SLC16A8 Antibody, HRP conjugated

What is SLC16A8 and what biological functions does it serve?

SLC16A8, also known as Monocarboxylate transporter 3 (MCT3), is a protein-coding gene located on chromosome 22 in humans. It functions as a proton-coupled monocarboxylate transporter that catalyzes the rapid transport of various monocarboxylates across the plasma membrane, including lactate, pyruvate, branched-chain oxo acids derived from leucine, valine, and isoleucine, as well as ketone bodies like acetoacetate, beta-hydroxybutyrate, and acetate. SLC16A8 serves as a high-affinity pyruvate transporter with expression restricted primarily to the retinal pigment epithelium (RPE) and choroid plexus epithelia, where it is specifically located on the basal membrane. This is in contrast to MCT1, which is found on the apical membrane of the same cells. The protein plays a crucial role in facilitating the transport of lactate and protons out of the retina, thereby contributing to the maintenance of pH and ion balance in the outer retina .

What are the key specifications of commercially available SLC16A8 Antibody, HRP conjugated?

SLC16A8 Antibody, HRP conjugated is typically a polyclonal antibody raised in rabbits against recombinant Human Monocarboxylate transporter 3 protein (specifically amino acids 432-504). The antibody is conjugated to Horseradish Peroxidase (HRP) for direct detection in immunoassays. It is supplied in liquid form with a buffer composition of 0.03% Proclin 300 as preservative, 50% Glycerol, and 0.01M PBS at pH 7.4. The antibody undergoes Protein G purification to achieve >95% purity. For storage, manufacturers recommend keeping it at -20°C or -80°C while avoiding repeated freeze-thaw cycles to maintain optimal activity . The antibody is designed to detect human SLC16A8 specifically and is validated primarily for ELISA applications .

How does SLC16A8 Antibody, HRP conjugated differ from non-conjugated forms?

SLC16A8 Antibody, HRP conjugated has the enzyme horseradish peroxidase directly attached to the antibody molecule, eliminating the need for a secondary antibody in detection systems. This direct conjugation provides several advantages over non-conjugated forms, including simplified experimental workflows, reduced background noise, and shorter assay duration. The conjugated antibody allows for direct detection in ELISA applications through enzymatic conversion of appropriate substrates, resulting in colorimetric, chemiluminescent, or fluorescent signals depending on the substrate used. In contrast, non-conjugated SLC16A8 antibodies require a secondary detection system, typically involving a species-specific secondary antibody that recognizes the primary antibody. While non-conjugated antibodies offer greater flexibility for detection methods and potential signal amplification through secondary systems, the HRP-conjugated version provides a more streamlined approach particularly suited for ELISA applications .

What are the validated research applications for SLC16A8 Antibody, HRP conjugated?

The primary validated application for SLC16A8 Antibody, HRP conjugated is ELISA (Enzyme-Linked Immunosorbent Assay) . While the HRP-conjugated form is specifically optimized for ELISA, related non-conjugated forms of the antibody have been validated for additional applications including Western Blot (WB) with recommended dilutions of 1:500-1:5000, Immunohistochemistry (IHC) with dilutions of 1:20-1:200, and Immunofluorescence (IF) with dilutions of 1:50-1:200 . The antibody demonstrates species reactivity with human samples and has been raised against recombinant Human Monocarboxylate transporter 3 protein, specifically the region corresponding to amino acids 432-504 . For researchers interested in different detection methods, alternative conjugates of the same antibody are available, including FITC-conjugated and Biotin-conjugated versions, with the latter also validated for ELISA applications .

How should SLC16A8 Antibody, HRP conjugated be incorporated into ELISA protocols?

When incorporating SLC16A8 Antibody, HRP conjugated into ELISA protocols, researchers should follow a systematic approach to ensure optimal results. For a sandwich ELISA configuration, the microplate should first be coated with a capture antibody specific to human SLC16A8. After blocking, add samples or standards to the wells where they will bind to the capture antibody. Then introduce the SLC16A8 Antibody, HRP conjugated as the detection antibody, which will bind to a different epitope on the target protein. After thorough washing to remove unbound antibodies, add the appropriate substrate solution, which will be converted by HRP to produce a detectable signal. The optical density can be measured spectrophotometrically at 450 nm (±2 nm), with the signal intensity being proportional to the concentration of SLC16A8 in the sample. For quantitative analysis, compare sample readings against a standard curve with a typical range of 0.63-40 ng/mL . The entire assay can be completed in approximately 1.5 hours, making it a rapid detection method compared to conventional ELISA techniques .

What sample types and preparation methods are compatible with SLC16A8 antibody detection?

SLC16A8 antibody detection is compatible with various biological sample types, including serum, plasma (with EDTA or citrate anticoagulants specifically validated), tissue homogenates, and cell culture supernatants . For optimal results, sample preparation should follow these methodological guidelines: For serum samples, allow blood to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at approximately 1000×g for 20 minutes to collect the serum. For plasma preparation, collect blood with appropriate anticoagulants and centrifuge at 1000×g within 30 minutes of collection. For tissue samples, thoroughly wash tissue with PBS to remove excess blood, homogenize in PBS (weight to volume ratio of 1:9), and then freeze-thaw the homogenate. After centrifugation at 5000×g for 10 minutes, collect the supernatant for analysis. For all sample types, if particulates are present, centrifuge again before testing. If the target protein concentration exceeds the standard curve range, dilute samples appropriately with the assay buffer provided in the ELISA kit. Store samples at -20°C or -80°C to avoid loss of bioactivity and contamination, and avoid repeated freeze-thaw cycles .

How can researchers optimize SLC16A8 antibody-based detection in retinal pigment epithelium studies?

Optimizing SLC16A8 antibody-based detection in retinal pigment epithelium (RPE) studies requires careful consideration of several methodological factors. Since SLC16A8 expression is restricted to the RPE and specifically localized to the basal membrane, tissue preparation techniques that preserve cellular polarity are crucial. For immunohistochemistry applications, use directionally-oriented sections or RPE flatmounts that allow clear distinction between apical and basal membranes. When performing co-localization studies, consider double-labeling with basolateral membrane markers to confirm proper localization, and utilize MCT1 (found on the apical membrane) as a control to demonstrate the differential distribution of monocarboxylate transporters in RPE. For quantitative assessment of SLC16A8 in RPE samples, tissue-specific protein extraction methods should be employed, focusing on membrane protein enrichment techniques. Additionally, when conducting functional studies, researchers should account for the unique microenvironment of the RPE, including pH gradients and the high metabolic activity of photoreceptors, which generate lactate that must be transported by SLC16A8. For optimized detection sensitivity in RPE-derived samples, consider signal amplification methods such as tyramide signal amplification when working with the HRP-conjugated antibody, particularly for specimens with potentially low expression levels .

What are the key considerations for troubleshooting false positives/negatives when using SLC16A8 Antibody, HRP conjugated in ELISA?

Troubleshooting false results when using SLC16A8 Antibody, HRP conjugated in ELISA requires systematic analysis of multiple experimental parameters. For false positives, examine potential cross-reactivity with other monocarboxylate transporters in the SLC16 family, particularly MCT1, MCT2, and MCT4, which share structural similarities with SLC16A8/MCT3. Implement more stringent washing procedures (increasing both duration and number of washes) to remove non-specifically bound antibodies. Consider adjusting blocking conditions (using different blocking agents or concentrations) to reduce non-specific binding. For false negatives, verify sample integrity by testing for general protein degradation through alternative protein detection methods. Examine whether storage conditions have compromised antibody activity—HRP conjugates are particularly sensitive to repeated freeze-thaw cycles and extended storage at suboptimal temperatures. Ensure the detection substrate is fresh and properly prepared, as oxidized or contaminated substrates can significantly reduce signal generation. Consider epitope masking in complex biological samples that may require additional sample preparation steps such as gentle detergent treatment or heat-induced epitope retrieval adapted for ELISA formats. Finally, validate results using alternative detection methods or antibodies targeting different epitopes of SLC16A8 to confirm findings and eliminate assay-specific artifacts .

How can researchers differentiate between SLC16A8/MCT3 and other monocarboxylate transporters in complex experimental systems?

Differentiating between SLC16A8/MCT3 and other monocarboxylate transporters in complex experimental systems requires a multi-faceted approach leveraging their distinct expression patterns, localization, and functional characteristics. At the protein detection level, researchers should utilize the antibody's specificity for the unique 432-504 amino acid region of SLC16A8, which differs from other MCT family members. Conduct competitive binding assays with recombinant SLC16A8 protein to confirm signal specificity. For tissue-specific differentiation, exploit SLC16A8's restricted expression pattern in retinal pigment epithelium and choroid plexus epithelia, contrasting with the broader distribution of other MCTs. In cellular localization studies, utilize the distinct basolateral membrane localization of SLC16A8/MCT3 versus the apical distribution of MCT1 in the same epithelial cells. For functional differentiation, design transport assays that capitalize on SLC16A8's substrate preferences and kinetic properties. While all MCTs transport lactate and pyruvate, substrate competition assays with specific inhibitors can help distinguish between transporters. Additionally, pH-dependent transport studies may reveal differences in proton coupling efficiency among MCT family members. For comprehensive analysis in complex systems, combine protein detection with functional assays and consider using siRNA knockdown or CRISPR-Cas9 gene editing to specifically modulate SLC16A8 expression, confirming the specificity of observed signals .

What quality control parameters should researchers verify before using SLC16A8 Antibody, HRP conjugated?

Before using SLC16A8 Antibody, HRP conjugated in experimental procedures, researchers should verify several critical quality control parameters to ensure reliable results. First, check the antibody's purity level, which should be >95% as determined by Protein G purification methods . Assess lot-to-lot consistency by reviewing the certificate of analysis provided by the manufacturer, which should include specific performance metrics for the particular lot. Verify HRP conjugation efficiency through a functional test using a small amount of antibody with an appropriate substrate to confirm enzymatic activity is within expected parameters. Examine antibody specificity documentation, including Western blot results showing a single band at the expected molecular weight for SLC16A8 (approximately 49 kDa). For quantitative applications, ensure the working concentration has been optimized—while manufacturers may provide recommended dilutions, validation in your specific experimental system is advisable. Verify the antibody's storage history, as repeated freeze-thaw cycles can significantly degrade HRP activity and antibody performance. Finally, check the antibody's expiration date and confirm it has been stored according to manufacturer recommendations (typically -20°C or -80°C) . Implementation of these verification steps before experimental use will substantially reduce technical variability and improve reproducibility of results with SLC16A8 Antibody, HRP conjugated.

What are the expected sensitivity and detection limits when using SLC16A8 Antibody, HRP conjugated in ELISA applications?

When using SLC16A8 Antibody, HRP conjugated in ELISA applications, researchers can expect a sensitivity of approximately 0.38 ng/mL with a standard curve range of 0.63-40 ng/mL, as demonstrated in validated sandwich ELISA systems . This sensitivity level enables reliable detection of SLC16A8 in various biological samples including serum, plasma, tissue homogenates, and cell culture supernatants. The detection limit is influenced by several factors including the specific ELISA format (direct, indirect, or sandwich), sample matrix complexity, and signal amplification methods employed. For optimal performance in quantitative applications, researchers should generate a standard curve using purified recombinant SLC16A8 protein diluted in the same matrix as experimental samples whenever possible. The linear dynamic range typically spans two orders of magnitude, allowing for quantification across diverse experimental conditions. When approaching the lower detection limit, signal-to-noise ratios become critical for reliable measurements; therefore, thorough blocking and washing steps are essential to minimize background signal. For enhanced sensitivity in detecting low abundance SLC16A8, particularly in complex biological samples, researchers might consider signal amplification strategies compatible with HRP, such as enhanced chemiluminescent substrates or tyramide signal amplification, which can potentially lower the detection limit by an order of magnitude .

What experimental controls should be included when studying SLC16A8 expression and function using antibody-based methods?

When studying SLC16A8 expression and function using antibody-based methods, a comprehensive set of controls is essential for ensuring data validity and addressing potential methodological artifacts. Positive controls should include samples known to express SLC16A8, such as retinal pigment epithelium or choroid plexus tissue/cells, where the protein is natively expressed at detectable levels . Negative controls should incorporate tissues or cell lines that do not express SLC16A8, such as cells from non-epithelial lineages. For specificity confirmation, include peptide competition assays where the antibody is pre-incubated with the immunizing peptide (amino acids 432-504 of human SLC16A8) before application to samples; this should abolish specific staining or signal. Technical controls should include isotype controls using non-specific IgG from the same host species (rabbit) and at the same concentration as the primary antibody to assess non-specific binding. For HRP-conjugated antibodies specifically, include substrate-only controls to assess endogenous peroxidase activity in samples. When investigating SLC16A8 function, include parallel experiments with pharmacological inhibitors of monocarboxylate transporters (such as α-cyano-4-hydroxycinnamate) to confirm that observed effects are due to transporter activity. For quantitative studies, create standard curves using recombinant SLC16A8 protein, and include internal reference standards across different experimental batches to normalize for technical variation. Additionally, when making comparative assessments of SLC16A8 expression, parallel quantification of housekeeping proteins or mRNA expression analysis can provide orthogonal validation of observed differences .

How can researchers design experiments to distinguish between SLC16A8 expression changes and functional alterations in disease models?

Designing experiments to distinguish between SLC16A8 expression changes and functional alterations in disease models requires a sophisticated multi-modal approach. Start with quantitative assessment of protein expression using ELISA with the SLC16A8 Antibody, HRP conjugated to establish baseline expression levels in control versus disease conditions. This should be complemented with mRNA quantification via qRT-PCR to determine whether any observed changes occur at the transcriptional or post-transcriptional level. For subcellular localization analysis, perform immunohistochemistry or immunofluorescence using membrane fraction separation techniques to assess whether the protein remains correctly positioned at the basolateral membrane of epithelial cells or if trafficking defects occur in disease states . To evaluate functional capacity independent of expression levels, implement transport assays using radiolabeled or fluorescently labeled monocarboxylates (e.g., lactate) to measure actual transport activity across membranes. Compare transport kinetics (Km and Vmax values) between control and disease models to identify changes in transporter efficiency that may occur even with stable expression levels. For mechanistic insights, design site-directed mutagenesis experiments targeting known functional domains of SLC16A8 to mimic disease-associated variants and assess both expression and function of these mutants. Additionally, employ pharmacological approaches using specific inhibitors at various concentrations to generate dose-response curves that can reveal functional differences in transporter activity that may not be apparent from expression studies alone. Finally, for comprehensive understanding in complex disease models, combine these approaches with metabolomic analysis of lactate and other monocarboxylate levels in the relevant microenvironment to connect molecular findings with physiological outcomes .