SLC10A7 Antibody

What is SLC10A7 and why is it significant in research?

SLC10A7 is a 358 amino acid multi-pass membrane protein belonging to the sodium:bile acid symporter family. Recent research has revealed that SLC10A7 plays a crucial role in glycosaminoglycan synthesis, particularly in skeletal development . Mutations in the human SLC10A7 gene have been associated with skeletal dysplasia with multiple dislocations and amelogenesis imperfecta . SLC10A7 deficiency has been demonstrated to lead to a proteoglycan synthesis defect and, more specifically, to decreased heparan sulfate (HS) content . Unlike other characterized members of the SLC10 family that transport bile acids, SLC10A7 appears to have distinct functions in glycosylation processes, making it an important target for understanding certain skeletal and developmental disorders.

What applications are SLC10A7 antibodies validated for?

Based on current validation data, SLC10A7 antibodies have been successfully employed in multiple research applications:

The variability in detected molecular weights (39 kDa calculated vs. 72 kDa observed in some experiments) may reflect post-translational modifications or alternative splicing .

How should I design experiments to determine SLC10A7 subcellular localization?

Determining SLC10A7 subcellular localization has proven challenging due to conflicting reports. While some studies have reported plasma membrane localization, others indicate endoplasmic reticulum, Golgi, or even nucleolar localization .

For reliable localization studies:

• Use epitope-tagged constructs (e.g., V5-SLC10A7) alongside commercial antibodies to confirm specificity

• Perform co-localization studies with established organelle markers:

  • Calnexin for endoplasmic reticulum

  • ERGIC-53 for ER-Golgi intermediate compartment

  • β-COP and SEC31A for COPI and COPII vesicles

  • Giantin for Golgi

  • TGOLN2 for trans-Golgi network

• Test in multiple cell types (HEK293, HeLa, fibroblasts) as localization may be cell-type dependent

• Include appropriate controls (untransfected cells, SLC10A7-knockout cells)

Recent research using V5-tagged SLC10A7 demonstrated predominant co-localization with cis-, medial-, and trans-Golgi network markers in both HeLa cells and fibroblasts , suggesting a role in the secretory pathway.

What is the optimal protocol for Western blot detection of SLC10A7?

For optimal Western blot detection of SLC10A7:

• Sample preparation:

  • Use RIPA buffer containing protease inhibitor cocktail for cell lysis

  • For tissues, homogenize in RIPA buffer (mouse liver tissue has shown good results)

• SDS-PAGE:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% polyacrylamide gels for optimal resolution

• Transfer and immunodetection:

  • Transfer to PVDF membrane at 100V for 1-2 hours

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary SLC10A7 antibody (1:500-1:2000 dilution) overnight at 4°C

  • Wash with TBST (3×10 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000) for 1 hour

  • Develop using ECL detection system

Note that some researchers have reported detecting SLC10A7 at 72 kDa rather than the calculated 39 kDa, which may represent post-translational modifications or alternative splicing .

How can I validate the specificity of an SLC10A7 antibody?

Rigorous validation is essential due to reported non-specific binding with some commercial SLC10A7 antibodies . A comprehensive validation approach includes:

• Genetic controls:

  • Test in SLC10A7 knockout or knockdown cells/tissues

  • Compare expression in HAP1-KOP7 (SLC10A7 knockout) versus wild-type cells

• Protein-level validation:

  • Peptide competition assays using the immunizing peptide

  • Mass spectrometry-based validation using SLC10A7-specific reference peptides (e.g., TEELTSALVHLK)

  • Test multiple antibodies targeting different epitopes

• Expression studies:

  • Compare antibody signal with mRNA expression data

  • Validate using overexpression systems (e.g., tetracycline-inducible SLC10A7 expression)

• Controls for non-specific binding:

  • Include isotype control antibodies

  • Test in tissues known to lack SLC10A7 expression

When publishing, report the antibody catalog number, lot number, dilution, and validation methods to enable reproducibility .

What could explain inconsistent results when detecting SLC10A7 by Western blot?

Several factors may contribute to inconsistent Western blot results:

• Protein size discrepancies:

  • Calculated molecular weight: 39 kDa

  • Observed molecular weight: Often 72 kDa in experiments

  • This discrepancy may reflect post-translational modifications, particularly glycosylation (SLC10A7 is involved in glycosylation pathways)

• Sample preparation issues:

  • Insufficient denaturation (try stronger reducing conditions)

  • Protein degradation (ensure fresh samples and complete protease inhibition)

  • Improper extraction of membrane proteins (consider specialized membrane protein extraction buffers)

• Antibody-specific factors:

  • Epitope accessibility may differ between applications

  • Some antibodies may preferentially recognize specific post-translational modifications

  • Lot-to-lot variability can affect performance

• Expression levels:

  • SLC10A7 is expressed at varying levels across tissues (highest in liver and lung, moderate in placenta, kidney, spleen, and thymus, low in heart, prostate, and testis)

  • Disease states may alter expression patterns

If experiencing inconsistent results, try multiple antibodies targeting different epitopes and include appropriate positive controls (e.g., liver tissue) .

How can I study the functional consequences of SLC10A7 mutations?

To investigate the functional impact of SLC10A7 mutations:

• Expression analysis:

  • Compare wild-type and mutant SLC10A7 expression in transfected cells using Western blot and immunofluorescence

  • Significant mutations (e.g., p.Leu74Pro) have shown reduced expression compared to wild-type

• Localization studies:

  • Analyze subcellular localization of tagged mutant constructs

  • Test if mutations affect trafficking to the correct cellular compartment

• Functional assays:

  • Analyze glycosaminoglycan synthesis in patient cells or cell models

  • Measure heparan sulfate levels using specific analytical methods

  • Assess N-glycosylation profiles using electrophoretic techniques

• Mouse models:

  • The Slc10a7-/- mouse model displays shortened long bones, growth plate disorganization, and tooth enamel anomalies, recapitulating the human phenotype

  • Analyze cartilage, bone, and tooth development in these models

• Patient-derived samples:

  • Analyze patient fibroblasts for glycosylation defects

  • Study abnormal N-glycoprotein electrophoretic profiles in patient blood samples

These approaches have revealed that SLC10A7 mutations affect proteoglycan synthesis and glycosylation pathways, providing insight into disease mechanisms .

What techniques can be used to analyze the relationship between SLC10A7 and immune function?

Recent research has suggested potential connections between SLC10A7 and immune function, particularly in cancer contexts . To investigate these relationships:

• Correlation analysis:

  • Use bioinformatics approaches to analyze correlations between SLC10A7 expression and immune cell infiltration in tumors

  • The TIMER algorithm can determine relationships between SLC10A7 and various immune cell populations

• Multiplexed immunohistochemistry:

  • Perform co-staining of SLC10A7 with immune cell markers

  • Investigate spatial relationships between SLC10A7-expressing cells and immune cells in tissue sections

  • Analyze correlation between SLC10A7 expression and immune checkpoint molecules (e.g., PD-1, PD-L1)

• Single-cell RNA sequencing:

  • Analyze SLC10A7 expression patterns across immune cell populations

  • Identify cell types with significant SLC10A7 expression

• Functional studies:

  • Investigate effects of SLC10A7 knockdown/knockout on immune cell function

  • Assess cytokine production and immune cell activation in models with altered SLC10A7 expression

When designing these experiments, consider tissue-specific expression patterns and potential splice variants of SLC10A7 that may have distinct functions in different cellular contexts.

How can I differentiate between genuine SLC10A7 signals and artifacts in immunofluorescence?

Distinguishing true SLC10A7 signals from artifacts in immunofluorescence requires careful controls and validation:

• Signal specificity verification:

  • Compare staining patterns between different SLC10A7 antibodies targeting distinct epitopes

  • Include SLC10A7 knockout or knockdown samples as negative controls

  • Perform peptide competition assays to verify epitope specificity

• Pattern analysis:

  • Authentic SLC10A7 localization has been reported primarily in the Golgi apparatus and plasma membrane

  • Nucleolar staining has been reported as non-specific with some commercial antibodies

  • Validate unusual localization patterns with tagged constructs and co-localization markers

• Technical considerations:

  • Use matched isotype controls to assess non-specific binding

  • Include single-antibody controls when performing multiple immunostaining

  • Adjust fixation protocols (PFA vs. methanol) if membrane proteins are poorly detected

• Alternative approaches:

  • Complement antibody-based detection with epitope-tagged constructs

  • Confirm localization with subcellular fractionation and Western blot

  • Consider proximity ligation assays for validating protein-protein interactions

Commercial SLC10A7 antibodies have shown variable specificity, with some research groups resorting to tagged constructs for reliable localization studies after finding that commercial antibodies produced non-specific signals in control and patient fibroblasts .

What is the significance of SLC10A7's role in glycosylation and how can this be studied?

SLC10A7 mutations have been linked to glycosylation defects, suggesting a crucial role in this pathway:

• Glycosylation abnormalities associated with SLC10A7 deficiency:

  • Decreased sialylation (49% in patients vs. 69-71% in controls)

  • Increased high-mannose glycans (14% in patients vs. 6-7% in controls)

  • Characteristic increase in glycans lacking GlcNAc (2.3% in patients vs. 0.7-0.8% in controls)

  • Decreased heparan sulfate levels in cartilage and patient fibroblasts

• Analytical approaches to study glycosylation:

  • Mass spectrometry analysis of N-glycan profiles

  • Transferrin glycoform analysis

  • ApoCIII mucin-type O-glycosylation profiling

  • Analysis of total plasma N-glycans

• Cellular models:

  • Compare glycosylation patterns in wild-type and SLC10A7-deficient cells

  • Express wild-type or mutant SLC10A7 in deficient cells to assess rescue of glycosylation defects

  • Analyze glycosylation in patient-derived fibroblasts

• Functional implications:

  • Altered glycosylation affects extracellular matrix mineralization

  • These defects likely explain the skeletal and dental abnormalities in patients with SLC10A7 mutations

Understanding SLC10A7's role in glycosylation provides insight into the molecular pathogenesis of skeletal dysplasia and may reveal novel therapeutic targets for these disorders.

How is SLC10A7 being investigated in cancer research?

While SLC10A7's role in cancer is still emerging, several research approaches are being employed:

• Expression analysis:

  • Analysis of SLC10A7 expression across cancer types using bioinformatics approaches

  • Correlation of expression levels with clinical outcomes and progression

• Immune correlation:

  • Investigation of relationships between SLC10A7 expression and immune cell infiltration in tumors

  • Potential associations with immune checkpoint molecules (PD-1, PD-L1)

  • Multiplexed immunohistochemistry to study SLC10A7 protein expression and immune cells in liver cancer

• Functional studies:

  • Effects of SLC10A7 knockdown/overexpression on cancer cell proliferation, migration, and invasion

  • Impact on glycosylation patterns of cancer-associated proteins

• Diagnostic potential:

  • Evaluation of SLC10A7 as a biomarker in specific cancer types

  • Correlation with other established cancer biomarkers

The SLC10 family genes have been particularly studied in liver cancer, with analyses of their clinical relevance and immune correlations suggesting potential roles in cancer biology that warrant further investigation .

What are the latest methodological advances in studying SLC10A7 function?

Recent technological advances have expanded the toolkit for SLC10A7 research:

• Gene editing approaches:

  • CRISPR/Cas9-based knockout models (e.g., HAP1-KOP7 cells)

  • Knock-in models for studying specific mutations

  • Base editing for introducing point mutations

• Advanced imaging techniques:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging to track SLC10A7 dynamics

  • Proximity labeling methods (BioID, APEX) to identify interaction partners

• Mass spectrometry advances:

  • Targeted proteomics using SLC10A7-specific reference peptides (e.g., TEELTSALVHLK)

  • Glycoproteomics for analyzing effects on specific glycosylated proteins

  • Spatial proteomics for tissue-specific analysis

• Inducible expression systems:

  • Tetracycline-regulated promoters for controlled SLC10A7 expression

  • Allows precise temporal studies of SLC10A7 function

• Organoid and 3D culture systems:

  • More physiologically relevant models for studying SLC10A7 in developmental contexts

  • Patient-derived organoids for personalized disease modeling

These methodological advances provide researchers with powerful tools to dissect SLC10A7's complex functions in normal physiology and disease contexts.