SLC25A5 Antibody

What is SLC25A5 and why is it significant in biological research?

SLC25A5, also known as ANT2 (Adenine Nucleotide Translocator 2), is a critical mitochondrial membrane protein involved in cellular energy metabolism. It belongs to the family of adenine nucleotide translocators, with four identified isoforms: ANT1 (encoded by SLC25A4) is highly expressed in different tissues; ANT2 (encoded by SLC25A5) is predominantly expressed in proliferating, regenerating, or undifferentiated cells; ANT3 is ubiquitously expressed; and ANT4 is mainly expressed in testis and germ cells . Research shows that SLC25A5 has significant prognostic value in cancer patients, as it attenuates cell proliferation, upregulates programmed cell death-related signatures, and inhibits the MAPK signaling pathway .

What applications are SLC25A5 antibodies suitable for?

SLC25A5 antibodies are validated for multiple research applications, with variations depending on the specific antibody:

These applications enable researchers to detect and quantify SLC25A5 protein expression, examine its intracellular localization, and study its interactions with other proteins.

What species reactivity is expected with commercially available SLC25A5 antibodies?

Different SLC25A5 antibodies exhibit varying species reactivity profiles:

This information is crucial for researchers to select appropriate antibodies based on their experimental model organism.

How should SLC25A5 antibodies be stored to maintain optimal activity?

For optimal preservation of antibody activity, storage recommendations typically include:

• Store at -20°C

• For fluorescently conjugated antibodies (like CL750-83104), avoid exposure to light

• Antibodies are generally stable for one year after shipment when properly stored

• For some antibodies like CL750-83104, aliquoting is unnecessary for -20°C storage

The storage buffer for CL750-83104 consists of PBS with 50% Glycerol, 0.05% Proclin300, and 0.5% BSA at pH 7.3 , which helps maintain antibody stability.

How can I validate the specificity of SLC25A5 antibodies given the sequence similarity with other ANT isoforms?

Validating antibody specificity is crucial since some antibodies recognize multiple ANT isoforms. For instance, CL750-83104 recognizes both ANT1 and ANT2 . Recommended validation approaches include:

• Western blot with isoform controls: Compare signals from tissues with differential ANT isoform expression (e.g., proliferating cells predominantly express ANT2/SLC25A5)

• RNA interference validation: Perform siRNA knockdown specific to SLC25A5 and observe decreased signal intensity

• Recombinant protein standards: Use purified ANT1 and ANT2 proteins as positive controls to assess cross-reactivity

• Immunoprecipitation-mass spectrometry: Identify all proteins captured by the antibody to confirm specificity and detect potential cross-reactivity

• Peptide competition assay: Pre-incubate antibody with the immunogenic peptide to block specific binding

What are optimal sample preparation protocols for detecting SLC25A5 in mitochondrial fractions?

As a mitochondrial membrane protein, SLC25A5 requires specific sample preparation techniques:

• Mitochondrial isolation:

  • Use differential centrifugation or commercial mitochondrial isolation kits

  • Maintain samples at 4°C throughout to preserve protein integrity

  • For tissue samples, mechanical homogenization in isotonic buffer is recommended

• Membrane protein extraction:

  • Use mild detergents (0.5-1% digitonin, CHAPS, or n-dodecyl β-D-maltoside)

  • Incubate on ice for 30-60 minutes with gentle agitation

  • Centrifuge at high speed (≥20,000 × g) to remove insoluble material

• Sample buffer considerations:

  • Add reducing agents (β-mercaptoethanol or DTT)

  • For membrane proteins like SLC25A5, avoid boiling samples (heat to 37-50°C instead)

  • Include protease inhibitors to prevent degradation

• Loading controls:

  • Use other mitochondrial proteins (VDAC, COX IV) as loading controls rather than cytosolic proteins

How does SLC25A5 expression correlate with clinical outcomes in cancer research?

Research indicates significant correlations between SLC25A5 expression and cancer outcomes:

These findings suggest SLC25A5 may function as a tumor suppressor in colon cancer, with potential as a prognostic biomarker.

What methodological approaches are recommended for distinguishing between ANT1 and ANT2 when using antibodies that recognize both isoforms?

When using antibodies like CL750-83104 that recognize both ANT1 and ANT2 , implementing these strategies can help distinguish between isoforms:

• Exploit differential expression patterns:

  • ANT1 (SLC25A4) is highly expressed in differentiated tissues

  • ANT2 (SLC25A5) predominates in proliferating, regenerating, or undifferentiated cells

  • Select experimental systems where one isoform naturally predominates

• Isoform-specific genetic manipulation:

  • Implement isoform-specific knockdown using validated siRNAs

  • Compare signal reduction to determine the contribution of each isoform

• Complementary techniques:

  • Supplement antibody-based detection with qRT-PCR using isoform-specific primers

  • Combine with mass spectrometry to identify peptides unique to each isoform

• Sequential immunoprecipitation:

  • Deplete one isoform first using a specific antibody

  • Then detect the remaining isoform with the dual-specificity antibody

What are the optimal conditions for flow cytometry detection of intracellular SLC25A5?

For detecting SLC25A5 by flow cytometry, consider these methodological optimizations:

• Fixation and permeabilization:

  • Use 2-4% paraformaldehyde for fixation (10-15 minutes at room temperature)

  • For mitochondrial proteins, thorough permeabilization is critical

  • Test commercial permeabilization reagents (saponin-based for membrane proteins show good results)

• Antibody parameters:

  • For CL750-83104, use 0.40 μg per 10^6 cells in a 100 μl suspension

  • Incubate 30-60 minutes at room temperature in the dark

  • Include 0.1-0.5% BSA in staining buffer to reduce background

• Essential controls:

  • Unstained cells for autofluorescence baseline

  • Isotype control at matching concentration

  • Fluorescence minus one (FMO) controls

  • Positive control (cell type with known high SLC25A5 expression)

• Mitochondrial co-staining:

  • Consider co-staining with MitoTracker dyes (applied pre-fixation)

  • This confirms mitochondrial localization and aids in proper gating

• Data analysis:

  • Gate on single, viable cells

  • For CL750-83104 (conjugated to CoraLite® Plus 750), use appropriate filters (excitation/emission maxima: 755 nm/780 nm)

How can I optimize Western blot protocols specifically for SLC25A5 detection?

Optimizing Western blot protocols for SLC25A5 detection requires attention to several parameters:

• Sample preparation refinements:

  • For total cell lysates, use RIPA buffer with 1% NP-40 or Triton X-100

  • For mitochondrial enrichment, consider sucrose gradient ultracentrifugation

  • Use freshly prepared samples whenever possible

• Gel selection:

  • 10-12% polyacrylamide gels provide optimal resolution for the 32-33 kDa SLC25A5 protein

  • Consider gradient gels (4-15%) for improved separation

• Transfer conditions:

  • For membrane proteins, semi-dry transfer at lower voltage for longer times often yields better results

  • Use PVDF membranes for stronger protein binding

• Blocking optimization:

  • Test both BSA and non-fat dry milk-based blocking solutions

  • 5% blocking agent in TBST for 1 hour at room temperature is typically effective

• Antibody incubation:

  • Primary antibody dilutions:

    • CL750-83104: 1:500-1:1000

    • E2B9D Rabbit mAb: 1:1000

  • Overnight incubation at 4°C generally provides the best signal-to-noise ratio

  • Include 0.05% sodium azide in primary antibody solution for preservation

• Detection system:

  • For standard Western blot, HRP-conjugated secondary antibodies work well

  • For increased sensitivity, consider fluorescent secondary antibodies or enhanced chemiluminescence substrates

What experimental considerations are important when studying SLC25A5 in relation to mitochondrial dysfunction?

When investigating SLC25A5 in mitochondrial dysfunction contexts:

• Functional assays:

  • Measure ATP/ADP exchange rates in isolated mitochondria

  • Assess mitochondrial membrane potential changes in relation to SLC25A5 expression

  • Monitor oxygen consumption rates as an indicator of mitochondrial function

• Context-dependent expression:

  • Compare SLC25A5 expression levels between normal and disease states

  • Assess changes in SLC25A5 localization during cellular stress

  • Investigate post-translational modifications that may alter function

• Interaction studies:

  • Identify binding partners using co-immunoprecipitation with E2B9D antibody

  • Investigate how these interactions change under pathological conditions

  • Consider proximity ligation assays to confirm in situ protein interactions

• Genetic approaches:

  • Implement CRISPR-Cas9 editing to create loss-of-function models

  • Use overexpression systems to rescue phenotypes

  • Generate point mutations that mimic disease-associated variants

• Pathway analysis:

  • Investigate connections to the MAPK signaling pathway as identified in cancer research

  • Assess relationships between SLC25A5 and programmed cell death pathways

  • Examine metabolic consequences of altered SLC25A5 function

How should researchers interpret contradictory results when analyzing SLC25A5 expression across different experimental platforms?

When faced with contradictory SLC25A5 expression data:

• Consider technical variables:

  • Antibody specificity (some detect both ANT1/ANT2)

  • Sample preparation differences (total lysate vs. mitochondrial enrichment)

  • Detection method sensitivity (chemiluminescence vs. fluorescence)

• Biological variables:

  • Cell/tissue-specific expression patterns of SLC25A5

  • Proliferation status affects SLC25A5 expression (higher in proliferating cells)

  • Disease state can alter normal expression patterns

• Validation approaches:

  • Confirm protein expression with multiple antibodies targeting different epitopes

  • Correlate protein data with mRNA expression (qRT-PCR)

  • Supplement with functional assays to assess biological relevance

• Data integration:

  • Compare with public database information (e.g., bioinformatic analyses showed both elevated and decreased expression depending on comparison datasets)

  • Consider statistical approaches to reconcile conflicting datasets

  • Evaluate methodological strengths and limitations of each experimental platform

What quality control measures should be implemented when using SLC25A5 antibodies for quantitative analyses?

For rigorous quantitative analyses with SLC25A5 antibodies:

• Antibody validation:

  • Confirm specificity using positive and negative controls

  • Perform peptide competition assays to verify epitope specificity

  • Validate lot-to-lot consistency with standard samples

• Standard curve generation:

  • Use recombinant SLC25A5 protein at known concentrations

  • Create calibration curves for each experimental batch

  • Ensure the detection system remains in the linear range

• Normalization strategies:

  • For Western blot, normalize to mitochondrial markers rather than whole-cell proteins

  • For flow cytometry, consider normalization to mitochondrial mass

  • Use housekeeping genes specific to mitochondria for qRT-PCR normalization

• Technical replicates:

  • Perform at least three technical replicates per biological sample

  • Calculate coefficient of variation to assess measurement precision

  • Establish acceptance criteria for replicate variability

• Statistical analysis:

  • Implement appropriate statistical tests based on data distribution

  • Consider power analysis to determine required sample sizes

  • Report effect sizes alongside statistical significance

How can SLC25A5 antibodies be employed in investigating cancer therapeutic responses?

SLC25A5 antibodies offer valuable tools for studying cancer therapeutic responses:

• Expression monitoring:

  • Track changes in SLC25A5 levels before and after treatment

  • Correlate with clinical responses and survival outcomes

  • Assess whether expression changes precede clinical response

• Predictive biomarker potential:

  • Stratify patients based on baseline SLC25A5 expression

  • Determine if expression levels predict response to specific therapies

  • Correlate with other established biomarkers

• Mechanism investigations:

  • Examine how therapeutics affect SLC25A5's inhibitory effect on the MAPK pathway

  • Investigate whether treatments alter the negative correlation between CD8 and SLC25A5

  • Assess changes in programmed cell death signatures in relation to SLC25A5 expression

• Resistance mechanisms:

  • Compare SLC25A5 expression in sensitive versus resistant tumors

  • Determine if alterations in SLC25A5 contribute to therapy resistance

  • Evaluate potential as a target to overcome resistance

• Combination therapy rationale:

  • Investigate whether modulating SLC25A5 enhances effects of standard therapies

  • Explore synergistic interactions between SLC25A5-targeted approaches and conventional treatments

  • Assess impact on tumor microenvironment and immune infiltration