3R2 and related proteins like NHERF1?

Distinguishing between SLC9A3R2 (NHERF2) and its closely related family member NHERF1 (SLC9A3R1) requires careful antibody selection and experimental design. Both proteins share structural similarities with two PDZ domains and function as scaffold proteins, but they have distinct tissue distribution patterns and interacting partners. Researchers should first select antibodies that have been specifically validated for lack of cross-reactivity with NHERF1, preferably those targeting unique regions that differ between the two proteins . Western blot analysis can be supplemented with immunoprecipitation followed by mass spectrometry to confirm protein identity. Additionally, researchers can design experiments that exploit the different molecular weights (NHERF1: ~50 kDa, NHERF2: ~37-45 kDa) or utilize cell lines with differential expression of these proteins . For definitive differentiation, implementing genetic approaches such as specific siRNA knockdown of each protein individually, followed by antibody detection, can confirm specificity and enable researchers to distinguish the unique functions of each NHERF family member in their experimental system.

What are the implications of the observed molecular weight discrepancy for SLC9A3R2?

The observed molecular weight of SLC9A3R2 in Western blot applications (approximately 45 kDa) differs from the expected theoretical weight of 37.414 kDa, which has important implications for research interpretation . This discrepancy likely reflects post-translational modifications such as phosphorylation, which is known to occur at multiple sites within SLC9A3R2 and affects its binding properties and function. Researchers should consider several analytical approaches to investigate this phenomenon: (1) phosphatase treatment of samples prior to Western blotting to determine if phosphorylation contributes to the observed shift; (2) mass spectrometry analysis to identify specific modifications; (3) comparison across different tissues and cell types to determine if the weight discrepancy is universal or context-dependent; and (4) correlation of different molecular weight forms with specific functions or cellular localizations . Understanding these modifications is crucial for accurately interpreting experiments, as they may represent different functional states of the protein rather than non-specific antibody binding.

How can SLC9A3R2 antibodies be effectively used in protein-protein interaction studies?

SLC9A3R2 functions as a scaffold protein that mediates numerous protein-protein interactions, making it an important target for interaction studies. When designing such experiments, researchers should consider several methodological approaches. Immunoprecipitation (IP) using SLC9A3R2 antibodies can be performed with 1-2 μg of antibody per 200-500 μg of total protein lysate, followed by Western blot analysis of co-precipitated proteins . To minimize interference from antibody heavy and light chains, researchers should consider using antibodies conjugated to beads or employing detection antibodies from different species. For proximity ligation assays (PLA), which allow visualization of protein interactions in situ, researchers should use the SLC9A3R2 antibody (2-5 μg/ml) in combination with antibodies against suspected interaction partners . When performing co-localization studies by immunofluorescence, careful optimization of antibody concentrations and blocking conditions is essential to minimize background. For more complex interaction networks, researchers might consider combining antibody-based pulldowns with mass spectrometry analysis to identify novel interaction partners in different cellular contexts.

What are common issues with SLC9A3R2 detection in IHC applications and how can they be resolved?

Immunohistochemical detection of SLC9A3R2 can present several challenges that require methodological optimization. Common issues include weak or absent staining, high background, or non-specific staining. Based on validated protocols, researchers should implement the following optimization strategies: (1) Ensure proper antigen retrieval using heat-mediated methods in EDTA buffer (pH 8.0) ; (2) Optimize blocking conditions using 10% goat serum to minimize non-specific binding ; (3) Titrate antibody concentration within the recommended 2-5 μg/ml range, testing both lower and higher concentrations if necessary ; (4) Extend primary antibody incubation to overnight at 4°C to enhance specific binding ; (5) Optimize secondary antibody concentration and incubation time (recommended 30 minutes at 37°C) ; (6) Include appropriate positive control tissues with known SLC9A3R2 expression (e.g., kidney, lung tissues for mouse and rat; thyroid, colorectal, or lung cancer tissues for human) ; and (7) Compare membrane versus cytoplasmic staining patterns, as SLC9A3R2 localization can vary depending on tissue type and physiological state.

How can specificity of SLC9A3R2 antibodies be verified in flow cytometry applications?

Verifying antibody specificity in flow cytometry applications requires rigorous control experiments and optimization procedures. For SLC9A3R2 detection by flow cytometry, researchers should follow this validation approach: (1) Prepare cells with 4% paraformaldehyde fixation and block with 10% normal goat serum ; (2) Use the recommended antibody concentration (1 μg per 10^6 cells) for primary antibody incubation (30 min at 20°C) ; (3) Apply fluorophore-conjugated secondary antibody (e.g., DyLight®488 conjugated goat anti-rabbit IgG at 5-10 μg per 10^6 cells) for 30 minutes at 20°C ; (4) Include multiple controls in each experiment: isotype control (rabbit IgG at equivalent concentration), unstained cells, and cells stained with secondary antibody only ; (5) For definitive specificity validation, compare staining in SLC9A3R2 knockdown/knockout cells versus wild-type cells; (6) Analyze the staining pattern in conjunction with cell permeabilization experiments to distinguish membrane versus intracellular protein pools; and (7) Consider dual staining with markers of subcellular compartments to verify expected localization patterns.

What approaches can resolve inconsistent Western blot results with SLC9A3R2 antibodies?

Inconsistent Western blot results can arise from various sources when detecting SLC9A3R2. Researchers facing such challenges should implement a systematic troubleshooting approach: (1) Optimize protein extraction methods, considering that scaffold proteins may require specialized lysis buffers containing appropriate detergents to solubilize membrane-associated fractions ; (2) Test fresh versus frozen samples, as protein degradation can affect detection; (3) Adjust protein loading (recommended 30 μg) and expose membranes for multiple time intervals to identify optimal signal-to-noise ratios ; (4) Optimize blocking conditions, testing BSA versus milk-based blockers if background is problematic; (5) Test a concentration gradient of primary antibody around the recommended 0.25-0.5 μg/ml range ; (6) Extend primary antibody incubation to overnight at 4°C as recommended in validated protocols ; (7) Verify transfer efficiency with reversible protein stains before immunodetection; and (8) Consider native versus reducing conditions, as some epitopes may be sensitive to strong reducing agents. If inconsistencies persist, researchers should also consider comparing results with an independent SLC9A3R2 antibody targeting a different epitope, as recommended by current antibody validation guidelines .

How should researchers interpret differential SLC9A3R2 expression patterns across tissue types?

When analyzing SLC9A3R2 expression across different tissues, researchers should consider several interpretative frameworks to extract meaningful biological insights. SLC9A3R2 demonstrates varied expression patterns across tissues, with notable detection in human cancer tissues (thyroid, colorectal, lung, laryngeal), mouse tissues (kidney, lung), and rat tissues (kidney, skin, lung) . These differential patterns likely reflect tissue-specific functions of this scaffold protein. When interpreting such data, researchers should: (1) Normalize expression to appropriate housekeeping proteins for quantitative comparisons; (2) Correlate protein expression with mRNA levels using orthogonal methods like RT-qPCR; (3) Consider subcellular localization differences, as SLC9A3R2 may redistribute between membrane and cytoplasmic compartments depending on tissue type or pathological state; (4) Analyze expression in the context of known interaction partners that may be tissue-specifically expressed; and (5) Interpret expression differences in relation to tissue-specific functions, such as ion transport in kidney versus other functions in cancer tissues. This contextualized interpretation approach will help distinguish biologically meaningful expression differences from technical artifacts.

What are the implications of SLC9A3R2 detection in various cancer tissues?

The detection of SLC9A3R2 in multiple cancer tissues, including thyroid cancer, colorectal adenocarcinoma, lung cancer, and laryngeal squamous cell carcinoma, suggests potential roles in cancer biology that merit careful investigation . When researching SLC9A3R2 in cancer contexts, several analytical considerations are important: (1) Compare expression levels between matched normal and cancer tissues from the same patients when possible; (2) Correlate expression patterns with cancer stage, grade, and patient outcomes to assess potential prognostic value; (3) Analyze subcellular localization changes that might reflect altered protein function in malignant versus normal cells; (4) Investigate co-expression with known oncogenic signaling partners, such as growth factor receptors that may interact with SLC9A3R2; (5) Evaluate the functional consequences of SLC9A3R2 knockdown or overexpression on cancer cell phenotypes (proliferation, migration, invasion); and (6) Consider how post-translational modifications of SLC9A3R2 (reflected in the observed molecular weight shift from 37 to 45 kDa) might relate to altered function in cancer contexts . This multifaceted approach can help distinguish whether SLC9A3R2 serves as a biomarker or plays a mechanistic role in cancer progression.

How can researchers reconcile contradictory results from different SLC9A3R2 antibody-based methods?

When faced with contradictory results from different antibody-based methods (e.g., Western blot showing one expression pattern and IHC indicating another), researchers should implement a systematic analytical approach. First, recognize that each method detects proteins in different states: Western blot analyzes denatured proteins, while IHC examines proteins in their tissue context with potential epitope masking . To reconcile discrepancies: (1) Validate each antibody independently using the five-pillar approach (orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry) ; (2) Compare antibody performance across multiple applications with standardized positive controls; (3) Evaluate epitope accessibility issues that might affect one method but not others; (4) Consider the possibility that different protein isoforms or post-translationally modified forms might be preferentially detected by different methods or antibodies; (5) Implement orthogonal, antibody-independent methods such as mass spectrometry or mRNA analysis to provide additional reference points ; and (6) Design experiments that can test specific hypotheses about the observed discrepancies, such as tissue-specific post-translational modifications or interaction-induced epitope masking.