SLC5A5 Antibody, FITC conjugated
Description
Recommended Dilutions
| Application | Dilution Range |
|---|---|
| Western Blot (WB) | 1:500 – 1:5,000 |
| Immunohistochemistry (IHC) | 1:500 – 1:1,000 |
| Immunofluorescence (IF) | 1:50 – 1:200 |
Target Biology and Relevance
SLC5A5 is a sodium/iodide symporter critical for iodide uptake in thyroid follicular cells, enabling thyroid hormone synthesis . Dysregulation is linked to thyroid dyshormonogenesis and cancer progression . The FITC-conjugated antibody enables visualization of SLC5A5 localization and expression levels in cellular studies.
Experimental Validation
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Immunohistochemistry:
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Western Blot:
While specific data for the FITC conjugate are not explicitly provided, its protocol compatibility is inferred from unconjugated antibody validations .
Comparison with Other Conjugates
| Conjugate | Product Code | Applications |
|---|---|---|
| APC | FAB8367A | Flow cytometry |
| HRP | CSB-PA835713LB01HU | ELISA |
| Biotin | CSB-PA835713LD01HU | ELISA |
The FITC conjugate is optimal for fluorescence-based assays like IF, whereas APC suits flow cytometry .
Research Context
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Functional Studies: SLC5A5 antibodies have been used to investigate iodide transport mechanisms in thyroid cancer models .
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Disease Link: Mutations in SLC5A5 cause congenital hypothyroidism (OMIM: 274400) .
Limitations
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Studies have shown that the iodide affinity in the binding pocket of human SMCT1 is significantly lower than that of human NIS. This finding suggests distinct mechanisms of iodide transport. PMID: 29131623
- The expression levels of NIS in gastric tissue of obese and control individuals are found to be remarkably similar, indicating that obesity may not significantly impact NIS expression in this tissue. PMID: 29631680
- Research has observed NIS localization in the follicular lumen of nodular goiter patients, suggesting a potential role of NIS in the development or progression of this thyroid condition. PMID: 28190186
- Studies have demonstrated that transgene induction under hypoxic conditions in tumor cell spheroid models can lead to enhanced radioiodide uptake facilitated by NIS. This finding holds potential for targeted radiotherapy strategies. PMID: 27458162
- Research suggests a complex regulatory mechanism for NIS expression in thyroid follicles involving thyroglobulin (TG) iodination and the TSH/TSHR signaling pathway. Lowerly iodinated TG upregulates NIS expression and PKA activity, while highly iodinated TG downregulates NIS expression and upregulates PKC activity. PMID: 28396984
- Data indicate that while less than 25% of cells express NIS, combining 131I with gemcitabine (GEMGEM/131I) resulted in substantial tumor regression in NIS-transduced breast cancer brain metastases. This suggests the potential for NIS-mediated targeted therapy. PMID: 27363025
- Studies using whole cell experiments have identified four crucial amino acid residues involved in NIS transport. Mutations at these conserved positions across the SLC5 family result in proteins that, despite membrane localization, exhibit impaired iodide transport, highlighting their importance in transporter function. PMID: 27562170
- Molecular evidence suggests that NIS is a direct target of wild-type p53. Activation of p53 upon Doxorubicin drug exposure leads to suppression of NIS expression and function in breast cancer cells. This finding has implications for understanding the interplay between p53 and NIS in cancer biology. PMID: 28528452
- Radiotracer accumulation in transfected cells was found to correlate with the induction of hNIS and the expression of specific miRNAs detected by real-time PCR. This observation suggests the potential use of NIS and miRNAs as biomarkers for targeted imaging and therapy. PMID: 28493972
- Results indicate that NIS can serve as an objective criterion for assessing the sensitivity of luminal B and basal breast cancer subtypes to neoadjuvant chemotherapy (NACT). This information could aid in tailoring treatment strategies for breast cancer patients. PMID: 28361851
- Research has shown that hTERT promoter-driven expression of the NIS gene in HeLa cells leads to 188Re uptake and therapeutic effects. This suggests that NIS-based gene therapy and imaging utilizing the hTERT promoter and 188Re may be viable therapeutic strategies. PMID: 27573304
- SLC5A5 has been found to be downregulated in follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC). Its expression can be modulated by hsa-let-7f-5p. Interestingly, this correlation between SLC5A5 and hsa-let7f-5p is lost in anaplastic thyroid carcinoma (ATC). These findings suggest potential implications for understanding and targeting thyroid cancer subtypes. PMID: 26960757
- Mutations in the C-terminal domain of the protein generally do not affect NIS function except for the creation of a diacidic motif. This motif leads to a decrease in total NIS protein levels without impacting its expression at the plasma membrane. PMID: 26831514
- Increasing the level of glycosylation, which enhances functional NIS activity, may be a promising therapeutic strategy for cancer patients who have not responded well to conventional radioiodine treatment. This approach could improve the efficacy of radioiodine therapy. PMID: 26599396
- Studies have confirmed that NIS overexpression enhances the sensitivity of ER-negative breast cancer cells to radioiodide therapy. This finding suggests that NIS could be a potential target for improving the efficacy of radioiodine treatment in certain breast cancer subtypes. PMID: 25955347
- miR-146b-3p has been found to bind to the 3'-untranslated region of PAX8 and NIS, leading to impaired protein translation and a subsequent reduction in iodide uptake. This suggests that miR-146b-3p may play a role in regulating thyroid hormone synthesis and potentially influencing thyroid function. PMID: 26282166
- Research has demonstrated an association between specific variants and haplotypes of the NIS gene with differentiated thyroid cancer. This finding suggests that genetic variations in NIS could be associated with an increased risk of developing thyroid cancer. PMID: 26160439
- Studies indicate that SLC5A5, SLC5A8 (sodium-coupled monocarboxylate transporter 1), and SLC26A4 (pendrin), the three known iodide transporters, are important in breast tissue metabolism during lactation and in breast neoplasms. PMID: 26285906
- Real-time PCR revealed that certain cell lines express mRNA for lactoferrin receptors. Flow cytometry and confocal microscopy confirmed that these cells efficiently internalize bLf, which in turn upregulates NIS expression. This suggests a potential role of bLf in regulating NIS expression and potentially influencing thyroid function. PMID: 26213306
- While the affinity of NIS for iodide is relatively low, it increases upon binding of sodium ions. NIS takes advantage of the high extracellular sodium concentration and the significant increase in its own affinity for iodide and for the second sodium ion, which is elicited by the binding of the first. This mechanism enhances NIS's efficiency in transporting iodide across the membrane. PMID: 24888603
- Research has concluded that miR-146b can modulate NIS-mediated radioiodide uptake. Therefore, miR-146b may serve as a potential target for enhancing the effectiveness of radioactive therapy against poorly differentiated thyroid carcinoma. PMID: 25960292
- In normal salivary glands, striated duct cells exhibit strong expression of NIS. In Warthin's tumors, eosinophilic epithelial cells show variable NIS expression. Technetium-positive specimens were found to be NIS-positive, while technetium-negative specimens were NIS-negative. These findings suggest a potential role of NIS in salivary gland function and the development of Warthin's tumors. PMID: 25199743
- Due to their low NIS expression, tall cell variant (TCV) and diffuse sclerosing papillary thyroid carcinoma (DSPTC) require higher cumulative doses of radioactive iodine therapy to achieve favorable outcomes. This highlights the importance of understanding NIS expression levels in guiding treatment strategies. PMID: 24096868
- Studies in the Guangxi Zhuang Region, China, have shown that the NIS mutation rate is very low. This suggests that mutations in other genes may play a more significant role in thyroid dyshormonogenesis in this population. PMID: 25465605
- Relatively weak melanoma-specific promoters can direct high NIS activity in melanoma cells, but weaker cancer-specific promoters only drive high NIS activity in certain melanoma cell lines. This observation indicates that the effectiveness of NIS-based targeted therapy may depend on the specific promoter used and the cancer cell type. PMID: 25842835
- The BRAF V600E mutation has been shown to inhibit NIS expression by upregulating its promoter methylation. This suggests that epigenetic modifications, specifically promoter methylation, can play a role in regulating NIS expression and potentially influencing the response to radioiodine therapy. PMID: 25378232
- Recent advancements in the study of NIS regulation provide a foundation for developing novel therapeutic approaches in extrathyroidal cancers. This research area holds promising potential for improving cancer treatments. PMID: 24884806
- The expression of the TSH receptor (TSHR) and NIS genes is regulated by a complex interplay of mechanisms, including epigenetic events induced by major signaling pathways involved in thyroid tumorigenesis. This understanding is crucial for developing targeted therapies that can modulate these pathways and potentially control thyroid cancer growth. PMID: 24353283
- NIS has been identified as a novel ovarian cancer marker, opening up possibilities for using radioiodide in diagnosing and treating ovarian cancer patients. This finding could lead to improved diagnostic tools and potentially more effective treatment options for ovarian cancer. PMID: 24708099
- Research has identified a novel distal enhancer, the NIS distal enhancer, which regulates gene expression through DNA methylation in thyroid cancer. This discovery provides further insights into the complex mechanisms regulating NIS expression and potential therapeutic targets for thyroid cancer. PMID: 24432988
- Studies have suggested that the delta-amino group of arginine at position 124 plays a crucial structural role in the maturation and cell surface targeting of the Na+/I- symporter. This finding highlights the importance of specific amino acid residues in protein structure and function. PMID: 23690546
- NIS expression is tightly regulated during the transition of intercalated to striated ducts and striated to excretory ducts in salivary ductal cells. However, NIS expression is decreased during inflammation and tumor formation. This observation suggests a potential role of NIS in salivary gland physiology and the development of salivary gland tumors. PMID: 23441638
- Data indicate that the Na+/I- symporter and type 3 iodothyronine deiodinase genes are expressed in the term placenta and amniotic membrane. This suggests a potential role of these genes in fetal development and the regulation of thyroid hormone metabolism during pregnancy. PMID: 23857380
- Research has established that the NIS gene is a direct target of the p53 family. This finding suggests that p53, a tumor suppressor gene, may play a role in regulating NIS expression and potentially influencing the response to radioiodine therapy. PMID: 24052075
- SLC5A5 comprises 13 transmembrane helices and 643 amino acid residues in humans. PMID: 23988430
- Studies have indicated that interhelical interactions are essential for the proper folding and activity of the Na+/I- symporter (NIS). This finding highlights the importance of protein structure and the interactions between different regions of the protein for proper function. PMID: 23650190
- Research using mutant recombinant proteins suggests that the proto-oncogene PBF/PTTG1IP (pituitary tumor-transforming 1 interacting protein) is a phosphoprotein. This study also emphasizes the importance of tyrosine residue Y174 in the interaction and co-localization of PBF/PTTG1IP with NIS. These findings suggest potential implications for understanding the role of PBF/PTTG1IP in thyroid cancer development. PMID: 23678037
- MEK inhibition has been found to lead to lysosome-mediated NIS protein degradation in human breast cancer cells. This observation suggests a potential strategy for targeting NIS degradation pathways in cancer therapy. PMID: 23404856
- Research has indicated a positive link between hNIS and estrogen receptor (ER) expression in breast cancer. This finding suggests a potential role of NIS in estrogen-dependent breast cancer growth and development. PMID: 23342072
- NIS is highly expressed in early human trophoblast at the feto-maternal interface. This suggests a potential role of NIS in placental function and fetal development. PMID: 23174149
- Studies have demonstrated that MV-NIS, an oncolytic measles virus encoding the human thyroidal sodium iodide symporter (NIS), can deliver targeted radiotherapy to the tumor site and promote a localized bystander effect. This approach has the potential to enhance the efficacy of radiotherapy for certain cancers. PMID: 23134812
- Data from various thyroid cancer cell lines suggest that PARP (poly(ADP-ribose) polymerase) inhibition increases NIS gene expression through specific modifications of transcriptional regulatory mechanisms, such as histone modifications. This finding provides insights into the intricate regulation of NIS expression and potential therapeutic targets. PMID: 22982218
- SLC5A5 expression in the placenta is upregulated during placentation. Its mRNA levels are low at 6 weeks of gestation and peak at 12 weeks. Furthermore, SLC5A5 protein expression increases with enhanced placental vascularization. These observations suggest a potential role of SLC5A5 in placental development and function. PMID: 22954554
- The drug nevirapine has been found to induce upregulation of NIS mRNA and TSHR mRNA in FRO anaplastic thyroid carcinoma cells. This finding suggests a potential therapeutic role of nevirapine in targeting NIS expression and potentially influencing thyroid cancer growth. PMID: 22781452
- Molecular imaging of luciferase signal and sodium-iodide symporter imaging may be valuable for optimizing bioenergetics in transplanted cells. This approach could improve the efficiency and success of cell transplantation therapies. PMID: 23255420
- Sodium/iodide symporter is expressed in the majority of seminomas and embryonal testicular carcinomas. This finding suggests a potential role of NIS in the development and progression of testicular cancer and may lead to improved diagnostic and therapeutic strategies. PMID: 23117572
- Research has established NIS as a carrier protein that interacts with a major cell signaling hub to facilitate tumor cell locomotion and invasion. This discovery sheds light on the intricate mechanisms by which NIS can contribute to cancer progression and metastasis. PMID: 22962269
- The BRAF(V600E) mutation has been associated with statistically significantly lower functional NIS protein expression in the classic variant of papillary thyroid carcinomas. This finding suggests a potential link between the BRAF mutation and reduced NIS activity, which could impact the response to radioiodine therapy. PMID: 23163107
- CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of sodium iodide symporter expression in thyroid cancer, acting at the transcriptional level. This suggests that targeting CREM expression could potentially modulate NIS activity and improve therapeutic outcomes. PMID: 22510021
- Research has focused on analyzing the subcellular distribution of the sodium iodide symporter in benign and malignant thyroid tissues. These studies provide valuable insights into the localization of NIS and its potential role in the development and progression of thyroid cancer. PMID: 22545753
Q&A
What is SLC5A5 and why is it a target for immunofluorescence studies?
SLC5A5 (Solute Carrier Family 5 Member 5) is the sodium iodide symporter/cotransporter protein responsible for active iodide transport in tissues such as the thyroid gland. This membrane protein plays a critical role in thyroid hormone biosynthesis and is implicated in various pathologies including thyroid disorders, cancer research, and metabolic regulation studies . The protein exists in multiple forms: a fully glycosylated 97kDa form, a non-glycosylated 68kDa form, and occasionally as higher-order structures at approximately 160kDa . The detection of SLC5A5 through fluorescent antibodies enables researchers to visualize its expression patterns, subcellular localization, and protein dynamics in experimental systems, making it valuable for both basic research and clinical investigations.
What spectral properties characterize FITC-conjugated SLC5A5 antibodies?
FITC-conjugated SLC5A5 antibodies possess specific optical properties that researchers must consider when designing multi-parameter experiments:
| Property | Specification | Practical Implication |
|---|---|---|
| Excitation maximum | 499 nm | Compatible with 488 nm argon laser lines |
| Emission maximum | 515 nm | Detected in green fluorescence channel |
| Laser line | 488 nm | Standard on most flow cytometers and confocal microscopes |
These spectral characteristics make FITC conjugates particularly suitable for multi-color applications where proper compensation and filter selection are implemented . When designing experiments, researchers should account for potential spectral overlap with other fluorophores, particularly those with emission in the 500-550 nm range.
How do clonality and host species affect experimental design with SLC5A5 antibodies?
The clonality and host species of SLC5A5 antibodies significantly impact experimental design and interpretation:
Polyclonal antibodies recognize multiple epitopes on the SLC5A5 protein, providing stronger signal amplification and greater tolerance to protein denaturation . They excel in applications where protein conformation may be altered, such as fixed tissue immunohistochemistry.
Host species considerations: Mouse monoclonal antibodies (like clone 14F) require anti-mouse secondary detection systems and may produce background when used on mouse tissues . Rabbit polyclonal antibodies offer advantages when working with murine samples but require anti-rabbit detection reagents . Cross-adsorbed secondary antibodies are recommended to minimize cross-reactivity in multi-species experiments.
What is the recommended protocol for optimizing SLC5A5 antibody dilution in immunofluorescence applications?
Optimization of SLC5A5 antibody dilution is critical for achieving optimal signal-to-noise ratio. The following titration protocol is recommended:
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Prepare serial dilutions of the FITC-conjugated antibody (e.g., 1:50, 1:100, 1:200, 1:500, and 1:1000) in appropriate antibody diluent .
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Apply dilutions to identical sample preparations, maintaining consistent incubation conditions.
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Process slides simultaneously using identical imaging parameters.
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Quantitatively assess signal intensity and background for each dilution.
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Select the dilution that provides maximum specific signal with minimal background fluorescence.
What sample preparation techniques enhance SLC5A5 detection in fixed samples?
Sample preparation significantly impacts SLC5A5 detection quality, particularly for this membrane-localized protein:
Fixation methods:
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4% paraformaldehyde (10-15 minutes) preserves membrane localization while maintaining fluorophore activity
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Avoid methanol fixation which can disrupt membrane protein conformation
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For tissues, 10% neutral buffered formalin with controlled fixation time (12-24 hours) followed by antigen retrieval optimizes epitope accessibility
Permeabilization considerations:
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Mild detergent treatment (0.1-0.2% Triton X-100 or 0.1% saponin) allows antibody access while preserving membrane structures
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Excessive permeabilization may disrupt membrane integrity and alter SLC5A5 localization patterns
Blocking strategy:
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Use 5-10% normal serum from the same species as the secondary antibody
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Include 1% BSA to reduce non-specific binding
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Consider adding 0.1% Tween-20 to reduce hydrophobic interactions
The preparation protocol should be systematically optimized based on the specific cellular or tissue system under investigation, as SLC5A5 expression levels and accessibility vary significantly across experimental models .
How should flow cytometry protocols be adapted for optimal SLC5A5 detection?
Flow cytometry analysis using FITC-conjugated SLC5A5 antibodies requires specific protocol adaptations:
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Cell preparation: Prepare single-cell suspensions while maintaining membrane integrity. Avoid harsh enzymatic dissociation methods that might cleave surface epitopes.
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Staining buffer optimization: Include 0.1% sodium azide to prevent internalization of the antibody-receptor complex and 2-5% serum to reduce non-specific binding.
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Titration: Perform antibody titration to determine the optimal concentration that provides maximum separation between positive and negative populations.
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Controls:
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Include unstained cells to establish autofluorescence baseline
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Use isotype-matched FITC-conjugated control antibodies at identical concentrations
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If available, use positive control cells with known SLC5A5 expression (e.g., HEK293 transfected with human SLC5A5)
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Consider including a blocking control using unconjugated antibody
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Instrument setup:
Experimental data from HEK293 cells transfected with human SLC5A5 and eGFP demonstrate the utility of this approach, where quadrant markers can be set based on control antibody staining . This methodology allows for accurate quantification of SLC5A5-positive populations in heterogeneous samples.
How can researchers differentiate between glycosylated and non-glycosylated forms of SLC5A5?
SLC5A5 exists in multiple forms, including the fully glycosylated 97kDa form and the non-glycosylated 68kDa form, presenting a challenge for specific detection . The following approaches can help differentiate these forms:
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Western blot analysis alongside immunofluorescence:
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Perform parallel western blot analysis to identify the molecular weight forms present in your sample
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Compare migration patterns before and after treatment with glycosidases (PNGase F or Endo H)
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Correlate western blot patterns with immunofluorescence intensity
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Subcellular localization assessment:
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The fully glycosylated form (97kDa) predominantly localizes to the plasma membrane
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The non-glycosylated form (68kDa) may show more intracellular/perinuclear distribution
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Use confocal microscopy with Z-stack imaging to precisely evaluate membrane vs. cytoplasmic localization
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Biochemical approaches:
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Treat samples with tunicamycin to inhibit N-linked glycosylation
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Compare antibody binding patterns before and after treatment
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Quantify changes in molecular weight distribution and fluorescence intensity
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Understanding the glycosylation state is particularly important when studying SLC5A5 trafficking, as proper glycosylation is required for membrane insertion and functional activity . Changes in the ratio between glycosylated and non-glycosylated forms may indicate alterations in protein processing pathways.
What strategies can resolve non-specific binding issues with FITC-conjugated SLC5A5 antibodies?
Non-specific binding can compromise experimental interpretation. The following systematic troubleshooting approach is recommended:
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Antibody validation:
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Confirm antibody specificity using positive control samples (e.g., thyroid tissue or transfected cell lines)
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Include appropriate negative controls lacking SLC5A5 expression
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Perform peptide competition assays to confirm epitope specificity
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Protocol optimization:
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Increase blocking duration and concentration (try 5-10% serum with 1-3% BSA)
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Include 0.1-0.3% Triton X-100 in blocking and antibody diluent solutions
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Increase washing stringency (more washes, higher salt concentration)
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Reduce antibody concentration based on titration results
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Signal-to-noise enhancement:
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Counter-stain nuclei with DAPI to facilitate cell identification
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Utilize spectral unmixing to separate true signal from autofluorescence
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Consider photobleaching techniques to reduce tissue autofluorescence
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Storage and handling:
If non-specific binding persists despite these interventions, consider switching to alternative detection methods (e.g., immunohistochemistry with enzymatic detection) or different antibody clones targeting distinct epitopes within the SLC5A5 protein.
How can SLC5A5 antibodies be integrated into multiplex immunofluorescence panels?
Multiplexed immunofluorescence incorporating SLC5A5 detection requires careful panel design:
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Spectral considerations:
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Antibody compatibility:
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Select complementary antibodies raised in different host species to avoid cross-reactivity
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When using multiple mouse monoclonals, consider sequential staining with direct conjugates
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Validate each antibody individually before combining in multiplex panels
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Multiplex staining protocol:
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Apply antibodies sequentially if raised in the same species
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Use Fab fragments to block potential cross-reactivity between detection systems
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Include single-stain controls for accurate spectral unmixing
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Analysis considerations:
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Employ spectral imaging and linear unmixing algorithms to separate overlapping signals
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Utilize image analysis software capable of cell-by-cell quantification
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Implement machine learning approaches for unbiased classification of complex expression patterns
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This approach allows simultaneous assessment of SLC5A5 expression alongside markers for cell phenotyping, signaling pathway activation, or functional state assessment, providing richer contextual information than single-marker studies.
What considerations are important when using SLC5A5 antibodies in high-resolution imaging techniques?
Advanced imaging techniques require special considerations for optimal SLC5A5 visualization:
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Super-resolution microscopy:
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FITC photobleaching can limit extended acquisition; consider oxygen scavenger systems
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For STORM/PALM applications, ensure appropriate labeling density for point localization
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For SIM applications, optimize grid pattern frequency based on structure size
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Live-cell imaging:
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FITC-conjugated antibodies are generally unsuitable for live-cell applications
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Consider using Fab fragments for reduced interference with protein function
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Implement photostability enhancers to reduce photobleaching during time-lapse imaging
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Tissue clearing techniques:
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Evaluate compatibility of FITC fluorescence with clearing protocols (CLARITY, CUBIC, etc.)
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Adjust imaging parameters to compensate for light scattering and signal attenuation
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Consider post-staining approaches after clearing for optimal antibody penetration
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Quantitative imaging:
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Include calibration standards for fluorescence intensity normalization
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Implement flat-field correction to account for illumination non-uniformities
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Standardize exposure settings and acquisition parameters across experimental groups
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By addressing these technical considerations, researchers can maximize the information obtained from SLC5A5 immunofluorescence studies and achieve reproducible, quantifiable results that advance our understanding of this important membrane transporter in normal physiology and disease states.
