rdh10a Antibody

What is RDH10 and why is it significant in developmental and disease research?

RDH10, or retinol dehydrogenase 10, is a critical enzyme responsible for catalyzing the first oxidative step in retinoic acid (RA) biosynthesis, converting retinol (vitamin A) to retinaldehyde. This 341-amino acid protein (38.1 kDa in humans) belongs to the short-chain dehydrogenase/reductase (SDR) family and is primarily localized to the endoplasmic reticulum . RDH10 has been established as the primary enzyme responsible for embryonic vitamin A oxidation, making it essential for proper developmental processes regulated by retinoic acid signaling .

Recent research has expanded our understanding of RDH10's significance beyond development, with studies revealing its potential role in cancer biology. High expression of RDH10 has been observed in human gliomas, where expression levels correlate with tumor grade and patient survival outcomes . Mechanistically, RDH10 appears to promote glioma cell proliferation and survival by modulating the TWEAK-NF-κB signaling axis . These findings position RDH10 as a protein of interest in both developmental biology and cancer research contexts.

What tissue distribution pattern does RDH10 exhibit, and how does this inform antibody selection?

RDH10 demonstrates a specific but widely distributed expression pattern across multiple tissues. High expression levels have been documented in retina, kidney, liver, small intestine, placenta, lung, heart, and skeletal muscle . Within the eye, RDH10 is notably present in both retinal pigment epithelium (RPE) and Müller cells of the retina, though expression is typically higher in the RPE .

When selecting antibodies for RDH10 detection, this expression pattern necessitates careful consideration. For studies focusing on ocular tissues, researchers should select antibodies validated specifically in retinal tissues, as demonstrated in publications that have successfully detected RDH10 in mouse eyecups and bovine retinal samples . Interestingly, RDH10 expression levels vary between mouse strains, with higher expression observed in BALB/c versus C57Bl/6 mice . This strain-dependent variation highlights the importance of including appropriate positive controls when working with RDH10 antibodies across different genetic backgrounds.

For studies examining RDH10 in multiple tissues, researchers should select antibodies with broad cross-reactivity profiles, ideally validated in each tissue of interest. Given RDH10's high sequence conservation (99% amino acid identity among human, bovine, mouse, and rat orthologs) , many commercial antibodies offer cross-species reactivity, facilitating comparative studies.

What applications are RDH10 antibodies most commonly used for?

RDH10 antibodies are employed across multiple experimental applications, with varying optimization requirements for each technique. Based on available commercial offerings and published research, the most common applications include:

The selection of application should be guided by the specific research question. For protein quantification, Western blot and ELISA provide reliable approaches. For spatial distribution studies, IHC and IF offer superior visualization of tissue and cellular localization patterns. When studying RDH10's enzymatic function, combining immunodetection with activity assays—measuring the conversion of all-trans retinol to all-trans retinal with NADP as the preferred cofactor—provides comprehensive functional characterization .

How should I validate the specificity of RDH10 antibodies in my experimental system?

Validating antibody specificity is critical for generating reliable results with RDH10 antibodies. A comprehensive validation approach should include:

• Positive and negative tissue controls: Compare tissues with known high RDH10 expression (retina, liver) against tissues with minimal expression. BALB/c mouse eyecups provide excellent positive controls due to their high RDH10 expression relative to C57Bl/6 mice .

• Genetic knockdown verification: Use RDH10-shRNA treated cells as negative controls, as demonstrated in studies where lentiviral-mediated RDH10 knockdown significantly reduced both mRNA and protein detection . This approach confirms signal specificity by demonstrating signal reduction following target depletion.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific signal should be blocked or substantially reduced.

• Multiple antibody comparison: Utilize antibodies recognizing different epitopes of RDH10. Concordant results with independently raised antibodies strongly support specificity.

• Western blot molecular weight verification: Confirm detection of a single band at the expected molecular weight (~38.1 kDa for human RDH10) .

• Subcellular fractionation: RDH10 localizes to the microsomal/ER membrane fraction. Enrichment in this fraction compared to cytosolic fractions supports antibody specificity .

A successful validation example was demonstrated in retinal studies where RDH10 antibody specificity was confirmed by detecting differential expression between mouse strains and specific localization to RPE and Müller cells, consistent with mRNA expression data . For knockdown studies, researchers achieved >80% reduction in RDH10 protein detection following shRNA treatment, validating both the knockdown efficiency and antibody specificity .

What are the optimal sample preparation protocols for detecting RDH10 using immunohistochemistry?

Optimized sample preparation for RDH10 immunohistochemistry varies by tissue type, with retinal tissue requiring particularly careful handling due to its delicate structure. Based on published methodologies, the following protocol elements are recommended:

• Fixation:

  • For paraffin sections: 4% paraformaldehyde fixation for 24 hours at 4°C provides optimal antigen preservation

  • For frozen sections: Brief fixation (10-15 minutes) with 4% paraformaldehyde followed by cryoprotection in sucrose gradients (15% to 30%)

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • For retinal tissues, gentle retrieval conditions are preferred to preserve tissue morphology

• Blocking conditions:

  • 5-10% normal serum (matching secondary antibody host) with 0.1-0.3% Triton X-100 for permeabilization

  • Include 1% BSA to reduce non-specific binding

• Antibody incubation:

  • Primary antibody: Dilutions typically range from 1:100 to 1:500 based on antibody source

  • Overnight incubation at 4°C maximizes specific signal while minimizing background

  • Secondary antibody: 1-2 hour incubation at room temperature

• Controls:

  • Include sections with primary antibody omitted

  • For retinal studies, compare RPE and Müller cell staining patterns, as both should show specific RDH10 localization

Successful RDH10 immunodetection has been demonstrated in retinal sections, where antibodies specifically labeled Müller cells in addition to RPE, confirming RDH10's expression in multiple retinal cell types . When optimizing protocols, it's advisable to test multiple antibody dilutions and incubation times, as commercial RDH10 antibodies vary considerably in their optimal working conditions.

What are the best practices for using RDH10 antibodies in Western blot applications?

Effective Western blot detection of RDH10 requires attention to several key experimental parameters:

• Sample preparation:

  • Enrichment of membrane fractions improves detection sensitivity, as RDH10 is primarily localized to the ER membrane

  • Include protease inhibitors in lysis buffers to prevent degradation

  • Use mild detergents (0.5-1% NP-40 or Triton X-100) to solubilize membrane-bound RDH10

• Gel electrophoresis conditions:

  • 10-12% polyacrylamide gels provide optimal resolution around the 38 kDa range

  • Load 20-50 μg of total protein depending on RDH10 abundance in the sample

• Transfer parameters:

  • Semi-dry or wet transfer at 100V for 60-90 minutes using PVDF membranes

  • Methanol-containing transfer buffers may improve transfer efficiency

• Blocking and antibody incubation:

  • 5% non-fat milk in TBST is typically effective for blocking

  • Primary antibody dilutions generally range from 1:500 to 1:2000 (optimize for each antibody)

  • Overnight incubation at 4°C typically yields best results

• Detection considerations:

  • Both chemiluminescent and fluorescent secondary antibodies have been successfully used

  • Expected molecular weight of human RDH10 is 38.1 kDa

  • Rat RDH10 shows similar molecular weight, with 99% sequence identity to human RDH10

• Controls and normalization:

  • Include positive controls from tissues with known high RDH10 expression (RPE, liver)

  • Use housekeeping proteins (β-actin, GAPDH) for normalization, though membrane protein controls may be more appropriate given RDH10's localization

In published studies, RDH10 Western blots have successfully detected expression differences between control and knockdown glioma cell lines, with shRNA treatment reducing RDH10 protein levels by approximately 80% . This approach provides both validation of antibody specificity and quantifiable knockdown efficiency measurement.

How can RDH10 antibodies be used to investigate the role of retinoic acid signaling in development?

RDH10 antibodies provide powerful tools for investigating retinoic acid (RA) signaling during embryonic development, particularly given RDH10's essential role as the primary enzyme responsible for the first oxidative step in RA synthesis . Several sophisticated approaches can be implemented:

• Temporal-spatial expression mapping:

  • Immunohistochemistry with RDH10 antibodies can map expression patterns throughout developmental stages

  • Co-staining with markers of specific developmental processes can reveal correlations between RDH10 expression and morphogenetic events

  • This approach has revealed that RDH10 expression patterns align with tissues requiring RA signaling during development

• Functional studies in developmental models:

  • In Rdh10 trex mutant embryos, which carry mutations affecting RDH10 function, immunodetection can confirm protein expression despite functional deficiency

  • Combined with retinaldehyde supplementation experiments, this approach has demonstrated that RDH10 is the primary dehydrogenase responsible for embryonic vitamin A oxidation

• Subcellular localization studies:

  • High-resolution immunofluorescence microscopy can reveal RDH10's membrane association

  • This has important functional implications, as the membrane-bound compartmentalization prevents inhibition by cytosolic cellular retinol binding protein (RBP1)

  • This compartmentalization is critical for regulated RA synthesis during development

• Cross-species comparative approaches:

  • Given the high conservation of RDH10 across species (99% amino acid identity among mammals) , antibodies can be used for evolutionary studies

  • RDH10 orthologs have been reported in mouse, rat, bovine, frog, chimpanzee, and chicken , enabling cross-species developmental comparisons

By combining RDH10 antibody detection with functional assays measuring retinol dehydrogenase activity (conversion of all-trans retinol to retinaldehyde with NADP as cofactor), researchers can establish direct links between RDH10 expression, enzyme activity, and developmental outcomes .

What is the significance of RDH10 in cancer research, and how can antibodies facilitate these studies?

Recent investigations have revealed RDH10's unexpected role in cancer biology, particularly in gliomas, where it appears to influence tumor development and progression . RDH10 antibodies are instrumental in elucidating these mechanisms through several sophisticated approaches:

• Expression correlation studies:

  • Immunohistochemical analysis using RDH10 antibodies has demonstrated that RDH10 expression correlates with glioma grade and patient survival outcomes

  • Higher expression levels are associated with more aggressive tumors and poorer prognosis

• Functional studies using knockdown models:

  • Western blot analysis with RDH10 antibodies has confirmed efficient knockdown in lentivirus-mediated shRNA models

  • This approach has revealed that RDH10 silencing reduces glioma cell proliferation, survival, invasion capability, and cell cycle progression

• Pathway analysis:

  • Immunoblotting for RDH10 and associated signaling molecules has helped elucidate the TWEAK-NF-κB mechanistic axis

  • RDH10 knockdown has been shown to alter expression of multiple components in this pathway, including TNFRSF12A (Fn14), TNFSF12 (TWEAK), TRAF3, and IKBKB (IKK-β)

• In vivo tumor models:

  • Immunohistochemical analysis of xenograft tumors allows quantification of RDH10 expression in relation to tumor growth characteristics

  • RDH10 knockdown reduces glioma growth in nude mice, with antibody detection confirming maintained knockdown throughout the experiment

• Clinical correlation approaches:

  • RDH10 antibodies enable tissue microarray analysis of patient samples

  • This approach has established correlations between RDH10 expression and clinical parameters including tumor grade and patient survival

The application of RDH10 antibodies in cancer research has revealed an unexpected function distinct from its canonical role in retinoic acid biosynthesis. These studies suggest RDH10 may serve as both a potential biomarker and therapeutic target in gliomas, highlighting the value of antibody-based approaches in uncovering novel cancer biology mechanisms.

How do we optimize detection of RDH10 across different species for comparative studies?

Optimizing RDH10 antibody detection across species requires careful consideration of sequence conservation, epitope selection, and validation strategies:

• Sequence conservation analysis:

  • RDH10 demonstrates remarkable conservation, with 99% amino acid sequence identity among human, bovine, mouse, and rat orthologs

  • This high conservation facilitates cross-species application of many RDH10 antibodies

  • Rat RDH10 cDNA encodes a protein of 341 amino acids, identical in length to human RDH10

• Epitope-specific considerations:

  • Antibodies targeting the most conserved regions offer the best cross-species reactivity

  • C-terminal targeted antibodies (like ARP53217_P050) demonstrate particularly broad reactivity across human, mouse, rabbit, rat, bovine, dog, guinea pig, horse, and zebrafish samples

  • Antibodies targeting species-specific regions may be necessary for distinguishing orthologs in closely related species

• Cross-reactivity validation:

  • Western blot comparison of multiple species samples run on the same gel

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Side-by-side staining of tissues from different species to compare localization patterns

• Protocol optimization by species:

  • Fixation requirements may vary by species and tissue type

  • Antigen retrieval conditions often require species-specific adjustment

  • Antibody dilution optimization should be performed for each species individually

• Control selection strategies:

  • Use tissues with known high RDH10 expression (retina, liver) from each species

  • Include RDH10-knockdown samples when available as negative controls

  • For mouse studies, consider strain differences, as RDH10 expression is higher in BALB/c than C57Bl/6 mice

A particularly useful approach is to test multiple commercial antibodies raised against different epitopes. Several suppliers offer broadly reactive RDH10 antibodies suitable for cross-species studies, including products that demonstrate reactivity to human, mouse, and rat RDH10 simultaneously . For novel species applications, researchers should begin with Western blot validation before attempting more complex applications like immunohistochemistry.

Why might I observe multiple bands when using RDH10 antibodies in Western blot analysis?

The detection of multiple bands in RDH10 Western blots can result from several biological and technical factors that require systematic investigation:

• Post-translational modifications:

  • RDH10 may undergo glycosylation, phosphorylation, or other modifications that alter its electrophoretic mobility

  • These modifications can be tissue or condition-specific, resulting in additional bands

  • Treatment with deglycosylation enzymes or phosphatases can help identify modification-dependent bands

• Alternative splicing:

  • While the canonical human RDH10 protein is 341 amino acids (38.1 kDa) , alternative splicing variants may exist

  • Comparison with RNA-seq or RT-PCR data can help identify potential splice variants

• Proteolytic processing:

  • As a membrane-associated protein, RDH10 may undergo proteolytic processing during sample preparation

  • Inclusion of multiple protease inhibitors and maintaining cold temperatures during lysis can minimize this issue

• Cross-reactivity:

  • Antibodies may detect related family members within the short-chain dehydrogenase/reductase (SDR) family

  • Sequence alignment analysis of detected bands by mass spectrometry can identify potential cross-reactive proteins

  • Comparison with RDH10 knockdown samples can distinguish specific from non-specific bands

• Sample preparation issues:

  • Insufficient denaturation or reduction can result in aggregates or multimers

  • Ensure complete sample denaturation with adequate SDS and reducing agent concentrations

  • Heat samples at 95°C for 5 minutes before loading

• Degradation products:

  • Partial degradation can generate fragments detected by the antibody

  • Fresh sample preparation and storage at -80°C until use can minimize degradation

In published studies, successful RDH10 Western blots typically detect a predominant band at approximately 38 kDa . When troubleshooting multiple bands, systematic comparison with positive controls (tissues with high RDH10 expression like retina or liver) and negative controls (RDH10 knockdown samples or tissues with minimal expression) can help identify the specific RDH10 band.

How can I optimize immunohistochemical detection of RDH10 in tissues with low expression levels?

Detecting RDH10 in tissues with low expression presents several challenges requiring specialized approaches:

• Signal amplification strategies:

  • Tyramide signal amplification (TSA) can increase sensitivity 10-100 fold

  • Polymer-based detection systems with multiple secondary antibodies per primary antibody binding event

  • Avidin-biotin complex (ABC) method with enzymatic amplification

• Sample enrichment approaches:

  • For tissues with subcellular compartmentalization, membrane fraction enrichment before immunohistochemistry can enhance signal

  • Thinner sections (4-5 μm) may provide better signal-to-noise ratio than standard (7-10 μm) sections

• Antigen retrieval optimization:

  • Extended retrieval times or higher temperatures may expose additional epitopes

  • Testing multiple retrieval buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0) to identify optimal conditions

  • Enzymatic retrieval with proteinase K may access epitopes resistant to heat-induced retrieval

• Antibody selection and incubation:

  • Polyclonal antibodies typically offer higher sensitivity than monoclonals due to recognition of multiple epitopes

  • Extended primary antibody incubation (48-72 hours at 4°C) can enhance detection

  • Higher antibody concentrations coupled with more stringent washing to control background

• Detection system enhancement:

  • Fluorescent detection with photomultiplier amplification for dim signals

  • Sequential multiple antibody layering techniques

  • Overnight substrate development for chromogenic detection (where applicable)

• Positive controls:

  • Include tissues with known high RDH10 expression (retina, kidney, liver) as positive controls

  • For mouse tissues, BALB/c strains show higher RDH10 expression than C57Bl/6 , making them better positive controls

When optimizing for low-expression tissues, a systematic approach testing multiple variables is recommended. Successful detection of RDH10 in Müller cells of the retina, which express lower levels than the retinal pigment epithelium, was achieved by researchers using extended antibody incubation times and polymer-based detection systems .

What controls should be included when studying the role of RDH10 in retinoic acid synthesis?

Comprehensive control strategies are essential when using RDH10 antibodies to investigate retinoic acid synthesis pathways:

• Genetic controls:

  • RDH10 knockdown cells or tissues provide essential negative controls

  • In developmental studies, Rdh10 trex mutant embryos with compromised RDH10 function serve as valuable controls

  • Comparison of RDH10 antibody signal between wild-type and knockdown/mutant samples confirms specificity

• Biochemical activity controls:

  • Parallel retinol dehydrogenase activity assays measuring conversion of tritiated all-trans retinol to all-trans retinal

  • HPLC analysis of reaction products confirms specificity for the all-trans isomer

  • Cofactor specificity testing (NADP vs. NAD) should match RDH10's known preference for NADP

• Subcellular fractionation controls:

  • Microsomal fraction enrichment should increase RDH10 detection and activity

  • Cytosolic fractions should show minimal RDH10 compared to membrane fractions

  • This compartmentalization is functionally significant, as it prevents inhibition by cytosolic cellular retinol binding protein (RBP1)

• Tissue/cell type expression controls:

  • Include tissues with known high expression (retina, kidney, liver)

  • For retinal studies, compare RPE (high expression) to retina (lower expression)

  • In mouse models, consider strain differences (BALB/c vs. C57Bl/6)

• Pathway perturbation controls:

  • Vitamin A deficiency or supplementation to modulate substrate availability

  • Retinaldehyde supplementation in RDH10-deficient models to bypass the first oxidative step

  • Inhibitors of subsequent enzymatic steps (RALDH inhibitors) to distinguish RDH10-specific effects

• Functional readout controls:

  • Retinoic acid responsive element (RARE) reporter constructs to monitor pathway activity

  • Expression of known RA-regulated genes as functional readouts

  • Phenotypic rescue experiments in developmental models

Studies have demonstrated that RDH10 is the primary enzyme responsible for the first step of embryonic vitamin A oxidation, with RDH10 deficiency causing developmental defects that can be rescued by retinaldehyde supplementation . This experimental approach exemplifies the power of combining antibody detection with functional perturbation and rescue strategies to establish mechanistic relationships.

How might emerging antibody technologies enhance RDH10 research?

The field of RDH10 research stands to benefit significantly from emerging antibody technologies that offer improved specificity, sensitivity, and functional capabilities:

• Recombinant monoclonal antibodies:

  • Molecularly defined antibodies eliminate batch-to-batch variation inherent to polyclonal antibodies

  • Engineered specificities can distinguish between highly similar family members in the SDR family

  • The ability to generate renewable antibody sources ensures experimental reproducibility

• Nanobodies and single-domain antibodies:

  • Smaller antibody fragments provide superior tissue penetration for imaging applications

  • Reduced steric hindrance allows access to epitopes within protein complexes

  • Improved access to conformational epitopes may better distinguish active vs. inactive RDH10

• Intracellular antibodies (intrabodies):

  • Expression of functional antibody fragments within living cells

  • Can provide real-time monitoring of RDH10 localization and activity

  • Potential for targeted inhibition of RDH10 function in specific cellular compartments

• Bifunctional antibodies:

  • Antibody-enzyme fusions for proximity-based labeling of RDH10 interaction partners

  • Antibody-fluorophore fusions optimized for super-resolution microscopy

  • PROTAC-conjugated antibodies for targeted RDH10 degradation in specific tissues

• Spatially-resolved antibody techniques:

  • Multiplexed immunofluorescence with spectral unmixing for co-localization studies

  • Mass cytometry and imaging mass cytometry for high-dimensional analysis of RDH10 in tissue context

  • Spatial transcriptomics combined with protein detection for integrated expression analysis

• Antibody-based biosensors:

  • FRET-based sensors incorporating RDH10 antibody fragments

  • Activity-based sensors that respond to local retinaldehyde concentration changes

  • Optogenetic tools coupled with antibody recognition elements

These technologies could address key knowledge gaps, including RDH10's dynamic regulation during development, its potential interaction partners in cancer contexts, and its precise subcellular localization in relation to other components of the retinoic acid synthesis pathway. The application of these advanced antibody technologies may particularly benefit efforts to understand RDH10's unexpected role in promoting glioma development through the TWEAK-NF-κB pathway .

What are the emerging connections between RDH10 and disease states beyond development?

Recent research has begun to uncover unexpected roles for RDH10 beyond its canonical function in retinoic acid synthesis during development, opening new avenues for antibody-based investigations:

• Cancer biology:

  • RDH10 is highly expressed in human gliomas, with expression levels correlating with tumor grade and patient survival

  • RDH10 knockdown impairs glioma cell proliferation, survival, invasiveness, and cell cycle progression

  • Mechanistic studies suggest RDH10 promotes glioma cell proliferation and survival by regulating the TWEAK-NF-κB signaling axis

  • Antibody-based tissue microarray studies could expand these findings to other cancer types

• Retinal diseases:

  • Given RDH10's expression in both retinal pigment epithelium and Müller cells , it may play roles in retinal pathologies

  • Immunohistochemical studies in models of retinal degeneration could reveal altered RDH10 expression or localization

  • Age-related macular degeneration, which involves retinoid cycle disruptions, represents a potential area for RDH10 investigation

• Metabolic disorders:

  • RDH10's role in vitamin A metabolism suggests potential involvement in metabolic conditions

  • Expression in liver and adipose tissue points to possible functions in energy homeostasis

  • Antibody-based studies in models of obesity or fatty liver disease could reveal new roles

• Inflammatory conditions:

  • The connection to NF-κB signaling identified in glioma studies suggests potential roles in inflammatory processes

  • Immunodetection of RDH10 in inflammatory disease models could reveal previously unrecognized functions

  • Potential interactions with immune cell retinoid metabolism warrant investigation

• Aging-related processes:

  • Retinoic acid signaling changes with age across multiple tissues

  • Antibody-based quantification of RDH10 across the lifespan could identify age-dependent regulation

  • Co-localization with senescence markers may reveal connections to cellular aging processes

The identification of RDH10's role in glioma through the TWEAK-NF-κB axis represents a paradigm shift in understanding this enzyme's function . This finding highlights the value of antibody-based approaches in uncovering unexpected roles for enzymes previously studied in limited contexts. Similar approaches could reveal additional non-canonical functions of RDH10 in various disease states, potentially identifying new therapeutic targets.