SLC25A12 Antibody, HRP conjugated

What is SLC25A12 and why is it important in research?

SLC25A12 is a mitochondrial electrogenic aspartate/glutamate antiporter that plays a critical role in the malate-aspartate shuttle. It favors the efflux of aspartate and entry of glutamate and proton within the mitochondria . SLC25A12 has gained significance in research due to its association with autism spectrum disorders (ASDs) and its crucial role in brain development and myelination . Understanding SLC25A12 function provides insights into mitochondrial energy metabolism and neurodevelopmental processes.

What types of SLC25A12 antibodies are commonly used in research?

Research commonly employs different types of SLC25A12 antibodies including rabbit recombinant monoclonal antibodies like EPR16294 and rabbit polyclonal antibodies . These antibodies vary in specificity and sensitivity depending on the application. Monoclonal antibodies offer high specificity to particular epitopes, while polyclonal antibodies recognize multiple epitopes of the SLC25A12 protein, potentially increasing detection sensitivity at the cost of potential cross-reactivity.

How do HRP detection systems work with SLC25A12 antibodies?

While primary SLC25A12 antibodies are typically unconjugated, they work effectively with HRP-conjugated secondary antibodies in detection systems. The primary antibody binds specifically to SLC25A12 in the sample, and the HRP-conjugated secondary antibody (typically anti-rabbit IgG-HRP) binds to the primary antibody . When exposed to a substrate, the HRP enzyme catalyzes a reaction producing a detectable signal, either colorimetric or chemiluminescent, allowing visualization of SLC25A12 in Western blots or immunohistochemistry applications.

What are the optimal conditions for Western blot detection of SLC25A12 using HRP systems?

For optimal Western blot detection of SLC25A12 using HRP systems, use the following protocol:

• Load 10-20 μg of tissue or cell lysate per lane

• Use recommended antibody dilutions (typically 1:1000-1:4000 for primary SLC25A12 antibody)

• Use appropriate HRP-conjugated secondary antibodies at 1:1000-1:5000 dilution

• Block with 5% non-fat dry milk in TBST (5% NFDM/TBST)

• For optimal results, short exposure times (3-10 seconds) are often sufficient for SLC25A12 detection with HRP-based chemiluminescence

The predicted band size for SLC25A12 is 74 kDa, though it is typically observed at approximately 75 kDa or 63 kDa depending on the antibody and sample type .

How should SLC25A12 antibodies be used for immunohistochemistry with HRP detection?

For immunohistochemistry applications with HRP detection:

• Perform antigen retrieval preferably with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0 may be used)

• Use SLC25A12 antibody at dilutions between 1:50-1:500

• Apply appropriate HRP-conjugated secondary antibody

• Develop using DAB (3,3'-diaminobenzidine) substrate for visualization

• For brain tissue, pay special attention to myelinated regions such as corpus callosum, anterior commissure, and internal capsule, where SLC25A12 plays significant functional roles

This method is particularly useful for studying SLC25A12 expression in tissues affected by neurodevelopmental disorders, with significant staining observed in human brain tissue, cancer tissues, and rodent models .

What sample types work best for SLC25A12 antibody detection?

SLC25A12 antibodies have demonstrated reactivity with multiple sample types:

Brain tissue samples are particularly important for SLC25A12 research due to its role in neurodevelopment and myelination processes . High expression has also been observed in heart tissue, reflecting the protein's role in mitochondrial energy metabolism .

How can background issues be minimized when using SLC25A12 antibodies with HRP detection?

To minimize background in SLC25A12 detection with HRP systems:

• Optimize blocking conditions using 5% non-fat dry milk in TBST (as demonstrated in effective protocols)

• Titrate antibody concentrations; start with manufacturer recommendations (1:1000-1:4000 for primary antibodies)

• Reduce exposure time for chemiluminescent detection; successful detection has been achieved with as little as 3-10 seconds exposure

• Include appropriate negative controls, such as isotype control antibodies (e.g., Rabbit IgG monoclonal [EPR25A] - Isotype Control ab172730)

• For immunohistochemistry, enhance washing steps between antibody incubations and consider adjusting antigen retrieval conditions

These optimization steps can significantly improve signal-to-noise ratio for specific detection of SLC25A12.

What are common issues when detecting SLC25A12 in different tissue types?

Common challenges when detecting SLC25A12 across tissue types include:

• Variation in observed molecular weight (74-75 kDa in most cases, but 63 kDa observed with some antibodies)

• Different expression levels across tissues (highest in brain, heart, and kidney)

• Antigen masking in fixed tissues requiring optimized antigen retrieval methods (preferably TE buffer pH 9.0)

• Potential cross-reactivity with other mitochondrial carriers in the SLC25 family

• Age-dependent expression differences, particularly relevant in developmental studies

To address these issues, validation in appropriate positive control tissues is essential, with brain, heart, and kidney tissue showing reliable SLC25A12 detection .

How can SLC25A12 antibodies be used to study neurodevelopmental disorders?

SLC25A12 antibodies can be effectively employed in neurodevelopmental disorder research through:

• Comparative expression analysis between normal and pathological tissues using Western blotting and immunohistochemistry

• Investigation of myelination defects by co-staining with myelin markers like MBP (myelin basic protein) and PLP (proteolipid protein)

• Analysis of neuron-oligodendrocyte interactions in slice cultures, where SLC25A12 function affects myelination processes

• Examination of neurofilamentous accumulations in neurons, which have been observed in SLC25A12 knockout models

• Correlation studies between SLC25A12 expression/function and autism spectrum disorder manifestations

Research has demonstrated that SLC25A12 disruption alters myelination and causes neurofilamentous accumulations, providing a model for studying autism spectrum disorders and related neurodevelopmental conditions .

What are the considerations for immunoprecipitation of SLC25A12 followed by HRP detection?

For successful immunoprecipitation of SLC25A12:

• Start with sufficient protein input (1mg of whole cell lysate has been demonstrated as effective)

• Use SLC25A12 antibody at approximately 1:40 dilution for immunoprecipitation

• Follow with Western blot detection using the same or different SLC25A12 antibody at 1:1000 dilution

• Use HRP-conjugated secondary antibodies specific to the non-reduced form of IgG at 1:1500 dilution

• Include appropriate controls, such as input lysate (10 μg) and isotype control antibody IP lanes

This approach has been successfully demonstrated for SLC25A12 immunoprecipitation from Jurkat cells with detection via Western blot using HRP-conjugated secondary antibodies .

How can SLC25A12 antibodies be used to investigate the malate-aspartate shuttle in mitochondrial research?

Advanced investigation of the malate-aspartate shuttle using SLC25A12 antibodies involves:

• Co-immunoprecipitation studies to identify protein interactions within the shuttle complex

• Subcellular fractionation with Western blot analysis to confirm mitochondrial localization and relative abundance

• Immunofluorescence microscopy with mitochondrial markers to study spatial distribution

• Investigation of expression changes under metabolic stress conditions (oxidative stress, glucose deprivation)

• Combination with functional assays measuring aspartate/glutamate exchange activity

This approach provides insights into how SLC25A12 functions within the malate-aspartate shuttle to support mitochondrial respiration, ATP production, and neuronal energy metabolism, all of which may be altered in neurodevelopmental disorders .

What methodologies combine SLC25A12 antibody detection with functional mitochondrial analyses?

Advanced methodologies combining SLC25A12 detection with functional analyses include:

• Correlative immunohistochemistry with respirometry measurements to link SLC25A12 expression to mitochondrial function

• Simultaneous assessment of SLC25A12 expression and NADH/NAD+ ratios to investigate shuttle activity

• Metabolomic profiling alongside SLC25A12 immunodetection to measure aspartate, glutamate, and N-acetyl aspartate (NAA) levels

• Rescue experiments using pyruvate supplementation in SLC25A12-deficient models with antibody verification of expression levels

• Multi-parametric analysis correlating SLC25A12 expression with myelination markers and energy metabolism indicators

Research has demonstrated that myelin deficits in SLC25A12 knockout models can be reversed by pyruvate administration, suggesting the critical role of this transporter in providing metabolic support for myelination through aspartate/NAA production and/or regulation of the NADH/NAD+ ratio .

What are the critical validation steps for SLC25A12 antibodies before experimental use?

Critical validation steps for SLC25A12 antibodies include:

• Western blot analysis with positive control tissues (brain, heart, kidney) to confirm the expected molecular weight (74-75 kDa or 63 kDa depending on the antibody)

• Negative controls using isotype-matched control antibodies to assess specificity

• Cross-validation with multiple antibodies targeting different epitopes of SLC25A12

• Testing in known SLC25A12 knockout or knockdown models

• Verification of reactivity in the intended experimental species (human, mouse, rat)

These validation steps ensure antibody specificity and reliability before proceeding with experimental applications, particularly important for studies of complex phenotypes associated with SLC25A12 dysfunction.

How do different fixation methods affect SLC25A12 epitope recognition in immunohistochemistry?

Different fixation methods can significantly impact SLC25A12 detection:

• Paraformaldehyde fixation (4%) has been successfully used for brain slice cultures and tissue sections

• For immunohistochemistry on paraffin sections, optimal antigen retrieval with TE buffer pH 9.0 is recommended (citrate buffer pH 6.0 as an alternative)

• Fixation time can affect epitope accessibility, with prolonged fixation potentially masking epitopes

• For immunofluorescence applications, shorter fixation times (10-20 minutes) may improve signal intensity

• Comparison of multiple fixation protocols may be necessary to optimize detection in specific tissue types

Optimization of fixation and antigen retrieval protocols is particularly important for detecting SLC25A12 in tissues with high lipid content like brain, where the protein's association with mitochondrial membranes can affect accessibility.