SLC5A10 Antibody

What is SLC5A10 and where is it primarily expressed?

SLC5A10, also known as SGLT5, is a sodium-dependent sugar transporter primarily expressed in the kidney, with high localization to the proximal straight tubules of the nephron . It is also detected at lower levels in the small intestine . SLC5A10 functions as a mannose (Km = 0.45 mM) and fructose (Km = 0.62 mM) transporter, showing the characteristic Na+-dependence typical of the SLC5 family . Immunohistochemistry studies have confirmed its apical expression in renal proximal tubules, suggesting its role in reabsorption of specific sugars from the glomerular filtrate .

What is the molecular weight of SLC5A10 protein and what should I expect to see in Western blot analysis?

The calculated molecular weight of SLC5A10 is 62 kDa (566 amino acids), but the observed molecular weight in experimental conditions is typically around 66 kDa . This slight discrepancy is common with membrane proteins due to post-translational modifications. When performing Western blot analysis, you should expect to detect a band at approximately 66 kDa in kidney tissue samples, particularly from mouse kidney which has shown consistent positive results .

Which tissue samples are recommended for positive controls when using SLC5A10 antibodies?

For Western blot applications, mouse kidney tissue is the recommended positive control . For immunohistochemistry applications, both mouse kidney tissue and human liver cancer tissue have shown reliable positive results . When establishing a new protocol, these tissues should be included as controls to verify antibody functionality before proceeding with experimental samples.

What are the optimal dilutions for SLC5A10 antibodies in different applications?

The optimal dilution ranges for SLC5A10 antibodies vary by application:

• Western Blot (WB): 1:500-1:1000

• Immunohistochemistry (IHC): 1:50-1:500

• ELISA: Follow manufacturer's recommendations

These dilutions should be optimized for each specific antibody and experimental system. The following table summarizes recommended starting dilutions:

What antigen retrieval methods are most effective for SLC5A10 immunohistochemistry?

For optimal immunohistochemical detection of SLC5A10, it is recommended to use TE buffer at pH 9.0 for antigen retrieval . Alternatively, citrate buffer at pH 6.0 may also be effective, though potentially with lower signal intensity . When optimizing your protocol, it's advisable to test both methods with positive control tissues to determine which provides the best signal-to-noise ratio for your specific experimental conditions.

What are the recommended storage conditions for SLC5A10 antibodies to maintain their reactivity?

SLC5A10 antibodies should be stored at -20°C for long-term preservation of activity . They are typically stable for one year after shipment when stored properly . For antibodies in solution, they are commonly supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, but for repeated use, small aliquots can prevent freeze-thaw cycles that may degrade antibody quality .

How can I address non-specific binding when using SLC5A10 antibodies in Western blot?

Non-specific binding can be minimized through several strategies:

• Optimize blocking conditions: Use 5% non-fat dry milk or BSA in TBST, and consider extending blocking time to 1-2 hours at room temperature.

• Adjust antibody concentration: If background is high, dilute the primary antibody further within the recommended range (1:500-1:1000) .

• Increase washing steps: Perform at least 3 washes of 5-10 minutes each with TBST after both primary and secondary antibody incubations.

• Pre-adsorb the antibody: If cross-reactivity with SLC5A1 is suspected (67% homology with some antibodies) , consider pre-adsorption against SLC5A1 peptides.

• Verify tissue specificity: Use mouse kidney tissue as a positive control and non-expressing tissue as a negative control to confirm specificity .

What are the potential cross-reactivity concerns with SLC5A10 antibodies?

Some SLC5A10 antibodies may show cross-reactivity with SLC5A1 (67% homology in some regions) . BLAST analysis of specific antibody immunogens can predict potential cross-reactivity. For example, the antibody targeting the cytoplasmic domain of human SLC5A10 shows no significant homology with other human proteins except SLC5A1 (67%) . When interpreting results, be aware of this potential cross-reactivity, especially in tissues where both transporters are expressed.

How can I validate the specificity of my SLC5A10 antibody results?

To validate antibody specificity, implement multiple approaches:

• Positive and negative tissue controls: Use known expressing tissues (kidney) and non-expressing tissues.

• Multiple detection methods: Confirm results using different techniques (IHC, WB, IF).

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to block specific binding.

• siRNA knockdown: Reduce target expression and confirm corresponding reduction in signal.

• Knockout models: If available, Slc5a10 knockout mice tissues provide excellent negative controls .

• Multiple antibodies: Use antibodies targeting different epitopes of SLC5A10 to confirm findings.

How can SLC5A10 antibodies be used to investigate the role of this transporter in fructose metabolism disorders?

SLC5A10 (SGLT5) has been identified as a mannose and fructose transporter in the kidney . Studies with Slc5a10 knockout mice showed increased fructosuria without affecting plasma fructose levels . To investigate its role in fructose metabolism disorders:

• Tissue expression analysis: Use SLC5A10 antibodies for IHC or WB to quantify expression levels in clinical samples from patients with fructose metabolism disorders versus healthy controls.

• Co-localization studies: Combine SLC5A10 antibodies with markers of kidney injury to assess correlation between transporter expression and tissue damage.

• Genetic variant correlation: In patients with identified SLC5A10 genetic variants, use antibodies to assess if protein expression or localization is altered.

• Functional studies: After pharmacological interventions targeting fructose metabolism, monitor changes in SLC5A10 expression.

• Animal models: Use antibodies to track SLC5A10 expression in animal models of metabolic syndrome or high-fructose diets.

What approaches can be used to investigate the relationship between SLC5A10 expression and 1,5-anhydroglucitol (1,5-AG) blood levels in diabetes research?

Genome-wide association studies have linked SLC5A10 genetic variations with impaired 1,5-anhydroglucitol (1,5-AG) blood levels, which are used as indicators of glycemic control . To investigate this relationship:

• Expression correlation analysis: Use quantitative IHC or WB with SLC5A10 antibodies to correlate transporter expression with 1,5-AG levels in patient samples.

• Variant-specific antibodies: For known functional variants, develop or use antibodies that can distinguish between normal and variant proteins.

• In vitro transport studies: After confirming SLC5A10 expression with antibodies, assess 1,5-AG transport in cell models.

• Pharmacological intervention studies: Investigate how SGLT2 inhibitors affect SLC5A10 expression and 1,5-AG levels, using antibodies to track protein changes.

• Cellular localization changes: Use confocal microscopy with SLC5A10 antibodies to determine if diabetes affects the subcellular localization of the transporter.

How can SLC5A10 antibodies be utilized in research exploring the broader SLC5 family and their roles in disease?

The SLC5 family comprises 12 members that transport various substrates using a sodium gradient-dependent mechanism . To investigate their collective roles:

• Comparative expression analysis: Use antibodies against multiple SLC5 family members to create expression maps across tissues, comparing patterns of co-expression.

• Regulatory mechanisms: Investigate how physiological or pathological conditions affect expression of multiple family members simultaneously.

• Functional compensation: In knockout models for one transporter, use antibodies to assess compensatory upregulation of other family members.

• Protein-protein interaction studies: Use SLC5A10 antibodies in co-immunoprecipitation experiments to identify interaction partners within or outside the SLC5 family.

• Therapeutic response markers: Investigate whether SLC5A10 expression (detected via antibodies) can predict response to SGLT inhibitors or other therapies.

What are the critical parameters for optimizing immunohistochemistry protocols with SLC5A10 antibodies?

For optimal IHC results with SLC5A10 antibodies, pay careful attention to these parameters:

• Antigen retrieval: TE buffer at pH 9.0 is recommended as the primary method, with citrate buffer at pH 6.0 as an alternative .

• Antibody dilution: Start with 1:50-1:500 dilution range and optimize based on signal intensity and background .

• Incubation conditions: Overnight incubation at 4°C typically provides better results than shorter incubations at room temperature.

• Detection system: For kidney tissues, HRP-polymer detection systems often provide better sensitivity than ABC methods.

• Counterstaining: Lighter hematoxylin counterstaining helps visualize the membrane localization without obscuring specific signals.

• Positive controls: Always include mouse kidney tissue or human liver cancer tissue as positive controls .

What are the recommended procedures for extracting and preparing kidney samples to maintain SLC5A10 integrity for Western blot analysis?

To preserve SLC5A10 integrity during sample preparation:

• Tissue harvesting: Flash-freeze kidney tissue in liquid nitrogen immediately after collection to prevent protein degradation.

• Homogenization buffer: Use a buffer containing 250 mM sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and protease inhibitor cocktail.

• Membrane protein enrichment: As SLC5A10 is a membrane protein, consider using a membrane protein extraction kit to enrich for the target.

• Sample denaturation: Heat samples at 37°C instead of boiling to prevent aggregation of membrane proteins.

• Loading controls: Use membrane protein-specific loading controls (e.g., Na+/K+ ATPase) rather than cytosolic proteins.

• SDS-PAGE conditions: 8-10% gels typically provide good resolution for the 66 kDa SLC5A10 protein .

How should experiments be designed to effectively compare SLC5A10 expression across different physiological conditions?

When designing comparative expression studies:

• Standardized sampling: Collect tissues from anatomically identical regions, particularly for kidney where expression may vary along the nephron.

• Time-matched controls: For conditions that may have circadian effects on transporter expression, match sampling times.

• Quantification methods: For IHC, use digital image analysis with standardized thresholds; for WB, use densitometry with appropriate normalization.

• Multiple detection methods: Confirm expression changes using both protein (antibody-based) and mRNA methods.

• Statistical approach: Design experiments with sufficient biological replicates (minimum n=5 per condition) to account for individual variation.

• Blinding procedure: Implement blinded analysis of samples to prevent observer bias, particularly for IHC scoring.