SLC22A5 Antibody, FITC conjugated

What is SLC22A5 and why is it an important research target?

SLC22A5 (Solute Carrier Family 22 Member 5) is an integral plasma membrane protein that functions as both an organic cation transporter and a sodium-dependent high-affinity carnitine transporter. It plays a critical role in the elimination of many endogenous small organic cations as well as various drugs and environmental toxins in the liver, kidney, intestine, and other organs. The protein is particularly important because mutations in SLC22A5 are associated with systemic primary carnitine deficiency (CDSP), which manifests as hypoketotic hypoglycemia and acute metabolic decompensation in early life and skeletal myopathy or cardiomyopathy later in life . Recent research has also demonstrated altered SLC22A5 expression in non-small cell lung cancer (NSCLC), making it a valuable target for cancer research . Its involvement in both metabolic disorders and potentially cancer pathways makes SLC22A5 a multifaceted research target spanning several disciplines.

What are the specific properties of FITC-conjugated SLC22A5 antibodies?

FITC-conjugated SLC22A5 antibodies typically consist of polyclonal or monoclonal antibodies directed against different regions of the SLC22A5 protein that have been labeled with fluorescein isothiocyanate (FITC). These antibodies are available in various configurations including those targeting the C-terminal region (such as ARP63405_P050-FITC) or specific amino acid sequences (such as antibodies targeting AA 1-180 or AA 42-142 regions) . The FITC conjugation provides a bright green fluorescence (excitation ~495 nm, emission ~520 nm) that enables direct visualization in fluorescence microscopy applications without requiring secondary antibody incubation. Most commercially available FITC-conjugated SLC22A5 antibodies are supplied in liquid form, often in buffers containing preservatives like Proclin 300 and stabilizers such as glycerol . These antibodies demonstrate species reactivity with human samples and, depending on the specific product, may cross-react with mouse, rat, cow, guinea pig, and other mammalian species .

What controls should be implemented when working with FITC-conjugated SLC22A5 antibodies?

When conducting experiments with FITC-conjugated SLC22A5 antibodies, a comprehensive control strategy is essential for ensuring reliable and interpretable results. Primary controls should include a negative control where the primary antibody is omitted but all other steps remain identical, allowing for assessment of background fluorescence and non-specific binding of components. An isotype control using a FITC-conjugated non-relevant antibody of the same isotype (typically IgG for polyclonal SLC22A5 antibodies) helps distinguish between specific binding and Fc receptor-mediated or other non-specific interactions . For validating antibody specificity, a blocking peptide control where the antibody is pre-incubated with its immunogen peptide should abolish specific staining .

Additionally, positive controls using tissues or cell lines known to express SLC22A5 (such as HeLa cells, as confirmed by BioGPS gene expression data) provide crucial validation of the antibody's performance . In multi-color immunofluorescence experiments, single-color controls are necessary to establish proper compensation settings and evaluate potential spectral overlap. For quantitative studies, calibration controls using standardized fluorescent beads allow for normalization between experiments and accurate comparison of fluorescence intensities across different experimental conditions or time points.

How should researchers optimize protocols for FITC-conjugated SLC22A5 antibody in immunofluorescence studies?

Optimizing immunofluorescence protocols for FITC-conjugated SLC22A5 antibody requires systematic adjustment of multiple parameters to achieve specific signal while minimizing background. Begin with fixation optimization—test both cross-linking (paraformaldehyde-based) and precipitating (methanol-based) fixatives to determine which best preserves SLC22A5 epitopes while maintaining cellular architecture. The membrane localization of SLC22A5 often necessitates careful permeabilization; test graded concentrations of detergents (0.1-0.5% Triton X-100 or 0.01-0.1% saponin) to allow antibody access while preserving membrane integrity .

Antibody concentration requires careful titration; begin with manufacturer recommendations (typically working dilutions are derived from the 0.5 mg/ml stock concentration) and perform a dilution series (1:50, 1:100, 1:200, 1:500) . Incubation conditions significantly impact staining quality—compare overnight incubation at 4°C versus 1-2 hours at room temperature. Given FITC's susceptibility to photobleaching, implement an antifade mounting medium containing DAPI for nuclear counterstaining .

For tissues with high autofluorescence (particularly liver and kidney where SLC22A5 is abundantly expressed), incorporate an autofluorescence quenching step using sodium borohydride or commercial quenching solutions. If background persists, implement additional blocking with 5-10% normal serum matching the host species of secondary antibodies plus 1% BSA. For quantitative studies, standardize image acquisition parameters including exposure time, gain, and offset settings across all experimental conditions to enable accurate comparisons .

What approaches can resolve inconsistent staining patterns when using SLC22A5 antibody?

Inconsistent staining patterns with FITC-conjugated SLC22A5 antibody can stem from multiple sources requiring systematic troubleshooting. First, examine antibody storage conditions—improper storage can lead to fluorophore degradation or antibody denaturation; FITC-conjugated antibodies must be stored in light-protected vials and should never be frozen, as freezing/thawing compromises both enzyme activity and antibody binding . For extended storage beyond 12 months, dilute with up to 50% glycerol and store at -20°C to -80°C .

Sample preparation variability often contributes to inconsistent results. Standardize fixation duration (typically 10-15 minutes for paraformaldehyde) and implement a controlled permeabilization protocol. The pH of buffers critically affects FITC fluorescence—maintain all solutions at pH 7.2-7.4 . Consider epitope retrieval methods for formalin-fixed tissues; test heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0).

If membrane staining appears punctate rather than continuous, this may reflect biological reality rather than technical artifact—SLC22A5 can localize to specific membrane microdomains. Validate patterns using multiple antibodies targeting different epitopes of SLC22A5. For tissue sections, thickness standardization (5-7 μm) improves consistency. In cells with variable SLC22A5 expression levels, consider normalizing to a housekeeping protein or implementing standardized exposure settings during image acquisition .

Implement batch processing where all samples undergo simultaneous staining to minimize technical variation, and consider automated staining platforms for high-throughput applications requiring maximum consistency across large sample sets.

What are the considerations for multiplexed immunofluorescence with FITC-conjugated SLC22A5 antibody?

Designing effective multiplexed immunofluorescence experiments with FITC-conjugated SLC22A5 antibody requires careful spectral planning and protocol optimization. The FITC fluorophore exhibits peak excitation at ~495 nm and emission at ~520 nm, necessitating selection of compatible fluorophores with minimal spectral overlap. Optimal companions include far-red fluorophores (such as Alexa Fluor 647) and red fluorophores (such as Alexa Fluor 594 or rhodamine derivatives) rather than yellow-orange fluorophores (like PE or Alexa Fluor 555) which have greater spectral overlap with FITC .

Sequential staining protocols often yield superior results compared to simultaneous antibody incubation, particularly when combining antibodies from the same host species. When examining SLC22A5 alongside other membrane transporters, careful titration of each antibody is essential to avoid competitive binding at the membrane. Order effects must be considered—typically begin with the weakest signal (often SLC22A5) followed by stronger markers .

Proper controls become increasingly critical in multiplex settings. Single-color controls for each fluorophore are mandatory for establishing compensation settings and identifying bleed-through. Fluorescence minus one (FMO) controls, where all fluorophores except one are included, help establish gating thresholds in flow cytometry applications or identify false positives in imaging .

For confocal microscopy applications, sequential scanning mode should be employed rather than simultaneous excitation to minimize crosstalk. If examining SLC22A5 subcellular localization alongside organelle markers, super-resolution techniques like structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy may be necessary to resolve closely associated but distinct structures .

How can FITC-conjugated SLC22A5 antibodies be utilized to investigate carnitine transport dysfunction?

FITC-conjugated SLC22A5 antibodies provide powerful tools for investigating carnitine transport dysfunction through multiple experimental approaches. For cellular models of systemic primary carnitine deficiency (CDSP), these antibodies enable visualization of mutant SLC22A5 protein trafficking defects through co-localization studies with ER markers (calnexin, PDI) or Golgi markers (GM130) to determine where mutant proteins are retained . Internalization kinetics studies using surface biotinylation followed by FITC-SLC22A5 antibody detection can quantify altered membrane residence time of mutant transporters.

In patient-derived fibroblasts or lymphoblasts, SLC22A5 antibodies can correlate protein expression levels with functional carnitine uptake assays, establishing genotype-phenotype relationships. High-content imaging platforms using FITC-SLC22A5 antibodies enable automated quantification of membrane localization changes in response to therapeutic compounds, facilitating drug screening approaches for CDSP treatment .

For in vivo studies, dual immunofluorescence using FITC-SLC22A5 antibodies alongside tissue-specific markers in cardiac or skeletal muscle biopsies from CDSP patients or animal models allows spatial mapping of transporter deficiencies that correlate with pathology. Time-course studies during development or disease progression require careful standardization of staining protocols to enable reliable quantitative comparisons across multiple timepoints .

When investigating drug-induced carnitine deficiency (a side effect of certain medications), FITC-SLC22A5 antibodies can visualize competitive binding or transporter downregulation mechanisms, offering insights into adverse drug reactions. The specific C-terminal region targeted by some FITC-conjugated antibodies is particularly valuable as this domain contains regulatory phosphorylation sites that modulate transporter activity in response to cellular signaling .

How does SLC22A5 expression correlate with clinical parameters in cancer research?

Recent research has revealed significant correlations between SLC22A5 expression and clinical parameters in cancer, particularly in non-small cell lung cancer (NSCLC). Studies utilizing quantitative analysis of SLC22A5 have demonstrated significant differences in expression levels between normal and cancerous tissues, with notable downregulation observed specifically in lung squamous cell carcinoma (LUSC) . This finding suggests SLC22A5 may serve as a potential biomarker or therapeutic target in specific NSCLC subtypes.

Statistical analyses have revealed significant associations between SLC22A5 gene expression and patient clinical characteristics. Sex-specific differences in SLC22A5 expression have been observed in NSCLC patients (P=0.002) and specifically in LUSC patients (P=0.001), indicating potential hormonal regulation of this transporter . Tobacco smoking status also correlates significantly with SLC22A5 expression levels (P=0.04), suggesting environmental factors may modulate transporter expression .

Particularly noteworthy is the significant negative correlation between SLC22A5 gene expression and standardized uptake value (SUV) in PET imaging for lung adenocarcinoma (LUAD) patients (r=−0.40, P=0.02) . This inverse relationship suggests that tumors with lower SLC22A5 expression demonstrate higher metabolic activity, potentially reflecting advanced disease or aggressive phenotypes. This correlation provides a potential mechanism to explain altered metabolic profiles in certain tumor types, as SLC22A5's role in carnitine transport directly impacts fatty acid metabolism and energy production.

For researchers investigating these relationships, FITC-conjugated SLC22A5 antibodies enable spatial correlation of transporter expression with histopathological features through digital pathology approaches. Quantitative immunofluorescence using standardized protocols allows correlation of protein expression with mRNA data and clinical outcomes across patient cohorts .

What methodological approaches can address epitope masking in SLC22A5 detection?

Epitope masking represents a significant challenge in SLC22A5 detection due to the protein's complex membrane topology with multiple transmembrane domains and potential post-translational modifications. When conventional immunostaining yields suboptimal results, researchers should implement a systematic approach to epitope retrieval and accessibility enhancement. For formalin-fixed specimens, comparative evaluation of heat-induced epitope retrieval (HIER) methods using citrate buffer (pH 6.0), Tris-EDTA buffer (pH 9.0), or commercial retrieval solutions can dramatically improve epitope accessibility .

Enzymatic retrieval methods provide an alternative approach—test proteolytic enzymes like proteinase K (1-5 μg/ml for 5-15 minutes) or trypsin (0.025-0.1% for 5-15 minutes) to cleave protein cross-links that may obscure epitopes. The optimal retrieval method often depends on the specific epitope targeted; antibodies directed against the C-terminal region (such as ARP63405_P050-FITC) typically respond differently to retrieval methods compared to those targeting N-terminal or transmembrane domains .

When investigating native (non-fixed) samples like fresh tissue sections or live cells, detergent selection becomes critical. Mild detergents (digitonin 0.001-0.01%) selectively permeabilize the plasma membrane while leaving intracellular membranes intact, allowing discrimination between surface and intracellular pools of SLC22A5. For accessing epitopes in specific membrane microdomains, test combinations of detergents (0.1% saponin with 0.01% Triton X-100) that can solubilize different lipid compositions .

For tissues with high endogenous biotin (liver, kidney), which can interfere with detection systems, implement a biotin-blocking step before antibody application. When phosphorylation status affects epitope accessibility, consider phosphatase inhibitors in buffers to preserve physiologically relevant modifications. If protein-protein interactions mask epitopes, gentle dissociation using low concentrations of SDS (0.01-0.05%) in the permeabilization buffer can improve antibody access while maintaining tissue architecture .

How should researchers validate FITC-conjugated SLC22A5 antibody specificity?

Comprehensive validation of FITC-conjugated SLC22A5 antibody specificity requires a multi-faceted approach combining molecular, cellular, and tissue-based methods. Western blotting serves as the foundation of validation, confirming that the antibody recognizes a protein of the expected molecular weight (approximately 63 kDa for human SLC22A5) . Validation should include samples from multiple species if cross-reactivity is claimed (human, mouse, rat, cow, etc.) and should evaluate potential cross-reactivity with other SLC family members, particularly closely related transporters like SLC22A4 (OCTN1) .

Genetic validation approaches provide definitive specificity confirmation. Testing the antibody on samples with SLC22A5 knockdown (siRNA or shRNA) or knockout (CRISPR-Cas9) should show corresponding reduction or elimination of signal. Conversely, overexpression systems can confirm signal increases proportional to expression levels. For research involving disease-associated mutations, testing cells expressing mutant SLC22A5 variants can reveal whether the antibody recognizes the mutated epitope .

Epitope-specific validation can be performed using competing peptide controls. Pre-incubation of the antibody with its immunogen peptide (such as the synthetic peptide for ARP63405_P050-FITC: TLFLPESFGTPLPDTIDQMLRVKGMKHRKTPSHTRMLKDGQERPTILKST) should abolish specific staining . Orthogonal validation comparing staining patterns with other validated SLC22A5 antibodies targeting different epitopes strengthens confidence in specificity.

Tissue expression validation should demonstrate expected expression patterns across multiple tissues. SLC22A5 is expected to show high expression in kidney, liver, intestine, heart, and skeletal muscle, with precise subcellular localization at the plasma membrane . For human tissues, correlation with RNAseq or microarray data from resources like BioGPS provides additional validation . When applied to pathological samples, staining patterns should correspond to known disease-associated changes in SLC22A5 expression or localization.

What are the optimal storage conditions for maintaining FITC-conjugated antibody performance?

Preserving the performance of FITC-conjugated SLC22A5 antibodies requires strict adherence to storage conditions that maintain both antibody binding capacity and fluorophore integrity. Light protection is absolutely essential as FITC is highly susceptible to photobleaching; antibodies should be stored in amber vials or regular vials wrapped in aluminum foil to prevent light exposure during storage . Temperature management is equally critical—these conjugated antibodies should be maintained at 2-8°C for short-term storage (up to 12 months) and should never be frozen in their original formulation as freezing/thawing cycles significantly compromise both enzyme activity and antibody binding .

For long-term storage (up to 24 months), FITC-conjugated antibodies can be diluted with up to 50% glycerol and then stored at -20°C to -80°C . This glycerol addition prevents ice crystal formation that could denature the antibody protein structure. Aliquoting the antibody upon receipt minimizes freeze-thaw cycles and reduces contamination risk. The optimal aliquot size should correspond to the amount needed for 1-2 experiments to avoid repeated freeze-thaw cycles of the same solution .

Buffer composition significantly impacts stability—FITC-conjugated SLC22A5 antibodies are typically supplied in PBS buffer (pH 7.4) with preservatives like 0.03% Proclin 300 and 50% glycerol . The pH must be maintained between 7.2-7.4, as FITC fluorescence is highly pH-dependent; acidic conditions significantly reduce fluorescence intensity. To prevent microbial contamination, some formulations include sodium azide (0.02-0.05%), though researchers should note this can inhibit HRP activity if the same samples will undergo peroxidase-based detection .

Stability testing should be performed periodically on stored antibodies by comparing current performance against baseline results using standardized positive controls. For quantitative applications, fluorescence intensity measurements of standard samples over time can track potential degradation. The table below summarizes optimal storage conditions for FITC-conjugated SLC22A5 antibodies:

How can SLC22A5 expression analysis contribute to personalized medicine approaches?

The analysis of SLC22A5 expression using FITC-conjugated antibodies offers significant potential for advancing personalized medicine approaches across multiple disease contexts. In primary carnitine deficiency (CDSP), quantitative immunofluorescence of SLC22A5 in patient-derived samples can stratify patients based on protein expression levels, distinguishing between those with trafficking defects versus catalytic site mutations . This distinction directly impacts treatment strategies—patients with trafficking defects may benefit from chemical chaperones that promote proper folding, while those with catalytic site mutations require different therapeutic approaches.

In oncology, the negative correlation between SLC22A5 expression and standardized uptake value (SUV) in lung adenocarcinoma suggests potential applications in treatment selection and monitoring . Patients with low SLC22A5 expression demonstrating high metabolic activity might benefit from metabolic-targeting therapies. The observed sex-specific differences in SLC22A5 expression patterns in NSCLC (P=0.002) and specifically in LUSC patients (P=0.001) suggest hormonal influences that could inform sex-specific treatment approaches .

For pharmacogenomic applications, FITC-conjugated SLC22A5 antibodies enable assessment of transporter expression in patient samples before administering medications that interact with or are transported by SLC22A5. This approach could identify patients at risk for adverse drug reactions or suboptimal drug responses due to altered transporter expression or localization. The correlation between SLC22A5 expression and tobacco smoking status (P=0.04) further suggests environmental factors may modulate drug transport capabilities, requiring personalized dosing adjustments .

Technically, the implementation of standardized FITC-SLC22A5 immunofluorescence protocols on automated staining platforms enables reproducible quantification across patient cohorts. Digital pathology approaches with machine learning algorithms can then correlate staining patterns with treatment outcomes, generating predictive models for patient stratification. Cell-type specific expression analysis using multiplexed immunofluorescence combining FITC-SLC22A5 with lineage markers provides higher resolution for personalized therapy selection based on the specific cells expressing the transporter .

What methodological innovations are enhancing SLC22A5 functional studies in complex disease models?

Recent methodological innovations have significantly advanced the capabilities for studying SLC22A5 function in complex disease models. Super-resolution microscopy techniques applied with FITC-conjugated SLC22A5 antibodies now enable visualization of transporter clustering in membrane microdomains at resolutions below 100 nm. This approach reveals how pathological conditions alter transporter organization rather than just expression levels, providing deeper mechanistic insights. These techniques include Stimulated Emission Depletion (STED) microscopy and Stochastic Optical Reconstruction Microscopy (STORM), which overcome the diffraction limit of conventional fluorescence microscopy .

Organoid systems derived from patient samples now allow for three-dimensional analysis of SLC22A5 expression and function in physiologically relevant contexts. FITC-conjugated antibodies, optimized for thick-section imaging with appropriate permeabilization protocols, enable visualization of transporter polarization in epithelial organoids. This approach is particularly valuable for studying intestinal and renal handling of carnitine and xenobiotics in patient-specific contexts .

Combinatorial approaches integrating functional assays with immunofluorescence have enhanced our understanding of transporter dynamics. For example, correlative light and electron microscopy (CLEM) techniques allow researchers to first identify SLC22A5-expressing structures using FITC-conjugated antibodies, then examine the same structures at ultrastructural resolution. This approach has revealed previously unappreciated membrane specializations associated with transporter clusters .

In cancer research, spatial transcriptomics combined with SLC22A5 immunofluorescence enables correlation of transporter expression with broader gene expression patterns at single-cell resolution within the tumor microenvironment. This approach has revealed how SLC22A5 expression in specific cellular niches correlates with metabolic adaptations and potentially treatment resistance .

For high-throughput screening applications, the development of flow cytometry protocols using FITC-conjugated SLC22A5 antibodies allows rapid quantification of transporter expression across large cell populations. When combined with functional carnitine uptake assays using fluorescent carnitine analogs, this approach enables correlation of expression with function at the single-cell level, revealing heterogeneity within seemingly homogeneous populations .