RDH10 Antibody

Basic Research Questions

• What is RDH10 and what are its known biological functions?

  RDH10 is a member of the short-chain dehydrogenase/reductase (SDR) family that plays an essential role in retinoic acid (atRA) synthesis, which is critical for embryonic development . It functions as the primary enzyme responsible for oxidizing retinol (Vitamin A) to retinaldehyde. It has dual roles in:

  • Embryonic development: Catalyzing the first step of Vitamin A oxidation

  • Visual cycle: Acting as both an all-trans retinol dehydrogenase and an 11-cis-retinol dehydrogenase that oxidizes 11-cis-retinol (11cROL) to 11-cis-retinaldehyde (11cRAL)

  RDH10 is a strictly NAD+-dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates .

• In which tissues and cell types is RDH10 expressed?

  RDH10 exhibits a diverse expression pattern across various tissues:

  Tissue/Cell Type	Relative Expression	Detection Method	Source
  Retinal pigment epithelium (RPE)	High	Immunohistochemistry
  Retinal Müller cells	Moderate	Immunohistochemistry, RT-PCR
  Liver	Moderate	Western blot
  Kidney	Moderate	Western blot, IHC
  Small intestine	Present	Immunoblotting
  Placenta	Present	Immunoblotting
  Lung	Present	Immunoblotting
  Heart	Present	Immunoblotting
  Skeletal muscle	Present	Immunoblotting

  RDH10 is notably expressed at higher levels in the eyecups of BALB/c mice compared to C57Bl/6 mice .

• What applications are RDH10 antibodies commonly used for in research?

  RDH10 antibodies have been validated for multiple research applications:

  Application	Publication Count	Dilution Range	Notes
  Western Blot (WB)	12+ publications	1:1000-1:4000	Detects ~39 kDa band
  Immunohistochemistry (IHC)	5+ publications	1:50-1:500	Antigen retrieval with TE buffer pH 9.0 recommended
  Immunofluorescence (IF)	4+ publications	Varies by antibody	Used for colocalization studies
  Immunoprecipitation (IP)	Validated	0.5-4.0 μg per 1.0-3.0 mg lysate	Used for interaction studies
  Knockdown validation	2+ publications	As per WB	Used to confirm RDH10 silencing

  These applications have been critical in investigating RDH10's role in visual cycle and cancer research .

• What are the expected molecular characteristics of RDH10 when detected by antibodies?

  When working with RDH10 antibodies, researchers should expect the following characteristics:

  • Calculated molecular weight: 38 kDa

  • Observed molecular weight in SDS-PAGE: Approximately 39 kDa

  • Human RDH10: 341 amino acids, shares high homology with other species

  • Rat RDH10: 341 amino acids, 99.4% homology with human, bovine, and mouse RDH10

  • Subcellular localization: Primarily in the endoplasmic reticulum (ER)

  When validating a new RDH10 antibody, these properties can serve as reference points for confirming specificity.

• What are the recommended storage conditions for maintaining RDH10 antibody activity?

  For optimal stability and performance of RDH10 antibodies, follow these evidence-based storage recommendations:

  • Store at -20°C for long-term stability

  • Stable for one year after shipment when properly stored

  • For antibodies in liquid form with 50% glycerol (pH 7.3), aliquoting is unnecessary for -20°C storage

  • Some formulations may contain 0.1% BSA and 0.02% sodium azide as preservatives

  • Avoid repeated freeze-thaw cycles to maintain antibody performance

  Always check manufacturer specifications as storage conditions may vary slightly between different commercial antibodies.

Advanced Research Questions

• How can I validate the specificity of an RDH10 antibody for my research?

  A comprehensive validation approach should include:

  1. Positive control tissues: Use tissues known to express RDH10 such as liver, kidney, or retinal tissue. Western blot analysis has confirmed RDH10 expression in HepG2 cells, A549 cells, mouse liver/kidney tissue, and rat kidney tissue .

  2. Knockdown/knockout validation: Compare antibody signal between wild-type samples and those where RDH10 has been knocked down via lentivirus-mediated shRNA . This approach has been successfully used in glioma studies.

  3. Recombinant protein: Test antibody against recombinant RDH10 protein.

  4. Peptide competition: Pre-incubate antibody with the immunizing peptide to confirm signal suppression.

  5. Cross-species reactivity: If working with non-human samples, validate the antibody in your specific species. The high conservation of RDH10 (99% sequence identity between rat, mouse, bovine, and human at amino acid level) suggests cross-reactivity is likely .

• What are the optimal protocols for immunohistochemical detection of RDH10?

  For successful IHC detection of RDH10:

  1. Tissue preparation:

     • Formalin-fixed, paraffin-embedded (FFPE) sections should be deparaffinized and rehydrated

     • For frozen sections, fix briefly with paraformaldehyde

  2. Antigen retrieval:

     • Primary method: TE buffer pH 9.0

     • Alternative method: Citrate buffer pH 6.0

     • Heat-induced epitope retrieval (HIER) has shown better results than enzymatic methods

  3. Blocking and antibody incubation:

     • Block with 5-10% normal serum from the species of secondary antibody

     • Primary antibody dilution: 1:50-1:500 (optimize for your specific tissue)

     • Incubate overnight at 4°C for best results

  4. Detection and visualization:

     • Use HRP-conjugated secondary antibodies with DAB substrate

     • For immunofluorescence, secondary antibodies conjugated to fluorophores

     • Counterstain nuclei with hematoxylin or DAPI

  5. Controls:

     • Include no-primary-antibody control

     • Include known positive tissue (mouse kidney recommended)

• How can I design experiments to investigate RDH10's role in the visual cycle?

  Based on published methodologies, a comprehensive experimental approach should include:

  1. Enzymatic activity assays:

     • Express human RDH10 in COS1 cells and prepare membrane fractions by differential centrifugation

     • Use both NAD+ and NADP+ as cofactors to compare activity (NAD+ confers more robust activity)

     • Conduct assays with 11-cis-retinol (11cROL) substrate in the presence/absence of purified CRALBP

     • Analyze retinoid products by HPLC

  2. Reconstitution of visual cycle in cell culture:

     • Co-express RDH10 with other visual cycle proteins (CRALBP, RPE65, LRAT) in HEK-293A cells

     • Treat with all-trans-retinol (atROL)

     • Quantify retinoid profiles by HPLC to determine conversion to 11cRAL

  3. Protein interaction studies:

     • Perform co-immunoprecipitation experiments to detect physical interactions between RDH10 and other visual cycle proteins like CRALBP and RPE65

     • Use immunocytochemistry to investigate co-localization patterns

  This multi-faceted approach has successfully demonstrated that RDH10 functions in the RPE retinoid visual cycle as an 11-cis-RDH .

• What is the evidence for RDH10's involvement in cancer progression, particularly in gliomas?

  Research has established RDH10 as a potential oncogenic factor in gliomas:

  1. Expression patterns:

     • RDH10 is highly expressed in human gliomas

     • Expression correlates with tumor grade and patient survival times

     • Higher expression in both LGG (lower-grade glioma) and GBM (glioblastoma multiforme) compared to normal tissue

     • Expression increases with advanced tumor grade in SCG (spinal cord glioma)

  2. Functional studies:

     • Lentivirus-mediated shRNA knockdown of RDH10 suppresses:

       • Glioma cell proliferation

       • Cell survival

       • Invasiveness

       • Cell cycle progression

     • In vivo, RDH10 knockdown reduced glioma growth in nude mice

  3. Molecular mechanisms:

     • RDH10 influences the TWEAK-NF-κB axis

     • RDH10 regulates EMT (epithelial-mesenchymal transition) process in SCG through PI3K-AKT pathway

     • RDH10 silencing reduces expression of TNFRSF12A (Fn14), TNFSF12 (TWEAK), TRAF3, IKBKB (IKK-β), and BMPR2

     • Enhanced invasion ability and increased EMT-related protein expression induced by RDH10 overexpression can be suppressed by PI3K-AKT pathway inhibitor (LY294002)

  These findings suggest RDH10 could potentially serve as a novel target for glioma treatment.

• How can I simultaneously assess both RDH10 protein levels and enzymatic activity in experimental samples?

  A comprehensive protocol combining protein detection and activity analysis:

  1. Sample preparation:

     • Divide tissue or cell samples for parallel protein and activity analysis

     • For activity assays: prepare membrane fractions by ultracentrifugation (100,000×g for 1 hour)

     • For protein detection: prepare lysates in SDS buffer with protease inhibitor cocktail

  2. Protein level assessment:

     • Western blot using validated RDH10 antibodies (1:1000-1:4000 dilution)

     • Normalize to housekeeping proteins (GAPDH recommended)

     • Include appropriate positive control tissues (liver or kidney)

  3. Activity assay protocol:

     • Membrane preparation: Resuspend membrane pellet in RDH activity buffer after washing

     • Substrate preparation: Under dim red light, prepare 11-cis-retinol or all-trans-retinol

     • Reaction conditions:

       • Use 32 μg of membrane proteins in 200 μl of RDH activity buffer

       • Include 1% BSA and 1 mM NADP+ or NAD+

       • Incubate under appropriate conditions (37°C)

     • Analysis: Quantify retinoid products by HPLC

  4. Correlation analysis:

     • Compare protein expression levels with enzymatic activity

     • Analyze relationship between protein/activity levels and biological outcomes

• What experimental approaches can be used to study RDH10's interaction with other visual cycle proteins?

  Based on successful published methods :

  1. Co-immunoprecipitation (Co-IP):

     • Prepare cell or tissue lysates under non-denaturing conditions

     • Use 0.5-4.0 μg RDH10 antibody per 1.0-3.0 mg of total protein lysate

     • Precipitate with protein A/G beads

     • Analyze precipitated complexes by western blot for interacting partners (CRALBP, RPE65)

     • Include appropriate controls (IgG control, input samples)

  2. Proximity ligation assay (PLA):

     • Visualize protein interactions in situ with single-molecule resolution

     • Use primary antibodies from different species against RDH10 and potential interacting partners

     • Follow with species-specific PLA probes

  3. Co-localization studies:

     • Perform double immunofluorescence staining

     • Use confocal microscopy to analyze co-localization patterns

     • Example: RDH10 co-localizes with RPE65 and CRALBP in primary bovine RPE cells

  4. Functional reconstitution assays:

     • Co-express RDH10 with other visual cycle proteins in cell systems

     • Assess functional outcomes (e.g., generation of 11cRAL from atROL)

     • Compare activity with and without interacting partners

• What are the recommended methods for RDH10 knockdown in functional studies?

  Based on successful approaches in glioma research :

  1. shRNA-mediated knockdown:

     • Design target-specific shRNAs against RDH10

     • Deliver via lentiviral vectors for stable knockdown

     • Include non-targeting shRNA controls

     • Validate knockdown efficiency by RT-PCR and western blot

  2. siRNA transfection:

     • Design siRNAs targeting conserved regions of RDH10

     • Optimize transfection conditions for your cell type

     • Validate knockdown at 48-72 hours post-transfection

  3. CRISPR/Cas9 genome editing:

     • Design guide RNAs targeting exonic regions of RDH10

     • Generate knockout cell lines through single-cell cloning

     • Validate by sequencing and western blot

  4. Functional validation assays:

     • Cell proliferation: MTT/CCK-8 assays

     • Migration: Wound healing assay (measure width changes at 0, 24, and 48h)

     • Invasion: Trans-well assays

     • In vivo validation: Xenograft studies using knockdown cells (5 × 10^6 cells per mouse)

  5. Rescue experiments:

     • Re-express RDH10 using constructs resistant to the knockdown method

     • Confirm that phenotypes are specifically due to RDH10 loss