SLC9A3R2 Antibody, Biotin conjugated

What is SLC9A3R2 and why is it significant in molecular research?

SLC9A3R2 (Solute Carrier Family 9 Member A3 Regulator 2), also known as NHERF-2 (Na+/H+ Exchanger Regulatory Factor 2) or SIP-1, is a PDZ domain-containing scaffold protein that plays critical roles in various cellular processes. This protein functions primarily in regulating membrane protein trafficking, localization, and signaling. The significance of SLC9A3R2 in research stems from its involvement in stimulating CFTR (Cystic Fibrosis Transmembrane conductance Regulator) channel activity, as indicated by developmental biology studies . This protein is expressed in multiple tissue types including kidney, lung, thyroid, and various cancer tissues, making it relevant to both physiological and pathological research contexts . Studying SLC9A3R2 provides insights into epithelial transport mechanisms, signal transduction pathways, and potential therapeutic targets in diseases where these processes are dysregulated.

What are the primary advantages of using biotin-conjugated antibodies for SLC9A3R2 detection?

Biotin-conjugated antibodies offer several significant advantages for SLC9A3R2 detection in research applications:

• Enhanced signal amplification: The biotin-streptavidin system provides one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), significantly enhancing detection sensitivity for proteins expressed at low levels .

• Versatility across detection platforms: Biotin-conjugated antibodies can be used with various detection systems including HRP, fluorescent dyes, or quantum dot-conjugated streptavidin, providing flexibility in experimental design .

• Improved stability: Biotin conjugates typically demonstrate better stability compared to direct enzyme or fluorophore conjugates, leading to more consistent results across experiments .

• Compatibility with multiplexed assays: The specific biotin-conjugated SLC9A3R2 antibody (CSB-PA01759D0Rb) is particularly recommended for ELISA applications, making it valuable for quantitative protein analysis .

• High-quality preparation: These antibodies undergo immunoaffinity chromatography using antigen coupled to sepharose beads followed by solid phase adsorption to remove unwanted reactivities, ensuring specificity in experimental applications .

Which experimental techniques are most compatible with biotin-conjugated SLC9A3R2 antibodies?

Biotin-conjugated SLC9A3R2 antibodies demonstrate compatibility with multiple experimental techniques:

• Enzyme-Linked Immunosorbent Assay (ELISA): The biotin-conjugated version (CSB-PA01759D0Rb) is specifically recommended for ELISA applications, providing sensitive quantitative analysis of SLC9A3R2 levels .

• Immunohistochemistry (IHC): While standard SLC9A3R2 antibodies have been extensively validated in IHC applications across various tissue types including human thyroid cancer, colorectal adenocarcinoma, lung cancer, and laryngeal squamous cell carcinoma, the biotin conjugate offers enhanced detection sensitivity through streptavidin-based amplification systems .

• Western Blotting: Though requiring optimization, recommended dilutions of 1:500-1:5000 for standard SLC9A3R2 antibodies suggest similar ranges would be effective for the biotin-conjugated version .

• Flow Cytometry: SLC9A3R2 antibody has demonstrated successful application in flow cytometry analysis of PC-3 cells, indicating the biotin-conjugated version could be effectively paired with streptavidin-fluorophore conjugates for this application .

• Dot Blot Analysis: Similar to other biotin-conjugated antibodies, SLC9A3R2 biotin conjugates would be suitable for dot blot applications using streptavidin detection systems .

What protocol modifications are necessary when using biotin-conjugated versus standard SLC9A3R2 antibodies?

When transitioning from standard to biotin-conjugated SLC9A3R2 antibodies, several protocol modifications are essential:

• Endogenous biotin blocking: For tissue samples (particularly kidney, liver, and brain), include a biotin/avidin blocking step before applying the biotin-conjugated antibody to prevent non-specific binding to endogenous biotin. This typically involves sequential incubation with avidin and biotin solutions .

• Detection system adaptation: Replace enzyme-conjugated secondary antibodies with streptavidin-conjugated detection reagents (HRP, fluorophores, or quantum dots) . The high affinity of streptavidin for biotin (Kd ≈ 10^-15 M) provides excellent sensitivity.

• Dilution optimization: While standard SLC9A3R2 antibodies are recommended at 1:20-1:200 for IHC and 1:500-1:5000 for Western blotting, biotin-conjugated versions typically require further optimization, often starting at 2-5 fold higher dilutions due to the signal amplification effect .

• Incubation time adjustments: Reduce streptavidin-conjugate incubation times compared to secondary antibodies to minimize background signal while maintaining specific detection.

• Washing stringency: Increase washing steps (both number and duration) following biotin-conjugated antibody and streptavidin-conjugate incubations to reduce background signal.

When properly optimized, these modifications enable more sensitive detection of SLC9A3R2 while maintaining specificity across experimental applications.

What are the optimal tissue preparation methods for SLC9A3R2 immunohistochemistry using biotin-conjugated antibodies?

Based on extensive validation studies, optimal tissue preparation for SLC9A3R2 immunohistochemistry follows this protocol:

• Fixation: Paraffin embedding following fixation in 4% paraformaldehyde provides consistent results across tissue types. This fixation method preserves SLC9A3R2 epitopes while maintaining tissue morphology .

• Sectioning: Thin sections (4-6 μm) ensure adequate antibody penetration and optimal signal-to-noise ratio.

• Antigen retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is consistently effective across diverse tissue types including human cancer tissues and rodent normal tissues. This specific retrieval condition appears critical for exposing SLC9A3R2 epitopes .

• Blocking: Two-step blocking is recommended:

  • First, block endogenous biotin using commercial avidin/biotin blocking kits

  • Second, block non-specific binding with 10% goat serum as consistently used in validation studies

• Primary antibody incubation: Incubate with biotin-conjugated SLC9A3R2 antibody (starting at approximately 2 μg/ml) overnight at 4°C for optimal binding kinetics .

• Detection: Incubate with streptavidin-HRP conjugate for 30 minutes at 37°C, followed by development using DAB chromogen .

This protocol has demonstrated consistent results across multiple tissue types including human thyroid cancer, colorectal adenocarcinoma, lung cancer, and laryngeal squamous cell carcinoma, as well as normal tissues from mouse and rat models .

How should researchers quantitatively analyze SLC9A3R2 expression across experimental conditions?

Quantitative analysis of SLC9A3R2 expression requires standardized methodologies to ensure reproducibility and accuracy:

• Flow cytometry quantification:

  • Fix cells with 4% paraformaldehyde

  • Block with 10% normal goat serum

  • Incubate with biotin-conjugated SLC9A3R2 antibody (approximately 1 μg/10^6 cells)

  • Detect with streptavidin-fluorophore conjugate

  • Include appropriate controls: isotype control antibody and unstained samples

  • Quantify mean fluorescence intensity and percent positive cells relative to controls

• Quantitative ELISA:

  • The biotin-conjugated SLC9A3R2 antibody (CSB-PA01759D0Rb) is specifically recommended for ELISA applications

  • Develop standard curves using recombinant SLC9A3R2 protein

  • Analyze samples in technical triplicates to ensure statistical reliability

  • Calculate protein concentration based on standard curve regression analysis

• Immunohistochemistry quantification:

  • Use digital image analysis software to measure:
    a) Staining intensity (integrated optical density)
    b) Percent positive cells/area
    c) Subcellular localization patterns

  • Include calibration standards in each batch to normalize for staining variability

  • Analyze multiple fields (minimum 5-10) per sample to account for tissue heterogeneity

• Western blot densitometry:

  • Use recommended dilutions (1:500-1:5000)

  • Include loading controls (β-actin, GAPDH)

  • Quantify band intensity using image analysis software

  • Normalize target protein to loading control for comparative analysis

For all quantitative analyses, statistical validation (t-tests, ANOVA) should be performed to determine significance of observed differences between experimental conditions.

How does SLC9A3R2 contribute to protein-protein interaction networks in cellular signaling?

SLC9A3R2 functions as a critical scaffold protein mediating multiple protein interactions through its PDZ domains. Research indicates several important aspects of its role in protein-protein interaction networks:

• Ion channel regulation: SLC9A3R2 stimulates CFTR channel activity, indicating a direct functional interaction with this cystic fibrosis-associated ion channel . This interaction likely occurs through the PDZ domains of SLC9A3R2 binding to the PDZ-binding motif (D-T-R-L) present in the C-terminus of CFTR.

• Experimental techniques for studying interactions:

  • Affinity purification using biotin-tagged peptides corresponding to interaction domains

  • Mass spectrometry for unbiased identification of binding partners

  • Formic acid elution (10%) for recovering protein complexes for further analysis

• Interaction dynamics: SLC9A3R2 may function as part of larger multiprotein complexes, mediating connections between membrane proteins and cytoskeletal elements. This organizational role positions SLC9A3R2 as a critical node in signaling networks.

• Methodological approach: Researchers investigating SLC9A3R2 interactions should consider:

  • Using biotin-conjugated SLC9A3R2 antibody for co-immunoprecipitation studies

  • Performing sequential purifications to identify components of multiprotein complexes

  • Employing proximity ligation assays to visualize interactions in situ

  • Utilizing pull-down assays with recombinant PDZ domains

Understanding these interaction networks provides insights into how SLC9A3R2 influences diverse cellular processes and potentially contributes to pathological conditions when dysregulated.

What expression patterns of SLC9A3R2 have been observed across normal and cancer tissues?

Immunohistochemical analyses reveal distinctive expression patterns of SLC9A3R2 across various tissue types:

• Cancer tissues with significant SLC9A3R2 expression:

  • Human thyroid cancer: Shows distinct SLC9A3R2 immunoreactivity following heat-mediated antigen retrieval in EDTA buffer

  • Human colorectal adenocarcinoma: Demonstrates SLC9A3R2 expression with defined cellular patterns

  • Human lung cancer: Exhibits notable SLC9A3R2 immunoreactivity

  • Human laryngeal squamous cell carcinoma: Shows specific SLC9A3R2 staining patterns

• Normal tissues with SLC9A3R2 expression:

  • Mouse kidney: Demonstrates consistent SLC9A3R2 expression

  • Mouse lung: Shows defined SLC9A3R2 immunoreactivity

  • Rat kidney: Exhibits notable SLC9A3R2 expression

  • Rat skin: Displays specific SLC9A3R2 staining patterns

  • Rat lung: Shows detectable SLC9A3R2 expression

• Cell line expression:

  • PC-3 cells (human prostate cancer cell line): Demonstrate detectable SLC9A3R2 expression by flow cytometry analysis

This expression data suggests SLC9A3R2 plays important physiological roles in epithelial and transport functions across multiple tissues. The consistent expression in kidney tissues across species (mouse and rat) particularly indicates an important role in renal physiology. The presence in multiple cancer types suggests potential involvement in pathological processes, though direct comparative analyses between matched normal and cancer tissues from the same patients would be necessary to determine expression changes in disease states.

What experimental design considerations are essential for multiplex detection including SLC9A3R2?

Multiplexed detection systems incorporating biotin-conjugated SLC9A3R2 antibody require careful experimental design:

• Endogenous biotin management:

  • Implement comprehensive biotin blocking (using commercial avidin/biotin blocking kits) prior to antibody application

  • Consider alternative detection strategies for tissues with exceptionally high endogenous biotin (liver, kidney, brain)

• Sequential versus simultaneous detection:

  • For biotin-based multiplex systems, sequential detection is preferable to minimize cross-reactivity

  • When using the biotin-conjugated SLC9A3R2 antibody alongside other primary antibodies, stagger detection steps with thorough washing between each

• Spectral compatibility:

  • When using fluorescent detection, consider quantum dot-labeled streptavidin as mentioned in research applications ("biotin-conjugated 20 nm-diameter quantum dots 605"), which allows multiplexing with minimal spectral overlap

  • Select complementary fluorophores with minimal spectral overlap for other detection channels

• Antibody compatibility:

  • Choose antibodies from different host species when possible to avoid cross-reactivity

  • When using multiple rabbit antibodies, consider tyramide signal amplification systems that allow sequential detection

• Validation controls:

  • Include single-stained controls to confirm specificity and rule out channel crosstalk

  • Process serial sections with individual antibodies to confirm staining patterns

• Image acquisition:

  • Collect spectral data sequentially rather than simultaneously when possible

  • Implement appropriate compensation algorithms during image analysis to correct for any spectral overlap

These considerations ensure reliable multiplexed detection including SLC9A3R2 without compromising data quality or introducing artifacts.

What are the most common technical issues when working with biotin-conjugated SLC9A3R2 antibody?

Researchers commonly encounter several technical challenges when working with biotin-conjugated SLC9A3R2 antibody:

• High background signal:

  • Cause: Endogenous biotin in tissues (particularly kidney and liver) or insufficient blocking

  • Solution: Implement dedicated avidin/biotin blocking step before antibody application; increase serum blocking concentration to 10-15%; include 0.1-0.3% Triton X-100 in blocking buffer

• Weak or absent signal:

  • Cause: Inadequate antigen retrieval or sample degradation

  • Solution: Strictly adhere to heat-mediated antigen retrieval in EDTA buffer (pH 8.0); extend primary antibody incubation to overnight at 4°C; consider signal amplification systems

• Non-specific staining:

  • Cause: Excessive antibody concentration or cross-reactivity

  • Solution: Titrate antibody concentration starting with recommended dilutions (1:500-1:5000 for WB, 1:20-1:200 for IHC); include appropriate blocking serums; extend washing steps

• Inconsistent results between experiments:

  • Cause: Variations in tissue processing or antibody handling

  • Solution: Standardize fixation conditions; maintain consistent antigen retrieval parameters; aliquot antibodies to avoid freeze-thaw cycles; include internal controls in each experiment

• Signal variability across tissue sections:

  • Cause: Tissue heterogeneity or processing artifacts

  • Solution: Analyze multiple fields per sample; maintain consistent section thickness; ensure uniform reagent distribution during incubations

Addressing these common issues through methodical optimization enables consistent and reliable results when working with biotin-conjugated SLC9A3R2 antibody.

What validation controls should be included in experiments using SLC9A3R2 antibody?

Comprehensive validation controls are essential for ensuring reliable results with SLC9A3R2 antibody:

• Technical controls:

  • Negative control: Omit primary antibody while maintaining all other steps (identifies non-specific binding of detection reagents)

  • Isotype control: Substitute primary antibody with non-specific antibody of the same isotype and concentration (rabbit IgG at 1 μg/10^6 cells)

  • Absorption control: Pre-incubate antibody with recombinant SLC9A3R2 protein to confirm specificity

• Biological controls:

  • Positive tissue controls: Include known positive tissues (kidney shows consistent expression across species)

  • Negative tissue controls: Include tissues with minimal expected expression

  • Cell line controls: PC-3 cells demonstrate detectable SLC9A3R2 expression by flow cytometry

• Antibody validation:

  • Western blot validation: Confirm antibody detects protein of expected molecular weight

  • Immunoelectrophoresis: Verify single precipitin arc against detection reagents

  • Cross-platform validation: Confirm consistent results across different detection methods (IHC, flow cytometry, Western blot)

• Quantitation controls:

  • Standard curves for quantitative assays

  • Internal reference standards for normalization between experiments

  • Technical replicates to assess methodological variability

Incorporating these validation controls provides crucial quality assurance and enables confident interpretation of experimental results when working with SLC9A3R2 antibody.

How should biotin-conjugated SLC9A3R2 antibody be stored to maintain optimal activity?

Proper storage and handling of biotin-conjugated SLC9A3R2 antibody is critical for maintaining its activity:

• Storage temperature:

  • Long-term storage: -20°C for lyophilized antibody or aliquoted solution

  • Working storage: 4°C for diluted antibody (use within 1 week)

  • Avoid repeated freeze-thaw cycles which damage both antibody structure and biotin conjugation

• Buffer composition:

  • Optimal storage buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, with 0.01% Sodium Azide and protein stabilizer (typically 10 mg/mL BSA)

  • Working dilutions should be prepared in buffer compatible with your application

• Reconstitution protocol:

  • Allow lyophilized antibody to reach room temperature before opening to prevent condensation

  • Reconstitute using sterile deionized water or recommended buffer

  • Gently mix without vortexing to avoid protein denaturation

• Aliquoting strategy:

  • Prepare single-use aliquots to avoid repeated freeze-thaw cycles

  • Use sterile microcentrifuge tubes for aliquoting

  • Document concentration and date on each aliquot

• Light protection:

  • While biotin conjugates are generally more stable than fluorophore conjugates, protection from extended light exposure is recommended

  • Store in amber tubes or wrap in aluminum foil

Following these storage guidelines ensures maximal retention of antibody activity and reproducibility across experiments.

What strategies can improve signal detection for low-abundance SLC9A3R2 samples?

When working with samples expressing low levels of SLC9A3R2, several signal enhancement strategies can be employed:

• Amplification systems:

  • Biotin-streptavidin system already provides significant signal amplification

  • For additional sensitivity, consider tyramide signal amplification (TSA) systems

  • Quantum dot conjugates (biotin-conjugated 20 nm-diameter quantum dots 605) offer enhanced brightness and photostability compared to conventional fluorophores

• Optimized antibody parameters:

  • Extended primary antibody incubation (overnight at 4°C as used in validation studies)

  • Higher antibody concentration (starting at 2 μg/ml as used in validation protocols)

  • Reduced washing stringency (shorter, gentler washes)

• Sample preparation enhancement:

  • Optimized antigen retrieval (heat-mediated in EDTA buffer, pH 8.0)

  • Extended retrieval time to maximize epitope exposure

  • Microwave-assisted or pressure cooker retrieval methods

• Detection optimization:

  • Extended chromogen development time (for IHC applications)

  • Use of high-sensitivity substrates (SuperSignal, ECL Prime)

  • Digital image acquisition with extended exposure times and signal integration

• Sample enrichment:

  • Concentration of protein samples before analysis

  • Immunoprecipitation to enrich target protein

  • Cell sorting to isolate populations with higher expression

These strategies should be systematically tested and optimized for specific experimental systems to achieve optimal signal-to-noise ratio while maintaining specificity.

How might SLC9A3R2 research contribute to understanding disease mechanisms?

SLC9A3R2 research has significant potential to advance understanding of disease mechanisms across multiple pathological conditions:

• Cancer biology:

  • The detection of SLC9A3R2 in multiple cancer types (thyroid cancer, colorectal adenocarcinoma, lung cancer, and laryngeal squamous cell carcinoma) suggests potential roles in oncogenic processes

  • Investigation of SLC9A3R2 expression correlation with tumor grade, stage, and patient outcomes could identify prognostic biomarkers

  • Exploration of SLC9A3R2's scaffold function in cancer cell signaling may reveal novel therapeutic targets

• Cystic fibrosis pathophysiology:

  • SLC9A3R2 stimulates CFTR channel activity, positioning it as a key regulator in the pathophysiology of cystic fibrosis

  • Research into how SLC9A3R2-CFTR interactions are affected by disease-causing CFTR mutations could identify new therapeutic approaches

  • Investigation of SLC9A3R2 as a potential compensatory target to enhance residual CFTR function

• Kidney disorders:

  • The consistent expression of SLC9A3R2 in kidney tissues across species highlights its importance in renal physiology

  • Research into SLC9A3R2's role in ion transport regulation could illuminate mechanisms of kidney diseases

  • Study of SLC9A3R2 interactions with renal transporters may identify novel therapeutic targets for kidney disorders

• Epithelial transport disorders:

  • As a scaffold protein regulating multiple transporters and channels, SLC9A3R2 likely plays important roles in epithelial transport

  • Investigation of SLC9A3R2 in absorptive and secretory epithelia could advance understanding of transport disorders

  • Exploration of compensatory mechanisms involving SLC9A3R2 may reveal adaptive responses in disease states

Biotin-conjugated SLC9A3R2 antibodies will facilitate these investigations through applications in tissue profiling, protein-protein interaction studies, and quantitative expression analysis across disease models.

What emerging techniques might enhance SLC9A3R2 research in the near future?

Several emerging technologies show particular promise for advancing SLC9A3R2 research:

• Advanced imaging modalities:

  • Super-resolution microscopy to visualize nanoscale organization of SLC9A3R2-containing protein complexes

  • Expansion microscopy for improved visualization of subcellular localization patterns

  • Live-cell imaging combined with optogenetic tools to study dynamic SLC9A3R2 interactions

• Proteomics approaches:

  • Proximity labeling methods (BioID, APEX) to identify proteins in close spatial proximity to SLC9A3R2

  • Cross-linking mass spectrometry to capture transient protein interactions

  • Quantitative interaction proteomics to measure changes in SLC9A3R2 interactome composition under different conditions

• Genomic engineering:

  • CRISPR/Cas9-mediated endogenous tagging of SLC9A3R2 for visualization and pulldown

  • Creation of conditional knockout models to study tissue-specific functions

  • Base editing or prime editing to introduce specific mutations modeling disease variants

• Multiplexed tissue analysis:

  • Highly multiplexed antibody-based imaging (CODEX, Imaging Mass Cytometry) to analyze SLC9A3R2 in the context of dozens of other markers

  • Spatial transcriptomics to correlate SLC9A3R2 protein expression with local transcriptional programs

  • Single-cell analysis combined with spatial information to understand cellular heterogeneity

• Computational approaches:

  • Molecular modeling of SLC9A3R2 interactions to predict functional consequences of mutations

  • Systems biology integration of SLC9A3R2 into protein-protein interaction networks

  • Machine learning applications for image analysis of SLC9A3R2 expression patterns

Biotin-conjugated SLC9A3R2 antibodies will interface well with many of these technologies, particularly those involving multiplexed detection systems and quantitative proteomics approaches.