RDH13 Antibody

What is RDH13 and what is its cellular function?

RDH13 (retinol dehydrogenase 13) is a mitochondrial short-chain dehydrogenase/reductase that catalyzes the reduction and oxidation of retinoids. Unlike other members of the RDH11-like group that are microsomal proteins, RDH13 is distinctively localized to mitochondria . Specifically, it is situated on the outer side of the inner mitochondrial membrane, facing the intermembrane space .

RDH13 functions primarily as an NADP+-dependent retinaldehyde reductase with significantly higher catalytic efficiency in reduction than oxidation reactions . The enzyme has a clear preference for NADPH over NADH as a cofactor and contributes to retinoic acid production . Additionally, RDH13 plays a crucial protective role against oxidative stress in mitochondria .

What is the tissue distribution pattern of RDH13?

RDH13 exhibits a wide tissue distribution with varying expression levels:

In the eye specifically, RDH13 is expressed in the photoreceptor inner segment layer, with no apparent expression in the retinal pigment epithelium (RPE), photoreceptor outer segments, or outer nuclear layer .

What types of RDH13 antibodies are available for research?

Several types of RDH13 antibodies are available:

• Based on host species:

  • Rabbit polyclonal antibodies

  • Mouse monoclonal antibodies

• Based on clonality:

  • Polyclonal antibodies: Offer broader epitope recognition

  • Monoclonal antibodies: Provide consistent specificity (e.g., E-2 clone)

• Based on conjugation:

  • Unconjugated primary antibodies

  • Conjugated formats (HRP, PE, FITC, Alexa Fluor®)

Each antibody type has specific applications where it performs optimally. When selecting an antibody, consider your experimental design, target tissue, and detection method.

What validation methods should be used to confirm RDH13 antibody specificity?

When validating RDH13 antibodies, consider the following methodological approaches:

• Knockout/knockdown validation:

  • Compare immunostaining in wild-type vs. RDH13 knockout tissues/cells

  • RDH13 expression was successfully abolished in knockout mice as determined by immunofluorescence and western blot

• Western blot analysis:

  • Verify a single band at the expected molecular weight (~36 kDa)

  • Compare band patterns across multiple cell/tissue types

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide to demonstrate specific binding

• Cross-reactivity testing:

  • Test reactivity with related proteins (e.g., RDH11, RDH12, RDH14)

  • Perform immunoprecipitation followed by mass spectrometry

What are the optimal conditions for using RDH13 antibodies in Western blotting?

For successful Western blotting with RDH13 antibodies:

• Sample preparation:

  • Use fresh tissue/cell lysates when possible

  • Include reducing agents in your buffer system (RDH13 requires reducing conditions to maintain activity)

  • Standard RIPA or NP-40 based lysis buffers are suitable

• Recommended dilutions:

  • Starting dilution of 1:1000 for Western blotting is typically effective

  • Optimize based on your specific antibody and sample type

• Detection considerations:

  • Expected molecular weight: 36 kDa (331 amino acids)

  • Use appropriate positive control tissues (kidney, heart, or lung show highest expression)

• Troubleshooting tips:

  • If signal is weak, ensure your sample contains mitochondrial fractions

  • RDH13 is more labile than related microsomal proteins, handle samples carefully

How can RDH13 antibodies be used for studying subcellular localization?

To accurately determine RDH13's subcellular localization:

• Immunofluorescence protocols:

  • Fixation: PFA-fixed samples are suitable for RDH13 detection

  • Permeabilization: Triton X-100 permeabilization allows access to mitochondrial targets

  • Co-localization: Use established mitochondrial markers (e.g., porin, an integral protein of the outer mitochondrial membrane)

• Cell fractionation approach:

  • Use sucrose gradient ultracentrifugation to separate cellular components

  • RDH13 typically appears in fractions containing mitochondria (corresponding to fractions where porin is detected)

  • Verify fractions using organelle-specific markers (e.g., lamin for nuclei, golgin for Golgi, calnexin for ER)

• Submitochondrial localization:

  • Protease protection assays can determine exact localization

  • RDH13 is protected from trypsin in intact mitochondria but degraded in mitoplasts (mitochondria with outer membrane removed)

How can RDH13 antibodies be used to investigate retinal protection mechanisms?

RDH13 plays a significant role in protecting the retina from light-induced damage. When studying these mechanisms:

• Light-damage experimental models:

  • Compare wild-type and RDH13 knockout mice exposed to intense light (e.g., 3,000 lx for 48h)

  • Analyze structural changes using H&E staining and functional changes via electroretinography

  • Measure thickness of outer-plus-inner-segment and outer nuclear layer

• Mitochondrial damage assessment:

  • Use RDH13 antibodies to examine localization before and after light exposure

  • Combine with TEM to observe mitochondrial morphological changes

  • In RDH13-/- mice, mitochondria in photoreceptor inner segments become distinctly swollen with disrupted cristae after light exposure

• Apoptotic pathway investigation:

  • Analyze cytochrome c release from mitochondria to cytosol

  • Evaluate expression of apoptosis-related proteins (CytC, Apaf-1, caspase 3, P65, Bax)

  • RDH13 deficiency leads to increased expression of these apoptotic markers following light exposure

What are the technical considerations when using RDH13 antibodies for studying mitochondrial oxidative stress?

When investigating RDH13's role in mitochondrial oxidative stress:

• Experimental design:

  • Compare oxidative stress markers between wild-type and RDH13-deficient systems

  • Consider using oxidative stress inducers (e.g., H₂O₂, paraquat) with varying intensities and durations

• Sample preparation:

  • Isolate intact mitochondria to preserve RDH13's natural environment

  • Maintain reducing conditions throughout sample handling

• Analytical approaches:

  • Co-immunoprecipitation to identify RDH13-interacting proteins involved in stress response

  • Combine with metabolic profiling of retinoids to correlate enzymatic activity with stress protection

• Data interpretation:

  • Consider that RDH13's protective effects may be tissue-dependent

  • The localization at the entrance to the mitochondrial matrix may serve as a barrier against oxidative damage

How can RDH13 antibodies be used in studying retinal diseases like Leber congenital amaurosis?

For investigating RDH13's role in retinal pathologies:

• Patient sample analysis:

  • Compare RDH13 expression patterns in control vs. disease tissues

  • Examine potential mutations in RDH13 using genetic analysis alongside protein expression studies

  • Defects in RDH13 have been linked to Leber congenital amaurosis (LCA) type 3, a common genetic cause of congenital visual impairment

• Functional validation:

  • Use antibodies to detect altered RDH13 protein expression or localization

  • Analyze correlation between RDH13 status and disease severity

• Therapeutic development monitoring:

  • Evaluate restoration of RDH13 expression or function after experimental therapies

  • Use antibodies to track changes in RDH13 and related pathway components

What are common issues when working with RDH13 antibodies and how can they be resolved?

How should RDH13 antibodies be stored and handled to maintain optimal performance?

For maximum antibody stability and performance:

• Storage conditions:

  • Store at -20°C for most antibodies, though some require -80°C storage

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

• Working dilutions:

  • Prepare fresh working dilutions for each experiment

  • For long-term storage of diluted antibodies, add stabilizing proteins (BSA)

• Special considerations for RDH13 antibodies:

  • RDH13 is more labile than related proteins; handle with care

  • Consider adding reducing agents to maintain activity during storage and use

How might RDH13 antibodies contribute to research on mitochondrial-targeted therapeutics?

The unique mitochondrial localization of RDH13 makes it valuable for developing targeted therapeutics:

• Biomarker applications:

  • Use RDH13 antibodies to evaluate mitochondrial integrity in response to experimental drugs

  • Monitor changes in RDH13 expression/localization as indicators of mitochondrial stress

• Drug delivery validation:

  • Verify targeting efficiency of mitochondria-directed compounds

  • Assess potential interactions between therapeutic agents and RDH13

• Future research opportunities:

  • Investigate whether RDH13's protective effects can be enhanced or mimicked

  • Explore if the RDH13 mitochondrial targeting sequence could be utilized for delivering therapeutic molecules to mitochondria

What new methodologies are being developed for studying RDH13's interactions with other proteins?

Advanced techniques for investigating RDH13's protein-protein interactions include:

• Proximity labeling approaches:

  • BioID or APEX2 fusions with RDH13 to identify neighboring proteins in the mitochondrial membrane

  • Help identify potential interaction partners involved in retinoid metabolism or mitochondrial protection

• Live-cell imaging techniques:

  • FRET-based assays to detect interactions between RDH13 and other mitochondrial proteins

  • Super-resolution microscopy to precisely localize RDH13 within mitochondrial subcompartments

• Proteomic strategies:

  • Quantitative proteomics comparing wild-type and RDH13-deficient mitochondria

  • Crosslinking mass spectrometry to identify direct binding partners