SLC5A4 Antibody

What is SLC5A4 and why is it important in research?

SLC5A4, also known as SGLT3, functions as a glucose sensor that depolarizes the plasma membrane in response to external glucose. Unlike other sodium-glucose transporters, SGLT3 mediates the influx of sodium ions into cells but does not transport sugars, serving primarily as a glucose-sensing mechanism . This makes it an important target for research in glucose homeostasis, metabolic disorders, and cellular signaling pathways. The protein has a calculated molecular weight of 72 kDa, though it is typically observed at 60-70 kDa in Western blot applications, likely due to post-translational modifications .

What species reactivity can be expected with commercial SLC5A4 antibodies?

Most commercially available SLC5A4 antibodies demonstrate reactivity with human samples, with several also showing cross-reactivity with mouse and rat tissues . For example, the polyclonal antibody from Proteintech (24327-1-AP) has confirmed reactivity with human, mouse, and rat samples in various applications including Western blot, immunohistochemistry, and immunofluorescence . When selecting an antibody for your research, it is critical to verify the validated species reactivity in the specific application you intend to use, as reactivity can vary between experimental contexts even with the same antibody.

What are the common applications for SLC5A4 antibodies in research?

SLC5A4 antibodies have been validated for multiple experimental applications including:

It's important to note that optimal dilutions may vary depending on sample type and experimental conditions, requiring optimization for each specific research setup .

What are the optimal conditions for Western blot detection of SLC5A4?

For effective Western blot detection of SLC5A4, researchers should consider the following optimized protocol based on validated experiments:

• Sample preparation: Use 30 μg of protein lysate under reducing conditions .

• Gel electrophoresis: Perform SDS-PAGE on a 5-20% gradient gel at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours .

• Transfer: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature .

• Primary antibody: Incubate with anti-SLC5A4 antibody at the appropriate dilution (0.5-2.5 μg/ml depending on antibody) overnight at 4°C .

• Washing: Wash with TBS-0.1% Tween 3 times, 5 minutes each .

• Secondary antibody: Probe with anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hours at room temperature .

• Detection: Develop signal using an enhanced chemiluminescent detection system .

The expected band size for SLC5A4 is approximately 72 kDa, though it may appear around 60-70 kDa or even 80 kDa depending on the cell type and post-translational modifications .

How should researchers optimize immunohistochemical detection of SLC5A4 in tissue samples?

For optimal immunohistochemical detection of SLC5A4 in tissue sections:

• Antigen retrieval: Use TE buffer pH 9.0 for optimal results. Alternatively, citrate buffer pH 6.0 may be used if TE buffer produces suboptimal results .

• Antibody dilution: Start with a dilution range of 1:20-1:200 for primary antibody and optimize based on signal-to-noise ratio .

• Tissue specificity: SLC5A4 antibodies show positive IHC detection in human kidney tissue, making this an appropriate positive control .

• Blocking: Ensure adequate blocking to minimize background, especially when working with kidney tissues that may have high endogenous peroxidase activity.

• Controls: Include both positive controls (kidney tissue) and negative controls (primary antibody omission) to validate staining specificity.

It is essential to titrate the antibody concentration in each experimental system to obtain optimal signal with minimal background .

What approaches can be used for flow cytometry detection of SLC5A4?

For flow cytometry applications with SLC5A4 antibodies:

• Cell fixation: Fix cells with 4% paraformaldehyde to maintain cellular architecture .

• Permeabilization: Use an appropriate permeabilization buffer to allow antibody access to intracellular targets .

• Blocking: Block with 10% normal goat serum to reduce non-specific binding .

• Antibody incubation: Incubate with anti-SLC5A4 antibody at approximately 1 μg per 10^6 cells for 30 minutes at 20°C .

• Secondary antibody: Use fluorophore-conjugated secondary antibody (e.g., DyLight®488 conjugated goat anti-rabbit IgG) at 5-10 μg per 10^6 cells for 30 minutes at 20°C .

• Controls: Include isotype control (rabbit IgG at equivalent concentration) and unlabelled sample as controls .

This approach has been validated in 293T cells and should be optimized for other cell types .

How can researchers address high background issues when using SLC5A4 antibodies?

High background is a common challenge when working with antibodies. For SLC5A4 detection, consider these specific approaches:

• Antibody dilution optimization: Test a broader dilution series than the recommended range (e.g., 1:250-1:4000 for WB applications) to identify the optimal concentration that provides specific signal with minimal background .

• Blocking optimization: Extend blocking time to 2 hours and test alternative blocking agents such as BSA or commercial blocking solutions if milk protein is causing high background.

• Washing stringency: Increase the number of washes (5-6 times instead of 3) and/or the duration of each wash (10 minutes instead of 5) with TBS-T (0.1% Tween) .

• Antibody incubation temperature: If overnight incubation at 4°C produces high background, try shorter incubation times (2-4 hours) at room temperature with more diluted antibody.

• Secondary antibody optimization: Ensure secondary antibody is used at appropriate dilution (1:5000-1:10000) and consider using more specific secondary antibodies with reduced cross-reactivity.

If high background persists specifically in kidney tissue samples, consider additional blocking steps with avidin/biotin blocking kit to reduce endogenous biotin interference.

What are the best practices for storing and handling SLC5A4 antibodies to maintain reactivity?

To maintain optimal antibody performance:

Most SLC5A4 antibodies are supplied in PBS with preservatives such as 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability .

How can researchers validate the specificity of SLC5A4 antibodies?

To ensure antibody specificity for SLC5A4:

• Positive controls: Use validated positive control samples such as mouse kidney tissue for Western blot and IP applications, or human kidney tissue for IHC applications .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight range (60-80 kDa, with 72 kDa being the calculated weight) .

• Knockout/knockdown validation: If possible, include SLC5A4 knockout or knockdown samples as negative controls to confirm specificity.

• Multiple antibody comparison: Use antibodies targeting different epitopes of SLC5A4 to confirm consistent detection patterns.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal reduction/elimination in positive samples.

• Cross-reactivity testing: Test the antibody against closely related proteins (other SLC5 family members) to confirm specificity within the protein family.

Published validations can provide additional confidence, with several SLC5A4 antibodies having demonstrated specificity in peer-reviewed publications .

How can SLC5A4 antibodies be used in co-immunoprecipitation experiments to identify protein-protein interactions?

For co-immunoprecipitation (co-IP) of SLC5A4 and its interacting partners:

• Lysate preparation: Prepare cell or tissue lysates (particularly kidney tissue) using non-denaturing lysis buffers that preserve protein-protein interactions .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody amount: Use 0.5-4.0 μg of anti-SLC5A4 antibody per 1.0-3.0 mg of total protein lysate .

• Incubation conditions: Incubate antibody with lysate overnight at 4°C with gentle rotation.

• Bead capture: Add protein A beads (for rabbit host antibodies) and incubate for 1-2 hours at 4°C.

• Washing: Perform 4-5 stringent washes with lysis buffer to remove non-specific interactions.

• Elution and analysis: Elute bound proteins and analyze by Western blot using antibodies against potential interacting partners.

This approach can help identify novel protein interactions involving SLC5A4, particularly with other membrane transporters, scaffolding proteins, or signaling molecules that may regulate its glucose-sensing function.

What strategies can be employed for multiplex immunofluorescence detection of SLC5A4 with other markers?

For multiplex immunofluorescence co-localization studies:

• Antibody host selection: Choose primary antibodies raised in different host species (e.g., rabbit anti-SLC5A4 combined with mouse antibodies against other targets) .

• Sequential staining: If multiple rabbit antibodies must be used, consider sequential staining with complete elution between rounds, or use directly conjugated primary antibodies.

• Blocking between rounds: Implement stringent blocking between sequential staining steps to prevent cross-reactivity.

• Spectral separation: Ensure sufficient spectral separation between fluorophores to avoid bleed-through (e.g., pair DyLight®488 for SLC5A4 with Cy5 for other markers) .

• Controls: Include single-stained controls for each antibody to confirm specificity and absence of cross-reactivity.

• Quantification: Use colocalization analysis software with appropriate thresholding to quantify spatial relationships between SLC5A4 and other proteins of interest.

This approach is particularly valuable for studying the relationship between SLC5A4 and other glucose transporters or signaling molecules in specialized tissues like kidney proximal tubules.

How can researchers apply SLC5A4 antibodies in tissue microarray analysis for expression profiling across multiple samples?

For tissue microarray (TMA) analysis of SLC5A4 expression:

• Antibody validation: First validate the SLC5A4 antibody on whole tissue sections of positive control samples (kidney) to establish optimal IHC conditions .

• Antigen retrieval optimization: Compare antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0) on control tissue before applying to valuable TMA sections .

• Dilution titration: Perform careful antibody dilution series (1:20-1:200) on representative cores to determine optimal concentration for TMA-scale analysis .

• Automated staining: Consider automated immunostaining platforms to ensure consistency across all TMA cores.

• Digital image analysis: Employ digital pathology software to quantify SLC5A4 expression levels and subcellular localization across multiple samples.

• Scoring system development: Develop a consistent scoring system that accounts for staining intensity and percentage of positive cells.

• Correlation analysis: Correlate SLC5A4 expression patterns with clinical or experimental variables to identify significant associations.

This approach is valuable for analyzing SLC5A4 expression across large sample cohorts in studies of metabolic disorders, diabetes, or kidney pathologies.

How should researchers interpret variations in SLC5A4 molecular weight observed in Western blot analysis?

Variations in observed molecular weight for SLC5A4 require careful interpretation:

• Expected vs. observed weight: While the calculated molecular weight for SLC5A4 is 72 kDa, it is typically observed between 60-80 kDa depending on the experimental system .

• Post-translational modifications: Glycosylation of SLC5A4 can increase apparent molecular weight, while proteolytic processing may produce lower molecular weight forms.

• Cell/tissue type differences: Different cell lines may process SLC5A4 differently, resulting in molecular weight variations. For example:

  • Human cell lines: Often show bands at approximately 80 kDa (293T cells, Jurkat cells)

  • Mouse kidney tissue: May show bands at 60-70 kDa

• Isoform consideration: Verify which isoform(s) your antibody recognizes, as alternative splicing can affect molecular weight.

• Denaturing conditions: Varying SDS-PAGE conditions and sample preparation methods can affect protein migration patterns.

When unexpected molecular weights are observed, researchers should confirm specificity through additional validation methods such as immunoprecipitation or using multiple antibodies targeting different epitopes .

What approaches can be used to quantify SLC5A4 expression levels in Western blot and immunohistochemistry?

For accurate quantification of SLC5A4 expression:

In Western blot analysis:

• Loading controls: Always include appropriate loading controls (β-actin, GAPDH) for normalization.

• Standard curves: Consider including a standard curve of recombinant SLC5A4 protein for absolute quantification.

• Densitometry: Use digital imaging software with background subtraction and region-of-interest selection.

• Technical replicates: Perform at least three technical replicates to account for transfer and detection variations.

• Linearity verification: Confirm signal linearity across a range of protein amounts (10-50 μg) to ensure quantification within the linear range.

In immunohistochemistry analysis:

• Digital pathology: Use digital image analysis software to quantify staining intensity and distribution.

• H-score method: Apply H-score (combining intensity and percentage of positive cells) for semi-quantitative analysis.

• Reference standards: Include reference tissue slides with known expression levels as internal controls.

• Blind scoring: Implement blind scoring by multiple observers to reduce bias.

• Machine learning approaches: Consider advanced image analysis using machine learning algorithms for objective quantification.

These approaches enable reliable comparison of SLC5A4 expression across experimental conditions or disease states.

How can researchers distinguish between specific and non-specific binding in immunofluorescence applications with SLC5A4 antibodies?

To differentiate between specific and non-specific signals:

• Negative controls: Include primary antibody omission controls and isotype controls at equivalent concentrations to the SLC5A4 antibody .

• Peptide competition: Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide to block specific binding.

• Subcellular localization assessment: Confirm that SLC5A4 staining localizes to expected cellular compartments (primarily cell membrane and potentially endosomes).

• Colocalization with markers: Perform co-staining with membrane markers to confirm appropriate localization of SLC5A4.

• Signal intensity thresholding: Apply appropriate thresholding to distinguish between specific signal and background autofluorescence.

• Knockout/knockdown validation: If possible, include samples with genetic knockdown of SLC5A4 as negative controls.

• Cross-validation: Compare immunofluorescence results with other detection methods such as Western blot or flow cytometry.

For flow cytometry specifically, compare staining patterns between test samples, isotype controls, and unlabeled controls to establish appropriate gating strategies that distinguish specific from non-specific signals .

How can SLC5A4 antibodies be applied in single-cell analysis techniques?

For single-cell applications with SLC5A4 antibodies:

• Flow cytometry optimization: Optimize antibody concentration and staining conditions for detecting SLC5A4 in individual cells, using 1 μg antibody per 10^6 cells as a starting point .

• Mass cytometry (CyTOF): Consider metal-conjugated SLC5A4 antibodies for high-dimensional analysis alongside other markers in heterogeneous cell populations.

• Single-cell Western blot: Adapt conventional Western blot protocols for microfluidic single-cell protein analysis platforms, starting with higher antibody concentrations and extended incubation times.

• Imaging flow cytometry: Combine flow cytometry with high-resolution imaging to analyze SLC5A4 expression and subcellular localization at the single-cell level.

• CITE-seq approaches: For multi-omic studies, optimize antibody-oligonucleotide conjugates targeting SLC5A4 for simultaneous protein and transcriptome analysis.

These approaches allow researchers to examine cellular heterogeneity in SLC5A4 expression and correlate it with other cellular parameters or disease states at unprecedented resolution.

What considerations are important when using SLC5A4 antibodies for super-resolution microscopy?

For successful super-resolution microscopy with SLC5A4 antibodies:

• Antibody penetration: Use optimized permeabilization protocols to ensure complete antibody access while preserving membrane structure.

• Signal-to-noise ratio: Implement more stringent blocking (10% normal serum) and washing steps to minimize background fluorescence .

• Secondary antibody selection: Choose secondary antibodies with bright, photostable fluorophores designed specifically for super-resolution applications.

• Sample preparation: Optimize fixation protocols to minimize autofluorescence while preserving antigen epitopes.

• Resolution validation: Use known membrane protein markers in multi-color experiments to validate resolution and colocalization analysis.

• Quantitative analysis: Develop quantitative approaches to measure nanoscale distribution patterns of SLC5A4 within the membrane microdomains.

These considerations enable visualization of SLC5A4 distribution and organization at the nanoscale level, potentially revealing novel insights into its spatial relationship with other membrane components.

How can researchers apply SLC5A4 antibodies in proximity ligation assays to study protein-protein interactions in situ?

For proximity ligation assay (PLA) applications:

• Antibody compatibility: Ensure primary antibodies against SLC5A4 and potential interacting partners are raised in different host species or use directly conjugated PLA probes.

• Optimization for membrane proteins: Modify standard PLA protocols to accommodate membrane protein detection, using mild fixation and optimized permeabilization conditions.

• Controls: Include both positive controls (known interacting proteins) and negative controls (proteins known not to interact with SLC5A4) to validate signal specificity.

• Antibody dilution: Start with manufacturer's recommended dilutions (e.g., 1:50-1:100 for IHC applications of anti-SLC5A4), but optimize specifically for PLA, which often requires higher antibody concentrations .

• Quantification: Develop robust quantification methods for PLA signals, accounting for three-dimensional distribution in tissue sections.

• Validation: Confirm PLA results with complementary methods such as co-immunoprecipitation or FRET microscopy.

This approach offers unprecedented ability to visualize and quantify SLC5A4 interactions with signaling partners in their native cellular context, potentially revealing new insights into glucose sensing mechanisms.