SLC25A14 Antibody

What is SLC25A14 and why is it a target of interest for researchers?

SLC25A14 (Solute Carrier Family 25 Member 14) is a mitochondrial carrier protein also known as UCP5 (Uncoupling Protein 5) or BMCP1 (Brain Mitochondrial Carrier Protein 1). It plays a crucial role in transporting metabolites across the inner mitochondrial membrane, contributing to energy production and cellular metabolism . This protein participates in the mitochondrial proton leak measured in brain mitochondria, separating oxidative phosphorylation from ATP synthesis with energy dissipated as heat .

SLC25A14 is of particular interest because dysregulation has been implicated in various metabolic disorders, making it a promising target for therapeutic intervention . It's widely expressed in many tissues with highest abundance in brain and testis, and alternative splicing results in multiple transcript variants . Understanding its function and regulation is essential for advancing research in metabolic diseases, mitochondrial dysfunction, and cancer metabolism.

What are the key characteristics of SLC25A14 antibodies currently available for research?

Most commercially available SLC25A14 antibodies share several common characteristics:

Most antibodies are validated for Western blot applications, with dilution ranges of 1:500-1:2000 being common for optimal results .

What is the relationship between SLC25A14 (UCP5) and other uncoupling proteins?

SLC25A14 (UCP5) belongs to the mitochondrial uncoupling protein (UCP) family, which are members of the larger family of mitochondrial anion carrier proteins (MACP) . Like other UCPs, SLC25A14:

• Facilitates the transfer of anions from the inner to the outer mitochondrial membrane

• Facilitates the return transfer of protons from the outer to the inner mitochondrial membrane

• Reduces mitochondrial membrane potential in mammalian cells

• Separates oxidative phosphorylation from ATP synthesis with energy dissipated as heat

While UCP1 is primarily expressed in brown adipose tissue and involved in thermogenesis, and UCP2/UCP3 have roles in various tissues, UCP5 (SLC25A14) shows highest expression in brain and is thought to have specialized functions in neuronal metabolism and protection against oxidative stress .

What applications are SLC25A14 antibodies best suited for, and what are the optimal conditions for each application?

Based on validated applications reported across multiple suppliers, SLC25A14 antibodies can be used in several experimental approaches:

Western Blot (WB):

• Recommended dilution: 1:500-1:2000

• Expected molecular weight: 36kDa

• Blocking buffer: 3% non-fat dry milk in TBST

• Secondary antibody: HRP-conjugated anti-rabbit IgG (1:10000)

• Positive controls: Mouse brain, rat brain tissue lysates

• Loading recommendation: 25μg protein per lane

Immunofluorescence (IF/ICC):

• Recommended dilution: 1:50-1:200

• Target visualization: Mitochondrial pattern (punctate cytoplasmic staining)

• Concentration for IF: 0.25-2 μg/mL

ELISA:

• Several antibodies are validated for ELISA applications

• Human SLC25A14 ELISA kits are available with sensitivity down to 1.0 pg/mL

How should researchers prepare samples for optimal detection of SLC25A14?

Sample preparation is critical for successful SLC25A14 detection:

For Western Blot:

• For tissue samples: Homogenize tissues in RIPA buffer containing protease inhibitors

• For cell lines: Lyse cells in RIPA or appropriate lysis buffer with protease inhibitors

• Centrifuge lysates at high speed (14,000g for 10 minutes) to remove debris

• Determine protein concentration using Bradford or BCA assay

• Mix samples with Laemmli buffer containing reducing agent

• Heat samples at 95°C for 5 minutes

• Load 25μg of protein per lane

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde (10-15 minutes)

• Permeabilize with 0.2% Triton X-100 in PBS (5-10 minutes)

• Block with 5% normal serum in PBS (1 hour)

• Incubate with primary antibody diluted in blocking buffer (overnight at 4°C)

• Use mitochondrial markers (like MitoTracker) for co-localization studies

Which species can be reliably studied using current SLC25A14 antibodies?

Different SLC25A14 antibodies have varying species reactivity profiles:

For studies in non-human species, researchers should carefully review the validation data specifically for that species before proceeding .

What are common issues encountered with SLC25A14 antibodies in Western blot applications and how can they be resolved?

When working with SLC25A14 antibodies in Western blot, researchers may encounter these common issues:

High Background:

• Increase blocking time (from 1 hour to overnight)

• Use 5% BSA instead of milk for blocking

• Increase washing steps (5-6 washes of 5-10 minutes each)

• Dilute primary antibody further (try 1:2000 instead of 1:500)

• Ensure secondary antibody is properly diluted (1:10000)

No Signal or Weak Signal:

• Use fresh positive controls (mouse brain or rat brain lysates)

• Increase protein loading (up to 50μg per lane)

• Increase primary antibody concentration (1:500)

• Extend primary antibody incubation (overnight at 4°C)

• Use fresh ECL substrate with appropriate sensitivity

Multiple Bands:

• SLC25A14 has alternative splice variants that may appear as multiple bands

• Isoform UCP5L runs at ~36kDa while UCP5S may appear at a slightly different size

• Increase transfer time for higher molecular weight proteins

• Use freshly prepared samples to avoid degradation products

How can researchers validate the specificity of SLC25A14 antibodies in their experimental system?

To ensure antibody specificity for SLC25A14 detection:

• Positive Controls:

  • Include known positive samples (mouse brain, rat brain)

  • Use cell lines with confirmed SLC25A14 expression

• Knockdown/Knockout Validation:

  • Compare signal between wildtype and SLC25A14 knockdown/knockout samples

  • siRNA treatment should reduce band intensity at 36kDa

• Peptide Competition:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced or eliminated with peptide competition

• Multiple Antibodies:

  • Use different antibodies targeting distinct SLC25A14 epitopes

  • Consistent results across antibodies increase confidence in specificity

• Subcellular Localization:

  • Confirm mitochondrial localization in IF/ICC experiments

  • Co-stain with established mitochondrial markers

What are the best storage and handling practices to maintain SLC25A14 antibody performance over time?

To preserve antibody functionality:

• Storage Conditions:

  • Store at -20°C in small aliquots to minimize freeze-thaw cycles

  • Most SLC25A14 antibodies contain 50% glycerol allowing short-term storage at 4°C

• Aliquoting Strategy:

  • Upon receipt, make 10-20μL aliquots in sterile microcentrifuge tubes

  • Return unused aliquots to -20°C immediately after use

• Handling During Experiments:

  • Thaw aliquots on ice

  • Avoid repeated freeze-thaw cycles (no more than 3-5 cycles)

  • Keep on ice when in use

  • Return to proper storage promptly after use

• Buffer Considerations:

  • Most SLC25A14 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Do not dilute stock antibody unless immediately using

  • Prepare working dilutions fresh on day of experiment

How can SLC25A14 antibodies be used to investigate mitochondrial dysfunction in metabolic diseases?

SLC25A14 plays a critical role in mitochondrial function, particularly in handling proton leakage. Researchers can employ these strategies:

• Comparative Expression Analysis:

  • Compare SLC25A14 expression levels between healthy and diseased tissues using Western blot

  • Quantify differences in expression levels in metabolic syndrome, obesity, or diabetes models

• Co-Immunoprecipitation Studies:

  • Use SLC25A14 antibodies to identify interaction partners in different metabolic states

  • Investigate how these interactions change during metabolic stress

• Tissue-Specific Expression:

  • Map expression patterns across different tissues in disease models using IHC/IF

  • Focus on brain regions where SLC25A14 is highly expressed

• Mitochondrial Isolation Studies:

  • Use antibodies to track SLC25A14 during mitochondrial isolation procedures

  • Compare functional parameters with SLC25A14 expression/localization

• Therapeutic Target Validation:

  • Monitor changes in SLC25A14 expression/localization in response to experimental therapies

  • Correlate with improvements in mitochondrial function and metabolic parameters

What approaches can be used to study the relationship between SLC25A14 and oxidative stress response?

As an uncoupling protein, SLC25A14 may protect against oxidative stress through regulation of mitochondrial membrane potential:

• Oxidative Stress Models:

  • Expose cells to oxidative stressors (H₂O₂, rotenone, hypoxia)

  • Monitor changes in SLC25A14 expression and localization using Western blot and IF

  • Correlate with ROS production and cell viability

• Brain-Specific Studies:

  • Investigate SLC25A14 expression changes in neurodegenerative disease models

  • Use brain tissue sections from models of Alzheimer's, Parkinson's, or stroke for IHC

  • Perform co-localization studies with oxidative stress markers

• Cellular Protection Mechanisms:

  • Manipulate SLC25A14 expression (overexpression/knockdown) and assess:

    • ROS production using fluorescent indicators

    • Mitochondrial membrane potential

    • Cellular resistance to oxidative stress

  • Validate findings using antibodies to track expression/localization changes

• Hypoxia Response Studies:

  • Examine SLC25A14 in hypoxic conditions (published studies show increased expression of UCP5 in high altitude hypoxia models)

  • Correlate with HIF-1α stabilization and other hypoxia-responsive proteins

How might researchers use SLC25A14 antibodies in the context of neurodegenerative disease research?

Given the enrichment of SLC25A14 in brain tissue, it represents an interesting target in neurodegenerative disease research:

• Comparative Analysis in Disease Models:

  • Use Western blot to quantify SLC25A14 expression changes in:

    • Alzheimer's disease models

    • Parkinson's disease models

    • Amyotrophic lateral sclerosis models

  • Compare results with mitochondrial function parameters

• Cellular Distribution in Neuropathology:

  • Perform IHC/IF on brain sections from patients with neurodegenerative diseases

  • Examine changes in cellular and subcellular distribution

  • Co-stain with neuronal/glial markers to identify cell type-specific changes

• Brain Region-Specific Analysis:

  • Compare SLC25A14 expression across different brain regions in disease models

  • Focus on regions particularly affected in specific neurodegenerative conditions

  • Use precise microdissection techniques followed by Western blot

• Mitochondrial Dynamics:

  • Investigate SLC25A14 localization during mitochondrial fission/fusion events

  • Examine potential correlations with mitochondrial transport defects in neuronal cells

  • Combine with live cell imaging using fluorescently tagged mitochondrial markers

• Therapeutic Response Monitoring:

  • Track SLC25A14 expression changes in response to neuroprotective agents

  • Correlate with improvements in mitochondrial function and neuronal survival

What are the molecular characteristics of various SLC25A14 isoforms and how can antibodies distinguish between them?

SLC25A14 exists in multiple isoforms due to alternative splicing:

• Known Isoforms:

  • Several transcript variants have been identified

  • Includes variants like UCP5L (long form) and UCP5S (short form)

  • A pseudogene of SLC25A14 has been identified on chromosome 4

• Epitope Selection for Isoform Specificity:

  • Available antibodies recognize different regions of SLC25A14:

    • Amino acids 25-200 of human SLC25A14 (NP_001269124.1)

    • Amino acids 51-100 in some antibodies

    • Amino acids 55-111 in others

    • Some use specific peptide sequences like "AAVIVSGHQKSTTVSHEMSGLNWKP"

• Detection of Specific Isoforms:

  • Select antibodies based on epitope location relative to splice junctions

  • Use recombinant isoform standards to confirm specificity

  • In Western blot, observe band patterns characteristic of each isoform

  • The main form typically appears at ~36kDa

What controls should be implemented when using SLC25A14 antibodies for quantitative research?

For quantitative applications such as expression level comparisons:

• Loading Controls:

  • Include mitochondrial markers (VDAC, COX IV) to normalize for mitochondrial content

  • Use established housekeeping proteins (β-actin, GAPDH) for whole cell lysates

  • Consider using total protein normalization methods (Ponceau S, REVERT stain)

• Standard Curves:

  • For ELISA applications, generate standard curves using recombinant SLC25A14

  • Ensure range covers expected physiological concentrations (detection limits reported down to 1.0 pg/mL)

• Reference Samples:

  • Include common reference samples across all experimental sets

  • Use mouse brain or rat brain lysates as positive controls

  • Consider commercial standardized lysates for inter-laboratory comparisons

• Technical Replicates:

  • Run at least 3 technical replicates for quantitative Western blots

  • Include multiple biological replicates to account for natural variation

  • Present data with appropriate statistical analysis

• Antibody Validation Controls:

  • Include antibody specificity controls in each experiment

  • Consider using knockdown/knockout samples alongside wildtype

  • For dual antibody approaches, confirm signal overlap from different epitope-targeting antibodies

What are the specialized applications of SLC25A14 antibodies beyond standard detection methods?

Beyond conventional applications, researchers can utilize SLC25A14 antibodies in specialized techniques:

• Proximity Ligation Assay (PLA):

  • Detect protein-protein interactions between SLC25A14 and other mitochondrial proteins

  • Investigate spatial relationships within mitochondrial membranes

  • Requires antibodies from different host species or directly conjugated antibodies

• Immunoelectron Microscopy:

  • Visualize precise submitochondrial localization of SLC25A14

  • Use gold-conjugated secondary antibodies for detection

  • Confirm inner mitochondrial membrane localization at ultrastructural level

• Chromatin Immunoprecipitation (ChIP):

  • For studying transcriptional regulation of SLC25A14

  • Investigate binding of transcription factors to SLC25A14 promoter regions

  • Combine with sequencing (ChIP-seq) for genome-wide binding profiles

• Mass Spectrometry-Based Approaches:

  • Use antibodies for immunoprecipitation followed by mass spectrometry

  • Identify post-translational modifications on SLC25A14

  • Discover novel interaction partners in different cellular contexts

• Super-Resolution Microscopy:

  • Apply optimized immunofluorescence protocols with super-resolution techniques

  • Study detailed mitochondrial distribution patterns of SLC25A14

  • Examine co-localization with other mitochondrial proteins at nanoscale resolution