SLC22A1 Antibody, Biotin conjugated

What is SLC22A1 and what experimental systems are most appropriate for its study?

SLC22A1 (solute carrier family 22 member 1), also known as organic cation transporter 1 (OCT1), is a 554 amino acid, 61 kDa transmembrane protein involved in the transport of organic cations across cellular membranes. SLC22A1 is predominantly expressed in the liver, with significant roles in drug uptake and metabolism .

The most appropriate experimental systems for studying SLC22A1 include:

• Mouse and rat liver tissues, which show high endogenous expression and are suitable for antibody validation

• Transfected cell lines (such as HEK293 cells) expressing human SLC22A1, which provide a controlled system for functional studies

• Liver-specific knockout mouse models, which enable in vivo functional analyses

Mouse liver tissue has proven particularly valuable as it shows consistent and strong expression patterns that align with expected cellular localization. For detecting human SLC22A1, the recommended approach involves transfected cell lines as demonstrated in flow cytometry analyses where SLC22A1-transfected HEK293 cells show clear membrane staining patterns compared to control transfected cells .

Which applications are validated for SLC22A1 antibody detection?

Based on extensive validation studies, SLC22A1 antibodies have been successfully used in multiple applications with specific optimization parameters:

When designing experiments, optimal results require tissue-specific antigen retrieval methods. For IHC applications with liver tissue, TE buffer at pH 9.0 is recommended for optimal antigen retrieval, though citrate buffer at pH 6.0 can serve as an alternative method . Publication data indicates consistent detection of SLC22A1 in Western blot applications across multiple studies, confirming antibody reliability .

How should sample preparation be optimized for SLC22A1 detection?

For optimal detection of SLC22A1, sample preparation is critical and varies by application:

For Western blot applications:

• Complete cell lysis is essential with RIPA or similar buffers containing protease inhibitors

• Samples should be processed quickly with minimal freeze-thaw cycles

• The observed molecular weight ranges from 61-67 kDa for endogenous expression and approximately 80 kDa in certain cell lines due to post-translational modifications

For immunohistochemistry applications:

• Freshly fixed tissues yield better results than long-term stored samples

• Antigen retrieval with TE buffer (pH 9.0) significantly improves signal detection

• Sections should be of optimal thickness (4-6 μm) for balanced signal intensity and morphological detail

For immunoprecipitation:

• Use of 0.5-4.0 μg antibody per 1-3 mg of total protein provides optimal binding efficiency

• Gentle wash conditions preserve protein-protein interactions

• Pre-clearing lysates minimizes non-specific binding

When troubleshooting detection issues, consider using positive controls such as mouse or rat liver tissue, which consistently display strong SLC22A1 expression.

How can SLC22A1 transport activity be functionally assessed in experimental models?

Functional assessment of SLC22A1 transport activity requires specialized assays that measure substrate movement across cellular membranes. Based on published protocols, the following methodological approach is recommended:

The efflux assay is particularly effective for measuring SLC22A1 transport function:

• Preload cells with radiolabeled substrates (such as [³H]-L-carnitine) for 30 minutes to label cellular pools of carnitine and acylcarnitines

• Wash cells thoroughly (2-3 times) with PBS to remove extracellular substrate

• Incubate in efflux media containing HBSS, 20 mM HEPES, and 20 μM carnitine

• Collect media at defined time points and lyse cells in 0.1N NaOH

• Measure radioactivity in both cell lysate and media, normalizing to protein content

• Calculate percent efflux as: (counts in media / [counts in media + counts in cell]) × 100

For SLC22A1-specific substrate production, adding 50 mM L-valine to the transport media drives the production of isobutyrylcarnitine, which has shown the strongest association with SLC22A1 activity in genetic studies . Control experiments using SLC22A1-null cells or SLC22A1 inhibitors are essential for distinguishing transporter-specific activity from background transport.

What are the key considerations when using SLC22A1 antibodies in co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) experiments with SLC22A1 antibodies require careful optimization to preserve protein-protein interactions while minimizing non-specific binding:

• Lysis conditions must preserve membrane protein interactions:

  • Use gentle detergents (0.5-1% NP-40 or Triton X-100)

  • Include protease inhibitors and perform all steps at 4°C

  • Avoid harsh detergents like SDS that may disrupt protein interactions

• Antibody selection criteria:

  • Choose antibodies validated for IP applications (such as 24617-1-AP)

  • Use 0.5-4.0 μg antibody per 1-3 mg of total protein lysate

  • Consider epitope location relative to potential interaction domains

• Control experiments are critical:

  • Include isotype controls to assess non-specific binding

  • Use SLC22A1-null samples as negative controls

  • Reverse Co-IP experiments can confirm specific interactions

Research has demonstrated that Co-IP approaches can successfully identify protein-protein interactions, as exemplified by studies with the related transporter SLC22A3, which was shown to directly interact with cytoskeletal proteins including α-actinin-4 (ACTN4) . Similar methodologies can be applied to SLC22A1 studies to identify novel interaction partners that may regulate its localization, activity, or degradation.

How do genetic variations and RNA editing affect SLC22A1 expression and function?

Genetic variations and RNA editing can significantly impact SLC22A1 expression and function, requiring specialized experimental approaches for comprehensive characterization:

RNA editing effects:

• A-to-I RNA editing can significantly alter transporter expression levels, as demonstrated in the related SLC22A3 transporter

• Edited transcripts may show reduced stability, leading to decreased protein expression

• Correlation analyses between editing levels and mRNA expression can quantify these effects

For investigating RNA editing effects:

• Use RNA sequencing to identify editing sites

• Perform site-directed mutagenesis to recreate edited variants in expression constructs

• Compare wild-type and edited variants in functional assays

• Measure mRNA stability through actinomycin D chase experiments

Research with the related SLC22A3 transporter showed that RNA editing negatively correlated with mRNA levels (Spearman's r = -0.394, p < 0.001), and edited transcript showed significantly reduced expression compared to non-edited forms . Similar methodologies can be applied to SLC22A1 research to understand how post-transcriptional modifications impact its expression and function.

What experimental models are available for studying SLC22A1 function in vivo?

Several experimental models have been developed for studying SLC22A1 function in vivo, with liver-specific knockout mice being particularly valuable:

The Slc22a1 conditional knockout mouse model:

• Generated using a gene targeting approach with LoxP sites flanking exons 2 and 3

• Liver-specific knockout achieved by crossing Slc22a1^fl/fl mice with Albumin-Cre transgenic mice

• Provides an excellent system for studying SLC22A1 function specifically in hepatocytes

Validation of knockout efficiency:

• RT-PCR using Slc22a1-specific primers (forward: 5′-AGGCTGATGGAAGTTTGGCA-3′; reverse: 5′-GTGGGGATTTGCCTGTTTGG-3′)

• Western blot analysis with validated SLC22A1 antibodies

• Functional transport assays comparing wild-type and knockout tissues

Alternative models include:

• Human cell lines with CRISPR/Cas9-mediated SLC22A1 knockout

• Xenograft models using cells with manipulated SLC22A1 expression

• Patient-derived samples with characterized SLC22A1 variants

When studying compensatory mechanisms, related transporters should be assessed, including SLC22A2 and SLC22A3, using validated primer sets:

• Mouse Slc22a2: forward 5′-TGGCATCGTCACACCTTTCC-3′, reverse 5′-AGCTGGACACATCAGTGCAA-3′

• Mouse Slc22a3: forward 5′-TCAGAGTTGTACCCAACGACATT-3′, reverse 5′-TCTGCCACACTGATGCAACT-3′

How can SLC22A1 antibodies be used to investigate changes in subcellular localization?

Investigating changes in SLC22A1 subcellular localization requires specialized imaging techniques and careful experimental design:

Immunofluorescence microscopy approach:

• Grow cells on glass coverslips or prepare tissue sections at 4-6 μm thickness

• Fix samples using 4% paraformaldehyde to preserve membrane structures

• Use optimized permeabilization protocols (0.1-0.5% Triton X-100 for 5-10 minutes)

• Block with appropriate serum (5-10% normal serum) to minimize background

• Incubate with validated SLC22A1 primary antibody at optimized dilutions

• Visualize using fluorophore-conjugated secondary antibodies

• Include co-staining with organelle markers to determine precise localization:

  • Na+/K+-ATPase for plasma membrane

  • Calnexin for endoplasmic reticulum

  • EEA1 for early endosomes

For studying dynamic changes in localization:

• Live-cell imaging with GFP-tagged SLC22A1 constructs

• Stimulus-response experiments to assess trafficking

• Time-course experiments following drug treatments

For quantifying changes in localization:

• Line-scan analyses across cellular compartments

• Colocalization coefficients with organelle markers

• Subcellular fractionation followed by western blotting

Research with related transporters has shown that protein-protein interactions, such as those between SLC22A3 and ACTN4, can significantly affect cellular localization and function . Similar approaches can be applied to SLC22A1 to understand how interacting proteins regulate its trafficking and membrane retention.

What are common pitfalls in SLC22A1 antibody experiments and how can they be resolved?

When working with SLC22A1 antibodies, researchers frequently encounter several challenges that can affect experimental outcomes:

The molecular weight of SLC22A1 can vary (61-67 kDa in mouse/rat liver; approximately 80 kDa in some cell lines) due to post-translational modifications . When troubleshooting, always include positive controls (mouse/rat liver tissue) and negative controls (non-expressing tissues or knockdown samples).

For immunohistochemistry applications, antigen retrieval is critical - TE buffer at pH 9.0 is recommended for optimal results, though citrate buffer at pH 6.0 can serve as an alternative . Additionally, sample-specific optimization may be necessary as expression levels vary significantly between tissues and cell types.

How can researchers distinguish between closely related SLC22 family members in experimental systems?

Distinguishing between closely related SLC22 family members (such as SLC22A1, SLC22A2, and SLC22A3) requires careful experimental design and validation:

Antibody specificity validation:

• Test antibodies on tissues with differential expression patterns:

  • SLC22A1 is predominantly expressed in liver

  • SLC22A2 shows strong expression in kidney

  • SLC22A3 is expressed across multiple tissues including placenta

• Validate using knockout or knockdown models for each transporter

• Perform peptide competition assays with the specific immunogens

For RT-qPCR analysis:

• Use validated primer sets with demonstrated specificity:

  • Human SLC22A1: Taqman primer Hs00427550_m1

  • Mouse Slc22a1: forward 5′-AGGCTGATGGAAGTTTGGCA-3′, reverse 5′-GTGGGGATTTGCCTGTTTGG-3′

  • Mouse Slc22a2: forward 5′-TGGCATCGTCACACCTTTCC-3′, reverse 5′-AGCTGGACACATCAGTGCAA-3′

  • Mouse Slc22a3: forward 5′-TCAGAGTTGTACCCAACGACATT-3′, reverse 5′-TCTGCCACACTGATGCAACT-3′

For functional discrimination:

• Use transporter-specific substrates or inhibitors

• SLC22A1 can be specifically assessed using transport assays with L-valine (50 mM) to drive isobutyrylcarnitine production

• Calculation of SLC22A1-specific activity by subtracting background transport in control cells

When interpreting results from different detection methods, consider that protein and mRNA levels may not directly correlate due to post-transcriptional regulation, including RNA editing, which has been shown to significantly impact expression levels of SLC22 family members .

How can SLC22A1 antibodies be used to investigate protein-protein interactions in transport complexes?

Recent advances in protein interaction studies provide powerful approaches for investigating SLC22A1 interactions with binding partners:

Advanced co-immunoprecipitation strategies:

• Crosslinking approaches can capture transient interactions:

  • Use membrane-permeable crosslinkers (DSP, formaldehyde)

  • Optimize crosslinking time to prevent over-fixation

  • Include proper reversal controls

• Proximity labeling techniques offer advantages for membrane proteins:

  • BioID or TurboID fusion constructs expressed with SLC22A1

  • APEX2-based proximity labeling in intact cells

  • Mass spectrometry identification of labeled proteins

• Pull-down strategy optimization:

  • Use epitope-tagged constructs (Flag, HA) to avoid antibody interference

  • Employ tandem affinity purification for higher specificity

  • Include appropriate detergent mixtures to solubilize membrane proteins while preserving interactions

Research with the related transporter SLC22A3 demonstrated successful identification of protein interactions using Flag-tagged constructs and mass spectrometry analysis. This approach identified ACTN4 as a key interacting partner, with functional consequences for protein activity . The interaction was confirmed through reverse co-IP experiments and through functional studies.

For SLC22A1, similar approaches could identify novel interaction partners that regulate its localization, stability, or transport activity. Special attention should be paid to cytoskeletal and scaffolding proteins that might facilitate membrane organization of transport complexes.

What is the role of SLC22A1 in disease models and how can antibodies help characterize pathological mechanisms?

SLC22A1 has emerging roles in various disease states, and antibody-based approaches provide valuable tools for investigating these pathological mechanisms:

Cancer research applications:

• Expression analysis in tumor versus normal tissues using IHC with standardized scoring

• Correlation of expression levels with clinical outcomes and treatment responses

• Investigation of regulatory mechanisms including epigenetic modifications and RNA editing

Metabolic disease applications:

• SLC22A1 has been linked to acylcarnitine transport and mitochondrial metabolism

• Antibody-based detection can assess expression changes in metabolic disease models

• Correlation studies between transporter expression and metabolite profiles provide mechanistic insights

Methodological approaches for disease studies:

• Tissue microarray analysis with validated SLC22A1 antibodies to assess expression across large sample cohorts

• Combined approaches linking expression data with functional assays:

  • Transport activity measurements in patient-derived samples

  • Correlation of expression levels with clinical parameters

  • Functional rescue experiments in model systems

Research has identified SLC22A1 as having roles in the transport of acylcarnitines, intermediate metabolites of mitochondrial oxidation, with potential implications for metabolic diseases . Additionally, studies with the related transporter SLC22A3 have demonstrated roles in cancer progression, with RNA editing leading to reduced expression and increased metastatic potential .

How can advanced imaging techniques enhance SLC22A1 localization and trafficking studies?

Advanced imaging approaches offer powerful tools for studying SLC22A1 localization, trafficking, and dynamic behavior in cellular systems:

Super-resolution microscopy techniques:

• Structured illumination microscopy (SIM) provides resolution enhancement (100-120 nm)

• Stimulated emission depletion (STED) microscopy offers resolution to approximately 50 nm

• Single-molecule localization methods (PALM/STORM) achieve 20-30 nm resolution

• These approaches can resolve SLC22A1 distribution within specialized membrane domains

Dynamic trafficking studies:

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measures lateral mobility within membranes

  • Quantifies immobile fractions indicating cytoskeletal tethering

  • Compares wild-type versus mutant mobility

• Pulse-chase experiments:

  • Antibody-based labeling of surface proteins

  • Tracking internalization and recycling kinetics

  • Quantifying endocytic trafficking rates

• Live-cell imaging with pH-sensitive fluorescent tags:

  • pHluorin-tagged constructs to monitor exocytosis events

  • Distinguishing between intracellular compartments based on pH

  • Real-time visualization of trafficking events

When analyzing trafficking data, quantitative approaches should include:

• Colocalization coefficients (Pearson's, Mander's) with organelle markers

• Object-based colocalization for punctate structures

• Trajectory analysis for vesicular transport

Studies with related transporters have demonstrated that protein interactions, such as those between SLC22A3 and ACTN4, can significantly affect localization patterns and influence cellular function . Similar advanced imaging approaches can reveal how SLC22A1 localization and trafficking are regulated under physiological and pathological conditions.