SLC25A23 Antibody, Biotin conjugated

What is SLC25A23 and what cellular functions does it perform?

SLC25A23 (Solute Carrier Family 25 Member 23) is a 467 amino acid mitochondrial inner membrane protein that functions primarily as a calcium-dependent mitochondrial solute carrier. It plays a vital role in shuttling metabolites, nucleotides, and cofactors across the mitochondrial inner membrane . Specifically, SLC25A23 operates as an ATP-Mg/P(i) exchanger, regulating the transport of magnesium-bound ATP in exchange for phosphate, which facilitates the net uptake or efflux of adenine nucleotides into or from mitochondria . This regulatory function is essential for maintaining mitochondrial energy metabolism and cellular homeostasis. The protein is predominantly expressed in brain, skeletal muscle, and pancreas, with lower expression levels detected in other tissues .

What is the structural organization of the SLC25A23 protein?

SLC25A23 contains several important structural domains that contribute to its functionality:

• Three EF-hand domains which likely participate in calcium sensing

• Three Solcar repeats which are critical for the protein's carrier function

• Four alternatively spliced isoforms have been identified, suggesting tissue-specific functional adaptations

The gene encoding SLC25A23 is located on human chromosome 19p13.3, and the protein is also known by several aliases including APC2, MCSC2, MGC2615, and SCaMC-3 . The structural organization of SLC25A23 enables its specialized function as a mitochondrial carrier protein that responds to calcium signaling.

How do biotin-conjugated antibodies differ from unconjugated antibodies in experimental applications?

Biotin-conjugated antibodies offer significant advantages over unconjugated variants in certain experimental applications:

Biotin-conjugated SLC25A23 antibodies, such as those targeting amino acids 1-188, are particularly valuable in ELISA applications where sensitivity and signal amplification are critical .

What are the optimal storage conditions for maintaining biotin-conjugated SLC25A23 antibody activity?

To maintain optimal activity of biotin-conjugated SLC25A23 antibodies:

• Store at -20°C for up to one year from the date of receipt

• Avoid repeated freeze-thaw cycles as these can degrade antibody quality and diminish activity

• Consider aliquoting the antibody upon receipt to minimize freeze-thaw cycles

• When working with the antibody, maintain cold chain conditions by keeping it on ice

• If brief storage is needed during experiments, the antibody can be kept at 4°C for 1-2 weeks, though this may result in some loss of activity

The antibody is typically formulated in a stabilizing buffer containing glycerol (≈50%), which helps prevent damage during freezing. For biotin-conjugated antibodies, protection from light is also recommended to prevent photobleaching of the biotin moiety.

What dilution ranges are recommended for biotin-conjugated SLC25A23 antibodies in various applications?

While specific dilution ranges for biotin-conjugated SLC25A23 antibodies aren't explicitly provided in the search results, we can make evidence-based recommendations based on related antibodies:

For all applications, preliminary titration experiments are strongly recommended to determine the optimal working dilution for your specific experimental conditions. The optimal dilution will depend on factors including the abundance of the target protein, the sample type, and the detection system used.

How can I optimize ELISA protocols when using biotin-conjugated SLC25A23 antibodies?

Optimizing ELISA protocols with biotin-conjugated SLC25A23 antibodies requires careful consideration of several parameters:

• Blocking agent selection:

  • Use 1-5% BSA in PBS or 5% non-fat dry milk in TBST

  • Consider adding 0.05% Tween-20 to reduce background

• Incubation conditions:

  • Primary antibody (biotin-conjugated anti-SLC25A23): Incubate at 4°C overnight or 1-2 hours at room temperature

  • Detection reagent (streptavidin-HRP/AP): Typically 30-60 minutes at room temperature

• Signal development:

  • For HRP conjugates: TMB substrate provides excellent sensitivity

  • For AP conjugates: pNPP substrate offers stable signal development

  • Monitor reaction kinetics to determine optimal stopping time

• Washing steps:

  • Perform 3-5 washes with PBST (PBS + 0.05% Tween-20) between each step

  • Thorough washing is critical to minimize background while maintaining specific signal

• Controls to include:

  • Positive control: Lysates from tissues with known high SLC25A23 expression (brain, skeletal muscle, pancreas)

  • Negative control: Omit primary antibody

  • Background control: Omit both primary and secondary detection reagents

When developing an ELISA for detecting endogenous SLC25A23, consider using recombinant SLC25A23 protein as a standard for quantitative analysis.

What are common causes of high background when using biotin-conjugated SLC25A23 antibodies, and how can they be addressed?

High background is a common challenge when working with biotin-conjugated antibodies. Here are the most frequent causes and solutions:

For tissues particularly rich in endogenous biotin (such as brain, kidney, and liver), consider using alternative detection methods or specialized biotin-blocking steps prior to applying the biotin-conjugated anti-SLC25A23 antibody.

How can I validate the specificity of SLC25A23 antibody binding in my experimental system?

Validating antibody specificity is crucial for generating reliable research data. For SLC25A23 antibodies, consider these validation approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (if available)

  • Reduced or eliminated signal indicates specificity

• Genetic validation:

  • Test in SLC25A23 knockout/knockdown systems

  • Compare with wild-type to confirm specificity

• Cross-species reactivity testing:

  • The antibody should detect SLC25A23 in species specified by the manufacturer (human, mouse)

  • Species-specific size variations may occur due to post-translational modifications

• Multiple antibody comparison:

  • Use antibodies targeting different epitopes of SLC25A23

  • Correlating results indicates higher likelihood of specificity

• Mass spectrometry validation:

  • Immunoprecipitate SLC25A23 and confirm identity by mass spectrometry

  • Particularly useful for confirming the identity of bands detected in Western blot

For SLC25A23 specifically, validation in tissues with known differential expression (high in brain, skeletal muscle, and pancreas; lower in other tissues) provides additional confidence in antibody specificity.

What is the expected band pattern for SLC25A23 in Western blot analysis?

When performing Western blot analysis with SLC25A23 antibodies, researchers should expect the following patterns:

• Primary band: Approximately 47-50 kDa corresponding to the full-length SLC25A23 protein (467 amino acids)

• Additional bands: May detect alternative splice isoforms, as SLC25A23 exists in four alternatively spliced forms

• Post-translational modifications: Potential detection of bands at higher molecular weights due to phosphorylation, ubiquitination, or other modifications

Tissue-specific patterns may be observed, with strongest signals expected in brain, skeletal muscle, and pancreatic tissues where SLC25A23 is predominantly expressed . When using reducing conditions, ensure complete denaturation of the protein to avoid artifactual bands resulting from incomplete reduction of disulfide bonds.

How can SLC25A23 antibodies be used to investigate mitochondrial calcium homeostasis mechanisms?

SLC25A23 plays a critical role in mitochondrial calcium homeostasis, making antibodies against this protein valuable tools for investigating these mechanisms:

• Co-immunoprecipitation studies:

  • Biotin-conjugated SLC25A23 antibodies can be used to pull down protein complexes

  • Identify novel interaction partners involved in calcium sensing and transport

  • Use streptavidin-coated beads for efficient pull-down of biotin-antibody complexes

• Calcium perturbation experiments:

  • Monitor SLC25A23 localization and expression under varying calcium conditions

  • Correlate SLC25A23 activity with mitochondrial calcium uptake measurements

  • Investigate how calcium modulates the ATP-Mg/P(i) exchange function

• Super-resolution microscopy:

  • Use biotin-conjugated antibodies with fluorescent streptavidin conjugates

  • Map the precise localization of SLC25A23 within the mitochondrial inner membrane

  • Analyze co-localization with other mitochondrial calcium transport proteins

• Functional measurements:

  • Combine antibody-based detection of SLC25A23 with measurements of:

    • Mitochondrial membrane potential

    • ATP production

    • Calcium flux across mitochondrial membranes

These approaches can help elucidate how SLC25A23, with its EF-hand domains, acts as a calcium sensor to regulate adenine nucleotide transport across the mitochondrial inner membrane in response to cellular calcium signals.

What methodological approaches can be used to study the interaction between SLC25A23 and other mitochondrial proteins?

Investigating protein interactions involving SLC25A23 requires sophisticated methodological approaches:

• Proximity ligation assay (PLA):

  • Combine biotin-conjugated SLC25A23 antibody with antibodies against potential interaction partners

  • Visualize interactions in situ with subcellular resolution

  • Quantify interaction dynamics under different physiological conditions

• FRET/BRET analysis:

  • Express SLC25A23 fused to donor fluorophore

  • Express potential interaction partners fused to acceptor fluorophore

  • Measure energy transfer as indication of protein proximity

• Cross-linking mass spectrometry (XL-MS):

  • Use chemical cross-linkers to stabilize transient interactions

  • Immunoprecipitate SLC25A23 complexes using biotin-conjugated antibodies

  • Identify cross-linked peptides by mass spectrometry to map interaction interfaces

• Split-reporter complementation assays:

  • Fuse SLC25A23 and potential partners to complementary fragments of reporters (luciferase, fluorescent proteins)

  • Reconstitution of reporter activity indicates protein interaction

• Mitochondrial sub-fractionation:

  • Isolate distinct mitochondrial compartments (outer membrane, intermembrane space, inner membrane, matrix)

  • Use biotin-conjugated SLC25A23 antibodies to track distribution

  • Identify co-fractionating proteins as potential interaction partners

These methods can reveal how SLC25A23 interacts with components of the mitochondrial calcium uniporter complex and other mitochondrial transport systems to integrate calcium signaling with metabolite transport.

How can SLC25A23 antibodies be used to investigate pathological conditions associated with mitochondrial dysfunction?

SLC25A23 antibodies provide valuable tools for investigating the role of this protein in various pathological conditions associated with mitochondrial dysfunction:

• Neurodegenerative diseases:

  • Analyze SLC25A23 expression and localization in brain tissues from neurodegenerative disease models

  • Correlate with markers of mitochondrial function and calcium homeostasis

  • Investigate potential alterations in SLC25A23-dependent ATP-Mg/Pi exchange

• Metabolic disorders:

  • Examine SLC25A23 expression in tissues from metabolic disease models

  • Analyze potential post-translational modifications in pathological states

  • Correlate findings with measurements of mitochondrial bioenergetics

• Aging-related pathologies:

  • Compare SLC25A23 expression and function across different age groups

  • Investigate relationship between SLC25A23 activity and markers of mitochondrial aging

  • Analyze tissue-specific changes in expression patterns

• Ischemia-reperfusion injury:

  • Monitor SLC25A23 levels during ischemia and subsequent reperfusion

  • Correlate with calcium overload and ATP depletion measurements

  • Investigate potential protective interventions targeting SLC25A23 function

Methodologically, a combination of immunohistochemistry, Western blotting, and functional assays can be employed using anti-SLC25A23 antibodies to comprehensively characterize the involvement of this carrier protein in pathological processes linked to mitochondrial dysfunction and calcium dysregulation.

What factors should be considered when choosing between different SLC25A23 antibodies for specific research applications?

When selecting an SLC25A23 antibody for your research, consider these critical factors:

For mitochondrial proteins like SLC25A23, epitope accessibility can be particularly challenging due to the protein's membrane localization. Antibodies targeting exposed regions typically perform better in applications where the protein maintains its native conformation (immunofluorescence, flow cytometry).

How does the choice of immunogen affect antibody performance in different applications?

The immunogen used to generate an SLC25A23 antibody significantly impacts its performance across applications:

• Peptide immunogens (e.g., synthetic peptides):

  • Advantages: High specificity for the targeted epitope, good for linear epitopes

  • Limitations: May not recognize native protein conformation

  • Best applications: Western blot, ELISA with denatured samples

  • Example: Antibodies generated against synthesized peptides derived from human SLC25A23

• Recombinant protein fragments:

  • Advantages: Recognition of multiple epitopes, better representation of protein folding

  • Limitations: Potential cross-reactivity with related proteins

  • Best applications: Immunoprecipitation, flow cytometry, native protein detection

  • Example: Antibodies generated against recombinant human SLC25A23 protein (AA 1-188)

• Full-length protein immunogens:

  • Advantages: Most comprehensive epitope coverage, best for conformational epitopes

  • Limitations: Challenging to produce due to transmembrane domains

  • Best applications: Most versatile across multiple techniques

When selecting an antibody, match the immunogen type to your application needs. For example, if studying native protein-protein interactions, prioritize antibodies raised against larger recombinant fragments or full-length proteins that are more likely to recognize the native conformation.

How might advances in antibody technology enhance SLC25A23 research in the coming years?

Emerging antibody technologies hold promise for advancing SLC25A23 research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows access to restricted epitopes in the mitochondrial inner membrane

  • Potential for improved penetration into mitochondrial compartments

  • Enhanced stability under varying experimental conditions

• Bifunctional antibodies:

  • Combine SLC25A23 targeting with proximity-based labeling (APEX, BioID)

  • Enable identification of transient interaction partners in native mitochondrial environment

  • Facilitate spatiotemporal tracking of SLC25A23 dynamics

• Antibody-based biosensors:

  • Development of conformation-sensitive antibodies that respond to SLC25A23 activity states

  • Integration with fluorescent reporters to monitor real-time changes in protein function

  • Potential for live-cell imaging of SLC25A23 activity in response to calcium fluctuations

• Engineered antibody fragments:

  • Creation of smaller Fab and scFv derivatives with enhanced mitochondrial penetration

  • Development of intrabodies that can track SLC25A23 in living cells

  • Expansion of species cross-reactivity for comparative studies

These technological advances could transform our ability to study SLC25A23's role in mitochondrial calcium homeostasis and adenine nucleotide transport, potentially revealing new therapeutic targets for mitochondrial disorders.

What experimental approaches could help elucidate the role of SLC25A23 in integrating calcium signaling with mitochondrial metabolism?

Understanding how SLC25A23 integrates calcium signaling with mitochondrial metabolism requires innovative experimental approaches:

• Optogenetic calcium modulation:

  • Use light-activated calcium actuators to precisely control calcium transients

  • Monitor SLC25A23-dependent ATP transport using fluorescent ATP analogs

  • Correlate with mitochondrial metabolic parameters in real-time

• Cryo-electron microscopy:

  • Determine high-resolution structures of SLC25A23 in different conformational states

  • Visualize calcium-binding to EF-hand domains and resulting structural changes

  • Map the transport pathway for ATP-Mg and phosphate

• Metabolic flux analysis:

  • Combine stable isotope labeling with antibody-based SLC25A23 quantification

  • Correlate SLC25A23 expression/activity with metabolic pathway utilization

  • Measure the impact of SLC25A23 modulation on mitochondrial substrate preference

• Single-molecule imaging:

  • Use biotin-conjugated antibodies with quantum dot-streptavidin for single-molecule tracking

  • Analyze SLC25A23 mobility and clustering in response to calcium signals

  • Correlate with functional measurements of ATP transport

• Tissue-specific conditional knockout models:

  • Generate tissue-specific SLC25A23 knockout animals

  • Analyze tissue-specific metabolic consequences using antibody-based techniques

  • Investigate compensatory mechanisms involving other SLC25 family members

These approaches would provide complementary insights into how SLC25A23's calcium-sensing capability through its EF-hand domains modulates its transport function to adjust mitochondrial metabolism according to cellular calcium signaling.