SLC26A4 Antibody, HRP conjugated

What is SLC26A4 and why is it important to study?

SLC26A4, also known as Pendrin or Sodium-independent chloride/iodide transporter, is a protein that functions as a transporter of chloride and iodide ions independently of sodium. Its significance stems from its critical physiological roles in multiple organ systems, particularly in the inner ear, thyroid, and kidney. Mutations in the SLC26A4 gene are associated with Pendred syndrome, a genetic disorder characterized by hearing loss and thyroid goiter. Studying SLC26A4 using specific antibodies enables researchers to investigate its expression patterns, localization, and functional properties in normal and pathological contexts .

What are the key characteristics of SLC26A4 Polyclonal Antibody, HRP Conjugated?

The SLC26A4 Polyclonal Antibody, HRP Conjugated is a primary antibody derived from rabbit hosts that specifically recognizes human SLC26A4 protein. Its key technical specifications include:

This antibody has been specifically designed for ELISA applications with a recommended dilution range of 1:500-1:1000 .

How does HRP conjugation enhance antibody functionality in SLC26A4 detection?

HRP (Horseradish Peroxidase) conjugation provides significant advantages for SLC26A4 detection by eliminating the need for a secondary antibody step in immunoassays. The HRP enzyme directly catalyzes the oxidation of chromogenic or chemiluminescent substrates, producing a detectable signal that corresponds to the presence and abundance of SLC26A4. This direct detection approach simplifies experimental workflows, reduces background noise that can occur from secondary antibody cross-reactivity, and typically enhances sensitivity. For quantitative ELISA applications, HRP-conjugated antibodies enable more precise measurement of SLC26A4 expression levels by providing a direct relationship between signal intensity and antigen concentration .

How should SLC26A4 Antibody, HRP Conjugated be stored and handled to maintain optimal activity?

For optimal preservation of the SLC26A4 Antibody, HRP Conjugated, researchers should implement a systematic storage and handling protocol. The antibody should be stored at -20°C for long-term storage, while avoiding repeated freeze-thaw cycles which can significantly degrade both antibody binding capacity and HRP enzymatic activity. For working solutions, store at 4°C for up to one week. When handling, always use clean, nuclease-free tubes and pipette tips to prevent contamination. Additionally, the antibody solution should never be vortexed vigorously, as this can denature the protein structure; instead, gentle inversion or mild pulse-vortexing is recommended. When preparing dilutions, use high-quality buffers free of reducing agents or sodium azide, as these chemicals can inhibit HRP activity. A stabilizing protein such as BSA (0.1-1%) in the dilution buffer can help maintain antibody performance .

What strategies can optimize specificity when using SLC26A4 Antibody, HRP Conjugated in tissues with low target expression?

Detecting SLC26A4 in tissues with low expression levels requires a multi-faceted optimization strategy. First, implement an antigen retrieval protocol tailored to SLC26A4's molecular characteristics—heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) is often effective for exposing the 592-655AA epitope region this antibody targets. Second, extend the primary antibody incubation time to 16-24 hours at 4°C using a higher antibody concentration (1:250-1:300 dilution) to enhance binding opportunities. Third, incorporate signal amplification systems like tyramide signal amplification (TSA), which can increase detection sensitivity by 10-50 fold while maintaining specificity. Fourth, reduce background by using specialized blocking solutions containing both serum (5%) matched to the host species of your tissues and 1% BSA. Finally, validate all signals using appropriate negative controls (sample processing without primary antibody) and positive controls (tissues known to express SLC26A4 at high levels). This comprehensive approach maximizes detection sensitivity while preserving the specificity required for confident interpretation of results .

How can SLC26A4 Antibody, HRP Conjugated be validated to ensure target specificity?

Comprehensive validation of SLC26A4 Antibody, HRP Conjugated requires a multi-method approach. Begin with positive and negative control tissues—thyroid, inner ear, and kidney tissues should show positive staining, while tissues not known to express SLC26A4 should remain negative. Implement peptide competition assays by pre-incubating the antibody with excess recombinant SLC26A4 protein (592-655AA region); this should abolish specific staining, confirming target specificity. For quantitative validation, perform western blotting with cell lines expressing recombinant SLC26A4 at different levels to establish a correlation between band intensity and known expression levels. Compare results with alternative antibodies targeting different epitopes of SLC26A4, as concordant results strengthen validation. Additionally, use siRNA knockdown or CRISPR knockout models to verify that signal reduction correlates with decreased SLC26A4 expression. The most rigorous validation includes mass spectrometry identification of immunoprecipitated proteins to confirm that the antibody captures authentic SLC26A4. Document all validation steps meticulously, including experimental conditions, to establish reproducibility and reliability standards .

What are the potential cross-reactivity concerns with SLC26A4 Antibody, HRP Conjugated, and how can they be addressed?

Cross-reactivity potential with SLC26A4 Antibody, HRP Conjugated primarily stems from sequence homology between SLC26A4 and other members of the SLC26 family, particularly SLC26A3 and SLC26A6, which share structural domains. Since this antibody targets the 592-655AA region of human Pendrin, researchers should conduct bioinformatic analyses to identify proteins with similar epitope sequences. To address these concerns methodologically, implement a tiered approach: first, perform pre-absorption studies by incubating the antibody with recombinant proteins of suspected cross-reactive targets; second, include western blot analysis comparing tissues with differential expression of SLC26 family members; third, use parallel staining with antibodies known to be specific for potential cross-reactive proteins and compare localization patterns. Additionally, validate results using orthogonal methods such as in situ hybridization to confirm that protein detection correlates with mRNA expression. When working with tissues from species other than human, conduct sequence homology analysis of the target epitope region (592-655AA) before experimentation to predict potential cross-reactivity issues. Document all controls systematically to support the specificity of observed signals .

How does epitope accessibility affect SLC26A4 detection, and what methods improve detection in fixed tissues?

Epitope accessibility for SLC26A4 detection is significantly affected by protein conformation, post-translational modifications, and fixation-induced chemical alterations that may mask the target epitope (592-655AA region). Formalin fixation often creates methylene bridges that cross-link proteins, potentially obscuring epitopes. To address this methodologically, implement a systematic optimization approach: first, compare multiple antigen retrieval methods, including heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0), Tris-EDTA (pH 9.0), and enzymatic retrieval using proteinase K or trypsin at varying concentrations and incubation times. For each method, establish a quantifiable readout of staining intensity and background. Second, optimize fixation protocols prospectively—reduce fixation time or use alternative fixatives such as zinc-based or alcohol-based solutions that cause less cross-linking. Third, incorporate detergents like Triton X-100 (0.1-0.3%) or saponin (0.01-0.1%) in antibody diluents to enhance membrane permeabilization and improve antibody access to transmembrane proteins like SLC26A4. Fourth, consider the use of section thickness—thinner sections (3-4 μm) often provide better reagent penetration. Finally, implement different detection systems with varying sensitivity thresholds, such as polymer-based detection versus avidin-biotin systems, to determine which best reveals SLC26A4 in your specific tissue context .

What is the optimal protocol for using SLC26A4 Antibody, HRP Conjugated in ELISA applications?

For optimal ELISA performance using SLC26A4 Antibody, HRP Conjugated, implement this standardized protocol: Begin by coating high-binding 96-well plates with target protein (recombinant SLC26A4 for standard curve) or sample proteins at 1-10 μg/ml in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. After coating, wash three times with PBS-T (PBS + 0.05% Tween-20) and block with 2% BSA in PBS for 2 hours at room temperature. Prepare antibody dilutions in 1% BSA/PBS-T at the recommended 1:500-1:1000 range, with serial dilutions for optimization if needed. Add 100 μl of diluted antibody to each well and incubate for 2 hours at room temperature or overnight at 4°C for maximum sensitivity. After incubation, wash five times with PBS-T, ensuring complete removal of unbound antibody. Add 100 μl of appropriate HRP substrate (TMB for colorimetric detection or luminol-based solutions for chemiluminescence) and monitor signal development. For TMB, stop the reaction with 50 μl of 2N H₂SO₄ when appropriate color development occurs (typically 5-30 minutes) and read absorbance at 450 nm. Include critical controls: blank wells (no antigen, with antibody), negative controls (irrelevant protein, with antibody), and positive controls (known SLC26A4-positive samples). For quantitative analysis, establish a standard curve using recombinant SLC26A4 protein at concentrations ranging from 0-1000 ng/ml. Calculate inter- and intra-assay coefficients of variation to establish reproducibility metrics for your specific experimental conditions .

Can SLC26A4 Antibody, HRP Conjugated be adapted for Western blotting, and what protocol modifications are necessary?

While the SLC26A4 Antibody, HRP Conjugated is primarily recommended for ELISA applications, it can be adapted for Western blotting with specific protocol modifications. First, during sample preparation, use a membrane protein extraction buffer containing 1% Triton X-100 or 0.5% SDS to efficiently solubilize SLC26A4, which is a transmembrane protein. For protein denaturation, heat samples at a lower temperature (70°C instead of 95°C) for 5 minutes to prevent potential aggregation of membrane proteins. Use a lower percentage gel (8-10% acrylamide) to better resolve the relatively large SLC26A4 protein (approximately 86 kDa). After transfer to PVDF membrane (preferred over nitrocellulose for hydrophobic proteins), perform blocking with 5% non-fat dry milk in TBS-T for 2 hours at room temperature. Since the antibody is already HRP-conjugated, dilute it in blocking buffer at 1:250-1:500 (higher concentration than for ELISA) and incubate overnight at 4°C. Perform more stringent washing steps: 5-7 washes with TBS-T, 10 minutes each, to reduce background. Use enhanced chemiluminescence (ECL) substrate with extended exposure times (starting at 30 seconds and increasing as needed). For validation, run parallel blots with conventional non-conjugated SLC26A4 antibodies followed by secondary antibody detection to compare band patterns. The expected molecular weight for human SLC26A4 is approximately 86 kDa, though post-translational modifications may result in higher apparent molecular weights. Include positive control lysates from cells known to express SLC26A4 (thyroid cell lines) and negative controls (SLC26A4-knockout or low-expressing cell lines) .

What strategies are recommended for multiplexing SLC26A4 Antibody, HRP Conjugated with other antibodies?

For successful multiplexing with SLC26A4 Antibody, HRP Conjugated, implement a sequential detection strategy that preserves signal specificity while enabling multiple target visualization. Since this antibody is already HRP-conjugated, it should be incorporated last in your multiplexing sequence to prevent signal interference. Begin with unconjugated primary antibodies against other targets of interest, followed by fluorophore-conjugated secondary antibodies with emission spectra distinct from the HRP detection channel. After thorough washing, apply the SLC26A4 Antibody, HRP Conjugated at its optimized dilution (1:500). For visualization, use tyramide signal amplification (TSA) with fluorescent substrates that emit in a non-overlapping wavelength (typically Cy5 or similar far-red fluorophores if your other targets are labeled with green/red fluorophores). If performing chromogenic multiplexing, utilize the HRP-conjugated SLC26A4 antibody with a brown DAB substrate, then employ alkaline phosphatase-conjugated antibodies with complementary substrates (Vector Blue, Vector Red) for additional targets. Critical optimization steps include: (1) thorough antibody stripping between detection cycles using glycine buffer (pH 2.2) or commercial stripping reagents; (2) implementing spectral unmixing during image acquisition and analysis to resolve potential signal overlap; (3) conducting single-staining controls in parallel to verify that multiplexing doesn't alter individual signal patterns; and (4) careful titration of each antibody in the multiplex panel to balance signal intensities. For automated systems, program extended washing cycles (minimum 5-7 washes) between antibody applications to minimize cross-reactivity and background accumulation .

How should researchers quantify and normalize SLC26A4 expression data from immunoassays?

For rigorous quantification and normalization of SLC26A4 expression data, implement a multi-layered approach that addresses both technical and biological variability. When using ELISA with the HRP-conjugated antibody, generate a standard curve using recombinant SLC26A4 protein (592-655AA region) at 8-10 concentrations ranging from 0-1000 ng/ml, and fit data to a 4-parameter logistic regression model to accurately interpolate unknown sample concentrations. For normalization, measure total protein concentration in each sample using BCA or Bradford assays and express SLC26A4 levels as pg of SLC26A4 per μg of total protein. If examining tissue sections or cell preparations, implement digital image analysis using software such as ImageJ or CellProfiler with consistent thresholding parameters across all samples. Quantify signal intensity as integrated optical density (IOD) and normalize to cell count using nuclear counterstains or to tissue area for histological sections. For western blotting adaptations, normalize SLC26A4 band intensity to established housekeeping proteins appropriate for your experimental context (β-actin for general normalization; Na⁺/K⁺-ATPase for membrane protein fraction). To address the common issue of non-linear detection ranges in chemiluminescent imaging, perform a dilution series of positive control samples to establish the dynamic range of detection and ensure all experimental measurements fall within this validated range. For time-course or treatment comparison studies, calculate fold change relative to baseline or control conditions after normalization. Report all quantitative data with appropriate statistical analyses including measures of technical replication (intra-assay coefficient of variation should be <15%) and biological replication (n≥3 independent biological samples) .

How can researchers distinguish between specific and non-specific signals when using SLC26A4 Antibody, HRP Conjugated?

Distinguishing specific from non-specific signals requires a systematic validation approach. First, implement comprehensive controls: (1) omission controls (exclude primary antibody but include all other reagents) to identify background from secondary detection systems, (2) isotype controls with matching concentration of irrelevant HRP-conjugated rabbit IgG to identify Fc receptor-mediated binding, and (3) peptide competition assays using excess recombinant SLC26A4 protein (592-655AA region) to confirm signal specificity. Second, examine signal localization patterns—SLC26A4 should predominantly localize to plasma membranes in polarized epithelial cells, particularly in thyroid follicular cells, inner ear structures, and kidney cells. Any nuclear, widespread cytoplasmic, or uniform staining across all cell types likely indicates non-specific binding. Third, compare signal intensity across tissues known to express varying levels of SLC26A4; signal strength should correlate with expected expression patterns (high in thyroid, moderate in inner ear, low/absent in tissues like skeletal muscle). Fourth, evaluate signal persistence across dilution series—specific signals should diminish in a concentration-dependent manner, while non-specific background may remain constant regardless of antibody dilution. Finally, validate findings with orthogonal detection methods such as in situ hybridization or independent antibodies targeting different SLC26A4 epitopes. Document all validation steps systematically, including imaging parameters, exposure times, and threshold settings to establish reproducible criteria for distinguishing specific from non-specific signals .

What are common sources of false positive and false negative results when detecting SLC26A4, and how can they be mitigated?

False positive and false negative results with SLC26A4 detection stem from distinct methodological issues requiring targeted interventions. For false positives: (1) Cross-reactivity with related SLC26 family members can be mitigated by pre-absorbing the antibody with recombinant proteins of suspected cross-reactive targets; (2) Endogenous peroxidase activity, particularly in tissues like liver and kidney, can be neutralized by pre-treatment with 0.3% H₂O₂ in methanol for 30 minutes; (3) Hydrophobic interactions causing non-specific binding can be reduced by including 0.1-0.3% Triton X-100 and 0.5% BSA in all antibody diluents; (4) Charge-based interactions can be minimized by increasing salt concentration in wash buffers to 150-300 mM NaCl. For false negatives: (1) Epitope masking due to fixation can be addressed through systematic optimization of antigen retrieval methods—compare heat-induced retrieval using citrate buffer (pH 6.0), Tris-EDTA (pH 9.0), and enzymatic retrieval with proteinase K; (2) Insufficient antibody concentration may require titration experiments with concentrations ranging from 1:100 to 1:1000; (3) Degraded SLC26A4 protein in samples can be prevented by adding protease inhibitor cocktails during sample preparation and processing tissues rapidly; (4) Competitive inhibition from endogenous biotin can be eliminated by using biotin-blocking kits prior to antibody application. For both scenarios, implement tissue microarrays containing positive and negative control tissues processed identically to experimental samples as internal controls for each assay. Additionally, include gradient controls (samples with known varying levels of SLC26A4 expression) to establish the detection threshold and dynamic range of your specific experimental conditions .

How should researchers interpret variable staining patterns of SLC26A4 across different tissue types?

Interpreting variable SLC26A4 staining patterns requires integration of biological context with technical considerations. SLC26A4 (Pendrin) exhibits tissue-specific localization patterns reflective of its diverse physiological functions. In thyroid follicular cells, authentic SLC26A4 staining typically appears as strong apical membrane labeling facing the follicular lumen, corresponding to its role in iodide transport. In the inner ear, particularly in the endolymphatic duct and spiral ligament, SLC26A4 localizes to basolateral membranes of epithelial cells, consistent with its function in maintaining ion homeostasis. In kidney, SLC26A4 expression is restricted to β-intercalated cells of the cortical collecting duct with distinctive apical membrane staining. When interpreting staining patterns, evaluate membrane localization specificity—cytoplasmic signals may represent newly synthesized protein in transit or potential artifacts. Consider polarization status of the cells, as SLC26A4 distribution changes with cellular differentiation and polarization. Staining intensity variations across tissues reflect natural expression differences and should correlate with known mRNA expression patterns. For quantitative comparisons, normalize signal intensity to positive control tissues processed in parallel. Variations in subcellular localization may have biological significance; for example, stress conditions can trigger redistribution from membrane to cytoplasmic compartments. When unexpected patterns emerge, validate with alternative antibodies targeting different epitopes and correlate with functional assays measuring SLC26A4-mediated ion transport. Document cellular context thoroughly, including tissue architecture and neighboring cell types, as microenvironmental factors influence SLC26A4 expression and localization .

What role does sample preparation play in SLC26A4 antibody performance, and how can it be optimized?

Sample preparation critically influences SLC26A4 antibody performance through multiple mechanisms affecting epitope preservation, accessibility, and specificity. For optimal results, implement a systematic approach addressing each preparation stage. During tissue collection, minimize ischemia time (<30 minutes) as prolonged ischemia can trigger protein degradation and translocation. For fixation, use 10% neutral-buffered formalin for 24-48 hours at room temperature—shorter periods risk inadequate fixation while longer periods cause excessive cross-linking that may mask the target epitope (592-655AA region). After fixation, thoroughly wash tissues in PBS to remove residual fixative that could interfere with antibody binding. During tissue processing, limit dehydration times to prevent protein denaturation and implement controlled paraffin infiltration temperatures (maximum 60°C) to preserve protein conformation. For sectioning, prepare 4-5 μm thick sections to balance structural integrity with reagent penetration. Prior to immunostaining, implement deparaffinization with fresh xylene followed by rehydration through an ethanol gradient to water. For antigen retrieval, systematically compare heat-induced methods (citrate buffer pH 6.0, EDTA buffer pH 9.0) at different temperatures (90-125°C) and durations (10-30 minutes) to determine optimal conditions for SLC26A4 detection. When working with frozen tissues, fix sections briefly (10 minutes) in 4% paraformaldehyde after cutting to stabilize membrane proteins. For cells in culture, optimize fixation conditions based on application—4% paraformaldehyde (10 minutes) for immunofluorescence or direct lysis in appropriately buffered detergent solutions for ELISA applications. Maintain consistent sample preparation protocols across experimental groups to ensure comparable results, and document all processing parameters to enable troubleshooting of unexpected staining patterns .