SLC14A1 Antibody, HRP conjugated

What is SLC14A1 and what is its biological significance?

SLC14A1 (Solute Carrier Family 14 Member 1) is one of two major mammalian urea transporters that plays a critical role in the urine-concentrating mechanism within the kidney. This 43-54 kDa multipass membrane protein is primarily expressed in erythrocytes but is also found in kidney cells, mesenchymal stem cells, and certain carcinomas. SLC14A1 contains the epitope identified as the Kidd (JK) blood group antigen, with antigenic forms differing at only one amino acid position (Asp280 represents Jk(a) and Asn280 represents Jk(b)). The protein's abundance is regulated by several hormones including vasopressin, glucocorticoids, and mineralocorticoids. These regulatory mechanisms are particularly significant in disease states such as diabetes, where altered urea transport may contribute to pathophysiology .

What are the structural characteristics of SLC14A1 Antibody, HRP conjugated?

SLC14A1 Antibody, HRP conjugated (catalog #bs-7639R-HRP) is a polyclonal antibody derived from rabbits immunized with a KLH-conjugated synthetic peptide from human SLC14A1/RACH1 (immunogen range: 151-250/389). The antibody is of IgG isotype with a concentration of 1μg/μl and has been purified using Protein A affinity chromatography. The horseradish peroxidase (HRP) conjugation enables direct detection without secondary antibodies, significantly streamlining immunodetection workflows. The antibody is stored in an aqueous buffered solution containing 0.01M TBS (pH 7.4), 1% BSA, 0.03% Proclin300, and 50% Glycerol to maintain stability and functionality .

What are the validated applications for SLC14A1 Antibody, HRP conjugated?

SLC14A1 Antibody, HRP conjugated has been validated for several applications with specific recommended dilutions:

These applications leverage the HRP conjugation for direct detection through colorimetric or chemiluminescent substrates. Each application requires specific optimization for the particular experimental conditions and sample types being used .

How should SLC14A1 Antibody, HRP conjugated be stored for optimal stability?

To maintain optimal stability and activity, SLC14A1 Antibody, HRP conjugated should be stored at -20°C. It is crucial to aliquot the antibody into multiple smaller volumes upon receipt to avoid repeated freeze-thaw cycles, which can degrade the antibody and reduce its performance. The storage buffer (aqueous buffered solution containing 0.01M TBS at pH 7.4 with 1% BSA, 0.03% Proclin300, and 50% Glycerol) is formulated to preserve antibody integrity during freezing. When working with the antibody, thaw aliquots completely before use and keep them on ice while preparing dilutions. Avoid exposing the antibody to strong light sources, as HRP is photosensitive. Additionally, record the number of freeze-thaw cycles for each aliquot to monitor potential degradation effects .

What controls should be included when using SLC14A1 Antibody for immunohistochemistry?

For rigorous immunohistochemical studies using SLC14A1 Antibody, multiple controls should be implemented:

• Positive tissue control: Erythrocyte-rich tissues or kidney sections known to express SLC14A1 should be included to confirm antibody functionality.

• Negative tissue control: Tissues known not to express SLC14A1 should be tested to evaluate potential non-specific binding.

• Isotype control: A non-specific IgG from the same host species (rabbit) at the same concentration as the primary antibody to assess non-specific binding due to Fc receptor interactions.

• Absorption control: Pre-incubating the antibody with excess antigenic peptide before application to verify binding specificity.

• Technical negative control: Omission of primary antibody while maintaining all other steps of the protocol.

Implementing these controls allows researchers to confidently interpret staining patterns and distinguish between specific SLC14A1 detection and potential artifacts or background .

How can SLC14A1 Antibody be used to investigate its role in renal cell carcinoma (RCC)?

Investigation of SLC14A1's role in RCC can be approached through multiple experimental methodologies:

What approaches can be used to study the interaction between SLC14A1 and TGF-β signaling pathways?

Investigating the interaction between SLC14A1 and TGF-β signaling can be accomplished through several sophisticated approaches:

• Co-immunoprecipitation and protein stability assays: Use SLC14A1 Antibody to immunoprecipitate protein complexes followed by Western blotting for TGF-β receptors (particularly TβRII) to confirm direct interaction. Research has shown that SLC14A1 interacts with TβRII and stabilizes it by impeding Smurf1-mediated K48-linked ubiquitination and degradation .

• Phosphorylation analysis: Employ the antibody alongside phospho-specific antibodies against Smad2 to evaluate TGF-β pathway activation following SLC14A1 modulation. Colorectal cancer research demonstrates that elevated SLC14A1 levels enhance TGF-β/Smad signaling .

• Reporter gene assays: Establish TGF-β responsive element reporters in cells with modulated SLC14A1 expression to quantify pathway activity.

• Chromatin immunoprecipitation (ChIP): Use this technique with transcription factors like Snail (identified as a regulator binding downstream of SLC14A1's transcription start site) to investigate the transcriptional regulation feedback loop between SLC14A1 and TGF-β signaling .

• Proximity ligation assay: Visualize and quantify protein-protein interactions between SLC14A1 and TGF-β pathway components in situ within cells or tissues.

These methodologies can comprehensively characterize the positive feedback loop established between SLC14A1 and TGF-β signaling that has been implicated in cancer progression .

How can researchers investigate SLC14A1's role in epithelial-mesenchymal transition (EMT) and metastasis?

Investigating SLC14A1's role in EMT and metastasis requires a multi-faceted experimental approach:

• Immunofluorescence co-localization: Use SLC14A1 Antibody alongside markers for epithelial (E-cadherin) and mesenchymal (N-cadherin, Vimentin) phenotypes in cancer cell models with modulated SLC14A1 expression to visualize EMT progression.

• Western blot analysis: Quantify changes in EMT markers following SLC14A1 overexpression or knockdown. Research in colorectal cancer has demonstrated that elevated SLC14A1 levels correlate with upregulated TGF-β/Smad signaling and enhanced EMT .

• Invasion and migration assays: Conduct Transwell or wound healing assays with cells expressing different levels of SLC14A1 to assess functional consequences. Studies show that SLC14A1 overexpression enhances CRC cell invasiveness and migratory abilities .

• In vivo metastasis models: Establish xenograft models with SLC14A1-modulated cells and use immunohistochemistry with the antibody to track metastatic spread and correlate with EMT marker expression.

• Clinical sample analysis: Perform multiplex immunohistochemistry on patient samples, particularly those with metachronous liver metastasis, to evaluate the correlation between SLC14A1, phosphorylated Smad2, and Snail expression. Research has shown these factors are markedly upregulated in CRC patients with metachronous liver metastasis .

This comprehensive approach can elucidate SLC14A1's mechanistic role in driving the metastatic cascade through EMT processes.

How can researchers address weak or inconsistent signals when using SLC14A1 Antibody, HRP conjugated?

When encountering weak or inconsistent signals with SLC14A1 Antibody, HRP conjugated, researchers should systematically evaluate and optimize several parameters:

• Antibody concentration optimization: Test a range of dilutions beyond the recommended range (1:100-5000 for WB, 1:100-500 for IHC). Create a dilution series and determine the optimal signal-to-noise ratio for your specific sample type .

• Antigen retrieval enhancement: For fixed tissues or cells, evaluate different antigen retrieval methods (heat-induced vs. enzymatic) and buffer compositions (citrate, EDTA, or Tris-based) to maximize epitope accessibility.

• Detection system amplification: Consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity or tyramide signal amplification (TSA) to boost signal without increasing background.

• Sample preparation refinement: Ensure proper protein extraction with protease inhibitors, optimize protein loading amounts, and verify sample integrity through Ponceau S staining of membranes.

• HRP activity assessment: The HRP conjugation may lose activity over time or with improper storage. Test the antibody on a known positive control to evaluate whether the issue is specific to your experimental system or indicates antibody degradation.

• Incubation conditions modification: Adjust primary antibody incubation time (extending to overnight at 4°C) or temperature to enhance binding kinetics while maintaining specificity.

Documenting each optimization step systematically will help establish reproducible protocols for consistent SLC14A1 detection across experiments .

What approaches should be taken when SLC14A1 antibody results contradict published literature?

When SLC14A1 antibody results appear contradictory to published literature, a methodical investigative approach is essential:

• Experimental system comparison: Carefully assess differences in experimental models (cell lines, tissue types, species), as SLC14A1 expression and function may vary substantially across different biological contexts.

• Technical validation: Verify SLC14A1 detection using complementary approaches:

  • Confirm protein detection with additional antibodies targeting different epitopes

  • Correlate protein data with mRNA expression via qRT-PCR

  • Validate knockdown or overexpression systems using multiple methodologies

• Physiological context consideration: Evaluate experimental conditions that could affect SLC14A1 expression or function, such as:

  • Hypoxia status (as SLC14A1 expression responds sensitively to hypoxia)

  • Hormonal influences (vasopressin, glucocorticoids, mineralocorticoids regulate SLC14A1)

  • Disease state progression (expression patterns may change during cancer evolution)

• Literature critical analysis: Re-examine published studies for methodology differences, statistical approaches, and potential limitations that might explain discrepancies.

• Collaborate and reproduce: Engage with authors of contradictory studies to share protocols and potentially exchange samples for direct comparison under identical conditions.

These apparent contradictions often lead to novel insights about context-dependent regulation or previously unrecognized functions of SLC14A1 .

What is the current understanding of SLC14A1's role in cancer progression?

Recent research has revealed complex and seemingly contradictory roles for SLC14A1 in different cancer types:

This differential role of SLC14A1 across cancer types highlights the need for context-specific therapeutic approaches targeting this protein. Future research should focus on elucidating the factors that determine whether SLC14A1 functions as a tumor suppressor or promoter in different cellular environments.

How does SLC14A1 influence mitochondrial function in disease models?

Emerging research has begun to uncover SLC14A1's impact on mitochondrial function, particularly in the context of renal cell carcinoma:

• Mitochondrial regulation: Studies have demonstrated that increased SLC14A1 gene expression affects mitochondrial function in A498 renal cancer cells. While the precise mechanisms remain under investigation, this finding suggests a novel role for SLC14A1 beyond its canonical function as a urea transporter .

• Hypoxia response connection: SLC14A1 gene expression responds sensitively to hypoxic conditions, and its overexpression under hypoxia affects mitochondrial parameters. This connects SLC14A1 to cellular adaptation to low oxygen environments, a critical factor in cancer progression .

• Mitochondrial-dependent cell death: Research indicates that SLC14A1 participates in the occurrence and development of hypoxia-induced renal cell carcinoma in a mitochondria-dependent manner. This suggests SLC14A1 may influence the intrinsic apoptotic pathway that depends on mitochondrial outer membrane permeabilization .

Future research directions should include detailed analysis of SLC14A1's effect on:

• Mitochondrial membrane potential

• Reactive oxygen species production

• Mitochondrial dynamics (fission/fusion)

• Mitochondrial biogenesis

• Metabolic reprogramming in cancer cells

These investigations may reveal novel therapeutic opportunities targeting the interaction between SLC14A1 and mitochondrial function in various disease states .

What potential exists for SLC14A1 as a therapeutic target or biomarker?

Based on current research findings, SLC14A1 shows significant potential as both a therapeutic target and biomarker in multiple disease contexts:

Future developments in this field should focus on validating these potential applications in larger clinical cohorts and developing targeted therapeutics that modulate SLC14A1 function or its interaction with key signaling partners .