SLC25A20 Antibody
Description
Introduction to SLC25A20 Protein
SLC25A20, also known as carnitine/acylcarnitine translocase (CACT), functions as a specialized mitochondrial-membrane-carrier protein. This crucial protein facilitates the transport of acylcarnitines into the mitochondrial matrix for oxidation . In humans, the canonical SLC25A20 protein consists of 301 amino acid residues with a molecular mass of approximately 32.9 kDa . The protein's subcellular localization is specifically within the mitochondria, where it plays an essential role in energy metabolism .
SLC25A20 mediates the electroneutral exchange of acylcarnitines (O-acyl-(R)-carnitine or L-acylcarnitine) of varying chain lengths with free carnitine ((R)-carnitine or L-carnitine) across the mitochondrial inner membrane through a ping-pong mechanism . This transport function is fundamental to fatty acid metabolism, as it enables long-chain fatty acids to enter the mitochondria for β-oxidation. The protein demonstrates notable expression in multiple tissues, with particularly high levels in metabolically active organs such as the duodenum and liver .
Biological Significance of SLC25A20
Recent research has revealed unexpected roles for SLC25A20 beyond its canonical metabolic functions. A study published in Nature's Cell Death & Disease journal identified SLC25A20 as a tumor suppressor in hepatocellular carcinoma (HCC) . The protein was found to suppress HCC growth and metastasis through multiple mechanisms, including inhibition of G1-S cell transition, suppression of epithelial-to-mesenchymal transition (EMT), and induction of cellular apoptosis .
Global genetic impairment of SLC25A20 significantly disrupts energy metabolism and has been linked to an inherited lethal syndrome in humans . This underscores the protein's critical importance in maintaining cellular metabolic homeostasis. Additionally, integrated proteogenomic analysis has identified SLC25A20 as one of the top three prognostic biomarkers in HCC, highlighting its potential clinical relevance .
SLC25A20 Antibodies: Types and Characteristics
SLC25A20 antibodies are immunological reagents specifically designed to recognize and bind to the SLC25A20 protein or its specific epitopes. These antibodies are available in various formats, each with distinct properties suitable for different experimental applications.
Classification by Clonality
SLC25A20 antibodies are available in both polyclonal and monoclonal formulations :
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Polyclonal Antibodies: These are derived from multiple B-cell lineages and recognize different epitopes on the SLC25A20 protein. Examples include ABIN7433891 and ABIN1536906, which are rabbit polyclonal antibodies .
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Monoclonal Antibodies: These are produced by a single B-cell clone and target a specific epitope. An example is the mouse monoclonal M2 unconjugated antibody (ABIN560147) .
Classification by Host Species
SLC25A20 antibodies are produced in different host animals, with the most common being:
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Rabbit: Most SLC25A20 antibodies are rabbit-derived, such as ABIN6738092 and ABIN6742017 .
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Mouse: Some monoclonal antibodies are produced in mice, like the M2 clone .
Classification by Target Epitope
Different SLC25A20 antibodies target various regions or epitopes of the protein:
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N-terminal Region: Antibodies targeting amino acids 1-211 or 1-301 .
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Middle Region: Antibodies targeting amino acids 143-192 or 216-265 .
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C-terminal Region: Antibodies targeting amino acids 235-264 .
Table 1: Examples of SLC25A20 Antibodies with Different Target Epitopes
| Catalog Number | Target Epitope | Host | Clonality | Application |
|---|---|---|---|---|
| ABIN6738092 | AA 216-265 | Rabbit | Polyclonal | WB |
| ABIN7433891 | AA 1-211 | Rabbit | Polyclonal | WB, IHC, IP, ICC |
| ABIN1536906 | AA 235-264 (C-Term) | Rabbit | Polyclonal | WB |
| ABIN560147 | AA 1-301 | Mouse | Monoclonal | ELISA, IF |
| ABIN6742017 | AA 143-192 | Rabbit | Polyclonal | WB |
Applications of SLC25A20 Antibodies
SLC25A20 antibodies serve as valuable tools in various experimental techniques, enabling researchers to investigate the expression, localization, and function of the SLC25A20 protein.
Western Blotting (WB)
Western blotting represents the most common application for SLC25A20 antibodies . This technique allows for the detection and semi-quantitative analysis of SLC25A20 protein expression in tissue or cellular lysates. Many commercially available SLC25A20 antibodies, such as ABIN6738092, have been validated specifically for WB applications .
Immunohistochemistry (IHC) and Immunocytochemistry (ICC)
These techniques enable the visualization of SLC25A20 localization within tissues (IHC) or cultured cells (ICC). Some antibodies, like ABIN7433891, have been validated for both applications . These methods provide valuable insights into the spatial distribution of SLC25A20 within cellular compartments and across different tissue types.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA allows for quantitative measurement of SLC25A20 protein levels in biological samples. Antibodies such as ABIN560147 are specifically validated for ELISA applications . This technique is particularly useful for high-throughput analysis of multiple samples.
Immunofluorescence (IF)
Immunofluorescence techniques, using antibodies like ABIN560147, enable high-resolution imaging of SLC25A20 localization . This approach is particularly valuable for co-localization studies with other mitochondrial markers.
Immunoprecipitation (IP)
Some SLC25A20 antibodies, such as ABIN7433891, are validated for immunoprecipitation applications . This technique allows for the isolation of SLC25A20 protein complexes, facilitating the study of protein-protein interactions involving SLC25A20.
Species Reactivity and Cross-Reactivity
SLC25A20 antibodies demonstrate varying degrees of species reactivity, with some antibodies recognizing the protein across multiple species due to the high conservation of SLC25A20 among vertebrates.
Multi-Species Reactivity
Certain SLC25A20 antibodies, particularly those targeting highly conserved epitopes, demonstrate broad species reactivity. For example, the antibody ABIN6738092 has been validated to recognize SLC25A20 from multiple species including human, mouse, rat, cow, sheep, dog, guinea pig, horse, rabbit, hamster, monkey, bat, chicken, and pig . This broad reactivity stems from the high sequence conservation in the target epitope (AA 216-265) across these species.
Predicted Cross-Reactivity Based on Sequence Analysis
BLAST analysis has been employed to predict potential cross-reactivity of SLC25A20 antibodies with orthologs from different species. For instance, the immunogen used for ABIN6738092 shows 100% sequence identity with SLC25A20 from human, chimpanzee, gorilla, orangutan, gibbon, monkey, galago, marmoset, mouse, rat, sheep, hamster, elephant, dog, bovine, bat, rabbit, horse, pig, opossum, guinea pig, turkey, chicken, and lizard . Additionally, it demonstrates 92% sequence identity with SLC25A20 from zebra finch, platypus, Xenopus, salmon, smelt, stickleback, and zebrafish .
Table 2: Species Reactivity of Selected SLC25A20 Antibodies
| Antibody Catalog | Mammalian Reactivity | Non-Mammalian Reactivity | Validation Method |
|---|---|---|---|
| ABIN6738092 | Human, Mouse, Rat, Cow, Sheep, Dog, Guinea Pig, Horse, Rabbit, Hamster, Monkey, Bat | Chicken, Pig | WB |
| ABIN7433891 | Rat | - | WB, IHC, IP, ICC |
| ABIN1536906 | Mouse | - | WB |
| ABIN560147 | Human | - | ELISA, IF |
| ABIN6742017 | Human, Mouse, Rat, Cow, Sheep, Hamster, Monkey | - | WB |
SLC25A20 in Disease Research
Research employing SLC25A20 antibodies has revealed significant associations between SLC25A20 dysregulation and various pathological conditions, particularly in cancer.
Role in Hepatocellular Carcinoma
A comprehensive study published in 2021 utilized SLC25A20 antibodies to investigate the protein's expression and function in hepatocellular carcinoma (HCC) . The research revealed that SLC25A20 expression is frequently down-regulated in HCC cells, primarily due to upregulation of miR-132-3p . This down-regulation was found to be associated with poor prognosis in HCC patients.
Mechanistic studies demonstrated that SLC25A20 suppresses HCC growth and metastasis through multiple pathways:
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Inhibition of G1-S cell transition
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Suppression of epithelial-to-mesenchymal transition (EMT)
At the molecular level, SLC25A20 down-regulation was shown to promote HCC growth and metastasis through suppression of fatty acid oxidation . These findings position SLC25A20 as a critical tumor suppressor in HCC pathogenesis and suggest its potential as a novel prognostic factor and therapeutic target.
Metabolic Disorders
Global genetic impairment of SLC25A20 has been linked to severe metabolic disorders . SLC25A20 deficiency leads to disrupted fatty acid metabolism, as the transport of long-chain acylcarnitines into mitochondria for β-oxidation is compromised. This metabolic dysfunction can result in an inherited lethal syndrome in humans, underscoring the critical importance of SLC25A20 in cellular energetics .
Selection Criteria for SLC25A20 Antibodies
When selecting an SLC25A20 antibody for a specific research application, several factors should be considered:
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Application Compatibility: Ensure the antibody has been validated for the intended application (WB, IHC, ELISA, IF, etc.) .
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Species Reactivity: Confirm the antibody recognizes SLC25A20 from the species under investigation .
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Epitope Specificity: Select an antibody targeting an appropriate epitope based on the research question .
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Clonality: Choose between polyclonal (broader epitope recognition) or monoclonal (specific epitope) based on the experimental requirements .
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Validation Data: Examine the validation data provided by the supplier to assess antibody performance .
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Citations: Consider antibodies with published citations as they have demonstrated utility in peer-reviewed research .
Table 3: Selection Guide for SLC25A20 Antibodies Based on Application
| Application | Recommended Characteristics | Example Catalog Numbers |
|---|---|---|
| Western Blot | Validated for WB, appropriate species reactivity | ABIN6738092, ABIN7433891, ABIN1536906 |
| IHC/ICC | Validated for IHC/ICC, low background | ABIN7433891 |
| ELISA | High specificity, validated for ELISA | ABIN560147 |
| Immunofluorescence | High specificity, low background | ABIN560147 |
| Immunoprecipitation | High affinity, validated for IP | ABIN7433891 |
Product Specs
Target Background
- Two novel cases of CACT Deficiency (CACTD) have been identified in mainland China. In addition to the known founder mutation c.199-10T>G, a novel c.1A>G mutation was discovered. Individuals with CACTD carrying the c.199-10T>G mutation typically exhibit a severe clinical phenotype. Early diagnosis and appropriate treatment are essential in this life-threatening disorder. PMID: 29137068
- Downregulation of microRNAs hsa-miR-124-3p, hsa-miR-129-5p, and hsa-miR-378 was observed to increase both expression and activity of CPT1A, CACT, and CrAT in malignant prostate cells. PMID: 28671672
- The antiport transport mechanism, characteristic of mitochondrial carriers like CAC, is driven by the coupling of uniport reactions in opposing directions, mediated by specific amino acid residues. PMID: 25325845
- Novel mutations in the CACT gene have been identified, including C.576G>A, c.106-2a>t, and c.516T>C. PMID: 24088670
- CPT2 and CACT are crucial for mitochondrial acylcarnitine formation and their export to the extracellular fluids in mitochondrial fatty acid beta-oxidation disorders. PMID: 23322164
- A comprehensive analysis of human SLC25A* genes, including functional information, is provided. PMID: 23266187
- Steroid Receptor Coactivator-3 (SRC-3) plays a critical role in long chain fatty acid metabolism by directly regulating the expression of the carnitine/acyl-carnitine translocase (CACT) gene. PMID: 22560224
- The transcriptional regulation of the CAC proximal promoter involves FOXA and Sp1 sites in HepG2 cells, and only the Sp1 site in HEK293 and SK-N-SH cells. PMID: 21130740
- Treatment of CACT deficiency involved a carnitine diet and the administration of medium-chain triglycerides. PMID: 15057979
- The clinical, biochemical, and molecular characteristics of six CACT-deficient patients from Italy, Spain, and North America are described, highlighting significant clinical heterogeneity. Five novel and three previously reported mutations were identified. PMID: 15365988
- Modulation of CACT expression impacts CPT 1 activity, while the biological effects of acetyl-carnitine are not directly related to a generic supply of energy compounds but rather to the anaplerotic properties of the molecule. PMID: 15515015
- The clinical outcome of two siblings with CACT deficiency is reported. PMID: 17508264
- Functional analysis of mutations in residues Pro278 and Ala279 in A. nidulans, alongside kinetic data in reconstituted liposomes, suggests a primary structural role for these amino acids. PMID: 18307102
- Peroxisome Proliferator-Activated Receptor alpha (PPARalpha) regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element. PMID: 19748481
Q&A
What is SLC25A20 and why is it significant in metabolic research?
SLC25A20, also known as carnitine/acylcarnitine translocase (CACT), is a mitochondrial-membrane-carrier protein involved in the transport of acylcarnitines into the mitochondrial matrix for oxidation . The protein plays a critical role in fatty acid metabolism and energy production. It mediates the electroneutral exchange of acylcarnitines (ranging from O-acetyl-(R)-carnitine to long-chain O-acyl-(R)-carnitines) with free carnitine across the mitochondrial inner membrane through a ping-pong mechanism . Dysregulation of SLC25A20 has been linked to various metabolic disorders, making it an important target for research in bioenergetics and mitochondrial diseases .
Experimental Applications
Optimal antibody dilutions vary by application technique:
| Application | Recommended Dilution |
|---|---|
| Western Blot (WB) | 1:1000-1:6000 |
| Immunohistochemistry (IHC) | 1:50-1:500 |
| Immunofluorescence (IF)/ICC | 1:10-1:100 |
It is recommended that researchers titrate the antibody in each testing system to obtain optimal results as sample dependence can affect performance .
Specimen Reactivity and Detection
For optimal antigen retrieval in IHC applications, the suggested protocol is:
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Primary method: Use TE buffer pH 9.0
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Alternative method: Use citrate buffer pH 6.0
These retrieval methods have been validated for SLC25A20 detection in tissues such as mouse heart, human heart, and human hepatocirrhosis tissue .
How is SLC25A20 expression altered in cancer, and what methodologies can detect these changes?
Research has shown that SLC25A20 expression is frequently down-regulated in hepatocellular carcinoma (HCC) cells, primarily due to the up-regulation of miR-132-3p . This down-regulation is associated with poor prognosis in HCC patients.
To investigate SLC25A20 expression in cancer:
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Use validated SLC25A20 antibodies for Western blot analysis to quantify protein levels
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Compare expression between normal and cancerous tissues
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Perform immunohistochemistry to visualize expression patterns within tissue sections
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Consider complementary techniques such as qRT-PCR to analyze transcript levels
SLC25A20 has been found to function as a tumor suppressor in HCC by:
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Suppressing G1-S cell transition
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Inhibiting epithelial-to-mesenchymal transition (EMT)
In vivo studies demonstrated that SLC25A20 overexpression significantly reduced tumor growth rates and metastatic potential in mouse models .
What functional assays can be used to study SLC25A20 activity?
To study SLC25A20 functional activity, researchers can employ several approaches:
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Fatty acid oxidation assays: Measure changes in fatty acid oxidation rates when SLC25A20 expression or activity is altered .
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Acylcarnitine transport assays: Monitor the transport of acylcarnitines across the mitochondrial membrane using labeled substrates .
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Inhibitor studies: Use known inhibitors such as ingenol mebutate (IngMeb) or omeprazole to block SLC25A20-mediated transport and observe metabolic effects .
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Metabolite profiling: Analyze changes in acylcarnitine levels using mass spectrometry when SLC25A20 is inhibited or its expression is altered. Elevated long-chain acylcarnitines can indicate impaired SLC25A20 function .
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Overexpression and knockdown studies: Examine the phenotypic consequences of modulating SLC25A20 levels in cellular models .
How can chemical proteomics approaches be used to study SLC25A20 interactions?
Chemical proteomics has successfully identified SLC25A20 as a functional target of drugs such as ingenol mebutate (IngMeb). The methodology involves:
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Using photoreactive, clickable analogues (e.g., Ing-DAyne) of the compound of interest
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Treating cells with the analogue, followed by UV cross-linking
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Cell lysis and conjugation to reporter tags (e.g., TAMRA-N3)
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Analysis by gel-based profiling or mass spectrometry
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Validation through competition experiments with the original compound
This approach revealed that IngMeb and its derivative IngDsx inhibit SLC25A20 activity in human cells, elevating long-chain acylcarnitines and disrupting fatty acid oxidation .
What protocols are recommended for validating SLC25A20 antibody specificity?
To validate SLC25A20 antibody specificity:
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Overexpression controls: Transfect cells with SLC25A20 expression vectors and compare antibody labeling between transfected and mock-transfected cells .
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Competition assays: Pre-incubate antibodies with the immunizing peptide before application to Western blot or IHC to demonstrate specific blocking.
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Knockdown/knockout validation: Use siRNA, shRNA, or CRISPR to reduce or eliminate SLC25A20 expression and confirm corresponding reduction in antibody signal.
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Multiple antibody comparison: Use antibodies targeting different epitopes of SLC25A20 and compare detection patterns.
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Cross-species reactivity: Test the antibody against predicted reactive species based on sequence homology. SLC25A20 antibodies have shown cross-reactivity with multiple species due to high sequence conservation .
What are the optimal storage conditions for SLC25A20 antibodies?
For long-term preservation of antibody activity:
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Store at -20°C for optimal stability
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Antibodies are typically stable for one year after shipment when properly stored
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Aliquoting is unnecessary for -20°C storage
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The standard storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3
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Some smaller package sizes (20μl) may contain 0.1% BSA as a stabilizer
What control samples are recommended for SLC25A20 Western blot experiments?
Based on validated positive controls in the literature, the following samples are recommended for Western blot experiments:
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Saos-2 cell lysate
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Mouse heart tissue lysate
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Human testis tissue lysate
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Mouse skeletal muscle tissue lysate
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Mouse testis tissue lysate
When validating a new antibody, it's advisable to include both positive control tissues with known high expression of SLC25A20 and negative controls where the protein has been knocked down.
How can researchers address non-specific binding when using SLC25A20 antibodies?
To reduce non-specific binding:
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Optimize blocking conditions (try 5% non-fat milk or 3-5% BSA in TBST)
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Increase washing steps (use at least 3 x 5 minutes with TBST)
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Titrate antibody concentration within the recommended range (1:1000-1:6000 for WB)
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For IHC applications, consider antigen retrieval optimization between TE buffer pH 9.0 and citrate buffer pH 6.0
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When persistent non-specificity occurs, consider testing alternative antibodies targeting different epitopes
What methodological adaptations are needed for detecting SLC25A20 in different experimental systems?
Depending on your experimental system, consider these methodological adaptations:
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For cancer tissue studies: Due to potential downregulation in cancer tissues such as HCC, more sensitive detection methods may be required. Consider using signal amplification systems or more concentrated antibody dilutions (e.g., 1:50-1:100 for IHC) .
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For co-localization studies: When performing immunofluorescence to determine mitochondrial localization, co-staining with established mitochondrial markers (e.g., TOMM20, COX IV) is recommended.
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For inhibitor studies: When using SLC25A20 inhibitors such as IngMeb or omeprazole, monitor acylcarnitine levels and fatty acid oxidation as functional readouts of inhibition .
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For detecting protein-protein interactions: Consider using proximity ligation assays (PLA) or co-immunoprecipitation studies with SLC25A20 antibodies to identify interaction partners.
