SLC16A8 Antibody, Biotin conjugated

What is SLC16A8 and what cellular functions does it perform?

SLC16A8, also known as Monocarboxylate transporter 3 (MCT3), is a proton-linked monocarboxylate transporter that catalyzes the rapid transport of various monocarboxylates across the plasma membrane . These substrates include lactate, pyruvate, branched-chain oxo acids derived from leucine, valine and isoleucine, as well as ketone bodies such as acetoacetate, beta-hydroxybutyrate and acetate . SLC16A8 plays a crucial role in cellular metabolism, particularly in contexts where rapid transport of metabolites is necessary. Recent research has identified SLC16A8 as a potential oncogene that promotes proliferation, epithelial-mesenchymal transition (EMT), metastasis, angiogenesis, and glycolysis in colorectal cancer under hypoxic conditions .

What are the optimal storage conditions for SLC16A8 Antibody, Biotin conjugated?

For maximum stability and activity retention, SLC16A8 Antibody, Biotin conjugated should be stored at -20°C or -80°C immediately upon receipt . It is critical to avoid repeated freeze-thaw cycles as these can compromise antibody integrity and performance . For short-term storage (less than one month), the antibody can be kept at 4°C . The antibody is typically supplied in a buffer containing preservatives such as 0.03% Proclin 300 and stabilizers including 50% Glycerol in 0.01M PBS at pH 7.4 . Proper aliquoting of the antibody upon receipt is recommended to minimize the need for repeated freeze-thaw cycles when conducting experiments over an extended period .

What applications has this antibody been validated for?

The SLC16A8 Antibody, Biotin conjugated has been primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) . Some SLC16A8 antibodies from other manufacturers have been validated for additional applications including Western Blot, Immunohistochemistry (IHC), and Immunohistochemistry-Paraffin (IHC-P) . For Western Blot, a recommended working concentration may be around 1.0 μg/ml, while for Immunohistochemistry applications, dilutions ranging from 1:10 to 1:500 may be appropriate . Researchers should perform preliminary experiments to determine the optimal working concentration for their specific experimental setup and should be aware that validation for one application does not guarantee performance in others.

How can SLC16A8 Antibody be utilized to investigate hypoxia-induced metabolic reprogramming in cancer?

Recent research has revealed SLC16A8's critical role in hypoxia-induced metabolic reprogramming, particularly in colorectal cancer (CRC) . To investigate this phenomenon, researchers can design experiments where cancer cell lines are cultured under normoxic and hypoxic conditions, followed by analysis of SLC16A8 expression using the biotin-conjugated antibody . The experimental approach should include:

• Establishment of hypoxic culture conditions (typically 1% O₂)

• Measurement of extracellular acidification rate (ECAR) and lactate production

• Assessment of glycolytic enzyme expression (PKM2, LDHA)

• Glucose consumption assays

• SLC16A8 detection via appropriate immunoassays

Studies have demonstrated that under hypoxic conditions, HIF-1α induces SLC16A8 expression, leading to enhanced metabolic reprogramming and increased lactate production . Using siRNA knockdown experiments in parallel with antibody detection can provide valuable insights into SLC16A8's role in the Warburg effect and cancer metabolism . This methodological approach allows researchers to establish cause-effect relationships between hypoxia, SLC16A8 expression, and metabolic changes.

What is the role of SLC16A8 in tumor angiogenesis and how can this antibody help elucidate these mechanisms?

SLC16A8 has been implicated in promoting angiogenesis within the tumor microenvironment, a critical aspect of cancer progression . To investigate SLC16A8's role in this process, researchers can employ a multi-faceted approach:

• Co-culture experiments with cancer cells and Human Umbilical Vein Endothelial Cells (HUVECs)

• Tube formation assays to assess endothelial cell function

• Evaluation of endothelial-mesenchymal transition (EndMT) markers

• In vivo xenograft models with SLC16A8 knockdown

Research has shown that SLC16A8 knockdown significantly inhibits hypoxia-induced EndMT in HUVEC cells and decreases their tube formation capacity . The biotin-conjugated SLC16A8 antibody can be used in these experimental setups to monitor SLC16A8 expression levels and localization, particularly in immunohistochemistry analyses of tumor sections from xenograft models . This allows researchers to correlate SLC16A8 expression with angiogenic markers and vascular density in tumor tissues, providing mechanistic insights into how this transporter influences the tumor microenvironment.

How should researchers design experiments to evaluate SLC16A8 as a potential therapeutic target?

Designing experiments to evaluate SLC16A8 as a therapeutic target requires a systematic approach that establishes both its functional role in disease progression and the effects of its inhibition. Based on recent findings, the following experimental design is recommended:

• Expression analysis in patient-derived samples comparing disease tissue with normal controls

• Correlation of expression levels with clinical outcomes and disease progression

• Functional studies using genetic knockdown approaches (siRNA, shRNA, or CRISPR-Cas9)

• Phenotypic assays measuring proliferation, migration, invasion, and metabolic parameters

• In vivo studies using xenograft models with SLC16A8 knockdown

• Combination studies with established therapeutic agents

Studies have demonstrated that SLC16A8 knockdown suppresses tumor growth, reduces proliferation marker Ki67 expression, and decreases HIF-1α levels in vivo . Additionally, SLC16A8 silencing has been shown to reduce serum lactate levels and alter the expression of proteins related to the Warburg effect . The biotin-conjugated antibody can be employed for monitoring treatment efficacy in these experimental contexts, particularly for assessing target engagement and expression changes following therapeutic interventions.

What are the optimal conditions for using SLC16A8 Antibody, Biotin conjugated in immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) samples?

While the biotin-conjugated SLC16A8 antibody has been primarily validated for ELISA, adaptation to IHC-P requires careful optimization. A methodological approach should include:

• Antigen retrieval optimization: Test both heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0), as well as enzymatic retrieval methods

• Antibody dilution optimization: Begin with a dilution range of 1:10 to 1:500 and perform a titration to determine optimal signal-to-noise ratio

• Blocking optimization: Use 5-10% normal serum from the same species as the secondary antibody

• Incubation conditions: Test both overnight incubation at 4°C and 1-2 hour incubation at room temperature

• Detection system: For biotin-conjugated antibodies, utilize streptavidin-HRP systems, being mindful of potential endogenous biotin in tissues

• Controls: Include positive control tissues known to express SLC16A8, negative controls (omitting primary antibody), and isotype controls

Researchers should be aware that biotin-conjugated antibodies may require additional blocking steps to prevent non-specific binding due to endogenous biotin in tissues. A biotin-blocking system or avidin-biotin blocking kit should be considered, especially when working with tissues known to have high endogenous biotin levels, such as liver, kidney, and some tumors.

What protein extraction and immunoprecipitation protocols are recommended when working with membrane proteins like SLC16A8?

SLC16A8 is a membrane-bound transporter protein, which presents specific challenges for extraction and immunoprecipitation. The following methodological approach is recommended:

• Sample preparation:

  • Fresh or frozen tissues should be homogenized in ice-cold lysis buffer containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), and protease inhibitor cocktail

  • For cultured cells, after washing with PBS, lyse directly in the membrane protein extraction buffer

• Membrane protein extraction:

  • Consider using commercial membrane protein extraction kits that contain specialized detergents

  • Alternatively, prepare buffers containing mild detergents like digitonin (0.5-1%) or n-dodecyl-β-D-maltoside (0.5-1%)

  • Incorporate sonication steps (3-5 short pulses) to enhance membrane disruption

• Immunoprecipitation:

  • Pre-clear lysates with Protein G beads to reduce non-specific binding

  • Incubate cleared lysates with SLC16A8 antibody (2-5 μg) overnight at 4°C

  • Since the antibody is biotin-conjugated, use streptavidin-coated beads instead of Protein G/A

  • Wash stringently (at least 4-5 times) with buffers containing decreasing detergent concentrations

  • Elute bound proteins with appropriate elution buffer or SDS sample buffer

• Western blot detection:

  • Resolve proteins on 10-12% SDS-PAGE gels

  • Transfer to PVDF membranes (preferred over nitrocellulose for membrane proteins)

  • Block with 5% BSA (preferred over milk for phosphoprotein detection)

  • Detect SLC16A8 at its expected molecular weight of approximately 55 kDa

This methodological approach accounts for the hydrophobic nature of membrane proteins like SLC16A8 and helps ensure efficient extraction and specific immunoprecipitation.

How can researchers effectively use this antibody in metabolic flux analysis experiments?

To effectively incorporate SLC16A8 Antibody, Biotin conjugated into metabolic flux analysis experiments, researchers should consider the following methodological approach:

Research has demonstrated that SLC16A8 expression significantly impacts glycolytic activities in cancer cells, particularly under hypoxic conditions . Increased SLC16A8 expression correlates with enhanced glucose consumption and lactate production, key metabolic shifts associated with the Warburg effect . By simultaneously measuring these metabolic parameters while monitoring SLC16A8 expression, researchers can establish functional relationships between this transporter and cellular energetics.

What are common issues encountered when using SLC16A8 Antibody, Biotin conjugated and how can they be resolved?

Researchers may encounter several challenges when working with SLC16A8 Antibody, Biotin conjugated. The following table presents common issues and recommended solutions:

Addressing these common issues requires systematic troubleshooting and careful optimization of experimental conditions specific to each laboratory's setup and sample types.

How should researchers interpret SLC16A8 expression patterns in the context of tumor heterogeneity?

Interpreting SLC16A8 expression patterns in heterogeneous tumor samples requires careful consideration of multiple factors:

• Spatial heterogeneity considerations:

  • Compare SLC16A8 expression between tumor core and periphery

  • Correlate expression patterns with hypoxic regions (use HIF-1α as a marker)

  • Assess relationship with vascular markers (CD31, CD34) to understand proximity to blood vessels

• Methodological approach for analysis:

  • Employ multi-staining approaches to simultaneously visualize SLC16A8 with hypoxia markers and vascular markers

  • Utilize digital pathology tools for quantitative spatial analysis

  • Consider single-cell approaches to resolve cell-type specific expression patterns

• Interpretation framework:

  • Higher SLC16A8 expression in hypoxic regions supports its role in metabolic adaptation

  • Correlation with invasive front may indicate involvement in metastatic processes

  • Co-expression with stemness markers might suggest association with tumor-initiating cells

Research has shown that SLC16A8 expression is significantly upregulated in colorectal cancer tissues compared to adjacent normal tissues and correlates with disease progression . Furthermore, under hypoxic conditions, HIF-1α has been shown to induce SLC16A8 expression, leading to enhanced metabolic reprogramming . These findings suggest that heterogeneous expression of SLC16A8 within tumors may reflect varying oxygen tensions and metabolic states across different tumor regions.

What are the best approaches for quantifying SLC16A8 expression in correlation with clinical outcomes?

To effectively quantify SLC16A8 expression and correlate it with clinical outcomes, researchers should implement a comprehensive methodological approach:

• Tissue analysis workflow:

  • Collect paired tumor and adjacent normal tissues from patients with detailed clinical data

  • Process tissues using standardized protocols (fixation, embedding, sectioning)

  • Perform immunostaining with optimized protocols for SLC16A8 detection

  • Implement rigorous scoring systems (H-score, Allred score, or digital image analysis)

• Expression quantification methods:

  • Semi-quantitative: Pathologist scoring (0, 1+, 2+, 3+) based on staining intensity and percentage of positive cells

  • Quantitative: Digital image analysis with automated algorithms for objective assessment

  • Complementary: Validate IHC findings with qRT-PCR or Western blot when possible

• Clinical correlation analysis:

  • Categorize expression levels (high vs. low) based on established cutoff values

  • Perform Kaplan-Meier survival analysis with log-rank tests

  • Use Cox proportional hazards models for multivariate analysis

  • Consider time-dependent ROC analysis to evaluate prognostic performance

Research has demonstrated that low SLC16A8 expression is associated with favorable prognosis and survival in colorectal cancer patients . Additionally, SLC16A8 expression increases with disease progression, suggesting its potential utility as a prognostic biomarker . When conducting these analyses, researchers should account for potential confounding factors such as tumor stage, grade, molecular subtypes, and treatment history to ensure robust and clinically meaningful correlations.

How might SLC16A8 Antibody be used in single-cell analysis workflows?

Incorporating SLC16A8 Antibody, Biotin conjugated into single-cell analysis workflows represents an emerging research direction with significant potential. A methodological approach would include:

• Sample preparation for single-cell analysis:

  • Optimize tissue dissociation protocols to maintain cell viability and surface protein integrity

  • Implement gentle fixation methods that preserve epitope accessibility

  • Consider cell sorting strategies to enrich for populations of interest

• Single-cell protein detection methods:

  • Mass cytometry (CyTOF): Conjugate SLC16A8 antibody with metal isotopes

  • Flow cytometry: Utilize streptavidin-fluorophore conjugates with the biotin-labeled antibody

  • Single-cell Western blot: Adapt protocols for detecting SLC16A8 in individual cells

  • Imaging mass cytometry: Combine with other antibodies for spatial context

• Integrated multi-omics approaches:

  • CITE-seq: Combine SLC16A8 protein detection with transcriptome analysis

  • Correlate SLC16A8 protein levels with metabolomic profiles at single-cell resolution

  • Integrate with spatial transcriptomics for contextual understanding

This methodological framework would enable researchers to investigate the heterogeneity of SLC16A8 expression within tumor tissues at unprecedented resolution. Such analyses could reveal distinct cellular subpopulations with unique metabolic profiles and potentially identify critical cell states associated with treatment resistance or metastatic potential.

What is the potential role of SLC16A8 in resistance to metabolically targeted cancer therapies?

Given SLC16A8's role in cellular metabolism, investigating its involvement in resistance to metabolically targeted cancer therapies represents an important research direction:

• Experimental model development:

  • Generate drug-resistant cell lines through prolonged exposure to metabolic inhibitors

  • Create patient-derived xenografts from treatment-resistant tumors

  • Develop isogenic cell line pairs with controlled SLC16A8 expression levels

• Resistance mechanism investigation:

  • Compare SLC16A8 expression between sensitive and resistant models

  • Analyze metabolic adaptations in resistant models, focusing on lactate handling

  • Perform gain/loss-of-function studies to directly assess SLC16A8's contribution

• Therapeutic strategy evaluation:

  • Test combination approaches targeting SLC16A8 alongside existing metabolic inhibitors

  • Evaluate temporal sequencing of treatments to prevent resistance development

  • Investigate synthetic lethality interactions with SLC16A8 inhibition

Research has shown that SLC16A8 is upregulated under hypoxic conditions and contributes to the Warburg effect in cancer cells . This metabolic reprogramming may provide cancer cells with alternative energy sources and metabolic flexibility, potentially contributing to resistance against therapies targeting glycolysis or mitochondrial metabolism. By understanding SLC16A8's role in therapy resistance, researchers may identify novel combination strategies to overcome metabolic adaptations in cancer.

How can SLC16A8 Antibody be used to investigate the interplay between tumor metabolism and immune cell function?

The tumor microenvironment represents a complex ecosystem where cancer cell metabolism can significantly impact immune cell function. To investigate SLC16A8's role in this interplay:

• Co-culture experimental systems:

  • Establish co-cultures of cancer cells with various immune cell populations

  • Modulate SLC16A8 expression in cancer cells using genetic approaches

  • Monitor immune cell phenotypes and functions under different conditions

• Metabolic analysis approaches:

  • Measure extracellular metabolites in the shared media

  • Track isotope-labeled metabolites to determine flux between cell types

  • Analyze lactate levels and pH changes in the microenvironment

• Immune function assessment:

  • Evaluate T cell proliferation, cytokine production, and cytotoxic activity

  • Assess dendritic cell maturation and antigen presentation capacity

  • Analyze macrophage polarization states (M1 vs. M2)

• Spatial analysis in tissues:

  • Perform multiplex immunofluorescence to visualize SLC16A8-expressing cells relative to immune cell populations

  • Correlate SLC16A8 expression with immune exclusion or infiltration patterns

  • Analyze the relationship between SLC16A8 expression and immune checkpoint molecule expression

Since SLC16A8 is involved in lactate transport, its expression and activity may contribute to the acidification of the tumor microenvironment, which can suppress immune cell function. High lactate levels have been associated with impaired T cell activation and dendritic cell maturation, suggesting that SLC16A8-mediated metabolite transport could represent an important immunomodulatory mechanism. By using the biotin-conjugated SLC16A8 antibody in these experimental contexts, researchers can gain insights into the metabolic regulation of anti-tumor immunity.

What are the critical validation steps when using SLC16A8 Antibody, Biotin conjugated in a new experimental system?

When implementing SLC16A8 Antibody, Biotin conjugated in a new experimental system, rigorous validation is essential to ensure reliable and reproducible results:

• Specificity validation:

  • Positive controls: Test on samples with confirmed SLC16A8 expression

  • Negative controls: Use samples known to lack SLC16A8 expression

  • Blocking peptide competition: Pre-incubate antibody with immunizing peptide

  • Genetic validation: Compare staining in wild-type versus SLC16A8 knockdown samples

• Technical optimization:

  • Antibody titration: Test multiple concentrations to determine optimal signal-to-noise ratio

  • Fixation optimization: Compare different fixatives and protocols

  • Incubation conditions: Optimize temperature, duration, and buffer composition

  • Detection system optimization: For biotin-conjugated antibodies, compare different streptavidin conjugates

• Reproducibility assessment:

  • Inter-assay variability: Repeat experiments multiple times under identical conditions

  • Intra-assay variability: Include technical replicates within each experiment

  • Operator variability: Have multiple researchers perform the same protocol

  • Lot-to-lot consistency: Test performance across different antibody lots if available

This methodical validation approach ensures that any findings related to SLC16A8 expression or function are robust and scientifically sound. Researchers should maintain detailed records of all validation steps and include appropriate validation controls in all subsequent experiments.

What considerations should be made when designing multiplexed immunofluorescence panels including SLC16A8?

Designing effective multiplexed immunofluorescence panels that include SLC16A8 requires careful planning and optimization:

• Panel design considerations:

  • Select complementary markers based on biological questions (e.g., hypoxia markers, other MCTs, glycolytic enzymes)

  • Consider antibody species to avoid cross-reactivity (the SLC16A8 antibody is raised in rabbit)

  • Plan spectral separation to minimize fluorophore overlap

  • Include essential controls for each marker

• Technical optimization for biotin-conjugated antibodies:

  • Address endogenous biotin: Implement biotin blocking steps

  • Select appropriate streptavidin-fluorophore conjugate with minimal spectral overlap

  • Determine optimal sequence in multiplexed staining (typically detect biotin-conjugated antibodies early in the sequence)

  • Consider tyramide signal amplification for weak signals

• Multi-round staining considerations:

  • If using sequential staining approaches, test antibody stripping efficiency

  • Validate epitope stability through multiple rounds of staining

  • Include registration markers for image alignment across rounds

  • Consider cyclic immunofluorescence or CODEX approaches for highly multiplexed panels

The biotin conjugation of the SLC16A8 antibody provides flexibility in detection strategies but requires specific considerations to prevent endogenous biotin interference and optimize signal detection. Through careful design and validation, researchers can effectively incorporate this antibody into multiplexed panels to study SLC16A8 in the context of related markers and pathways.

What is the recommended approach for quantitative analysis of SLC16A8 expression in tissue microarrays?

Quantitative analysis of SLC16A8 expression in tissue microarrays (TMAs) requires a standardized methodological approach:

• Staining protocol standardization:

  • Perform all TMA staining in a single batch to minimize technical variability

  • Include positive and negative control tissues on each TMA slide

  • Implement rigorous quality control for each step of the staining protocol

  • Use automated staining platforms when possible to ensure consistency

• Image acquisition parameters:

  • Standardize microscope settings (exposure time, gain, offset)

  • Capture images at appropriate resolution (typically 20x or 40x objective)

  • Include color/intensity calibration standards in each imaging session

  • Consider whole slide scanning for comprehensive analysis

• Quantitative analysis workflow:

  • Implement digital pathology software for automated or semi-automated analysis

  • Define precise scoring algorithms (consider intensity, percentage positive cells, H-score)

  • Validate automated scoring against pathologist assessment

  • Establish clear cutoffs for categorizing expression levels (e.g., low vs. high)

• Statistical analysis considerations:

  • Account for missing or damaged TMA cores

  • Implement appropriate statistical tests based on data distribution

  • Consider multiple testing corrections for correlations with clinical parameters

  • Validate findings in independent cohorts when possible