RDH14 Antibody

What is RDH14 and what is its primary function in cellular biology?

RDH14 (Retinol Dehydrogenase 14) is an enzyme that exhibits oxidoreductive catalytic activity towards retinoids. It functions most efficiently as an NADPH-dependent retinal reductase and displays high activity toward 9-cis, 11-cis, and all-trans-retinol, while showing very weak activity towards 13-cis-retinol. Notably, RDH14 has no steroid dehydrogenase activity detected . The protein is expressed in brain tissue and has been implicated in neurological function, with mutations potentially associated with intellectual disability and cerebellar atrophy .

What types of RDH14 antibodies are currently available for research?

Current research-grade RDH14 antibodies are available in both monoclonal and polyclonal formats with various species reactivity profiles:

What is the difference between RDH14 monoclonal and polyclonal antibodies?

Monoclonal RDH14 antibodies (like clone 3E1) are derived from single B-cell clones and recognize a single epitope, offering high specificity but potentially limited sensitivity. These antibodies are typically produced using full-length recombinant protein of human RDH14 (NP_065956) expressed in HEK293T cells as the immunogen . Polyclonal RDH14 antibodies are derived from multiple B-cell lineages and recognize multiple epitopes on the target protein, potentially offering higher sensitivity but with increased risk of cross-reactivity. The choice between them depends on the experimental requirements for specificity versus sensitivity .

What are the validated applications for RDH14 antibodies?

RDH14 antibodies have been validated for several experimental applications:

• Western Blot (WB): Most RDH14 antibodies are validated for WB, with recommended dilutions typically between 1:200-1:500 for monoclonal antibodies .

• Flow Cytometry (FACS): Selected monoclonal antibodies like clone 1A7 and 3E1 are validated for flow cytometry with typical working dilutions of 1:100 .

• ELISA: Some polyclonal antibodies have been validated for ELISA applications .

• Immunohistochemistry (IHC): Selected antibodies, particularly from the Prestige Antibodies collection, are validated for IHC with recommended dilutions of 1:500-1:1000 .

• Immunofluorescence (IF): Some antibodies have been validated for IF with recommended concentrations of 0.25-2 μg/mL .

How should RDH14 antibodies be stored and handled to maintain optimal activity?

Based on manufacturer recommendations, proper storage and handling of RDH14 antibodies includes:

• Store antibodies at -20°C for long-term storage .

• For unconjugated antibodies stored in glycerol solutions, avoid repeated freeze/thaw cycles by preparing small aliquots before freezing .

• DyLight 755 conjugated antibodies should be stored at 4°C in the dark to prevent photobleaching .

• Some preparations may require brief centrifugation if small volumes become entrapped in the vial cap during shipping and storage .

• Most RDH14 antibodies are supplied in buffers containing preservatives like 0.02-0.05% sodium azide, which should be noted when designing experiments sensitive to these components .

What are the recommended protocols for RDH14 antibody validation?

To validate RDH14 antibody specificity for research applications:

• Western blot validation: Compare lysates from cells transfected with recombinant RDH14 against empty vector controls. A validated antibody should show a specific band at approximately 36.8 kDa in RDH14-expressing samples but not in controls .

• Flow cytometry validation: Test using HEK293T cells transfected with either recombinant RDH14 protein or empty vector. A positive shift should be observed only in RDH14-expressing cells .

• Cross-reactivity testing: Evaluate potential cross-reactions with other retinol dehydrogenase family members, especially those with structural similarity to RDH14.

• Peptide competition assay: Pre-incubate antibody with immunizing peptide before application to confirm that binding is specifically blocked.

How can RDH14 antibodies be used to investigate its potential role in intellectual disability?

Recent research has implicated RDH14 in intellectual disability with cerebellar atrophy. Researchers could:

• Use RDH14 antibodies for immunohistochemistry of brain sections, particularly focusing on cerebellar tissue, to examine expression patterns in normal versus affected tissues.

• Employ co-immunoprecipitation (Co-IP) with RDH14 antibodies to investigate its reported interaction with proton-activated chloride channel 1 (PACC1/TMEM206). This interaction was found to be diminished by a frameshifting mutation in RDH14 associated with intellectual disability .

• Design immunofluorescence experiments with RDH14 antibodies to examine its subcellular localization, which has been reported to be primarily in the nucleoplasm in HEK293 cells .

• Combine RDH14 antibodies with markers for neuronal development to investigate potential mechanisms through either disrupted retinoic acid signaling or chloride ion homeostasis in the brain .

What methods can be used to study the subcellular localization of RDH14?

For effective subcellular localization studies of RDH14:

• Use immunofluorescence with RDH14 antibodies alongside organelle markers, particularly for the nucleoplasm, endoplasmic reticulum, mitochondria, and lysosomal membranes, as RDH14 has been reported to localize to these compartments .

• Employ subcellular fractionation followed by Western blotting with RDH14 antibodies to quantitatively assess distribution across cellular compartments.

• Consider utilizing confocal microscopy with fluorescently labeled RDH14 antibodies to achieve high-resolution imaging of localization.

• For live-cell imaging applications, consider using recombinant expression systems with fluorescent protein tags, validated against fixed-cell immunofluorescence using RDH14 antibodies.

How can protein-protein interactions of RDH14 be studied using these antibodies?

To investigate RDH14 protein interactions:

• Co-immunoprecipitation (Co-IP): Use RDH14 antibodies to pull down the protein complex, followed by Western blotting or mass spectrometry to identify interacting partners. This approach has been used successfully to confirm RDH14's interaction with PACC1/TMEM206 .

• Proximity ligation assay (PLA): Combine RDH14 antibodies with antibodies against suspected interacting proteins to visualize and quantify protein-protein interactions in situ with single-molecule sensitivity.

• FRET/BRET analysis: Though not directly using the antibodies, these techniques can validate interactions identified through antibody-based methods.

• Immunofluorescence co-localization: Use RDH14 antibodies alongside antibodies for potential interacting partners to examine spatial co-localization in fixed cells or tissues.

What are common issues with Western blotting using RDH14 antibodies and how can they be resolved?

Common challenges and solutions when using RDH14 antibodies for Western blotting:

• Non-specific binding: May be reduced by:

  • Optimizing antibody dilution (recommended range: 1:200-1:500 for monoclonal; 0.04-0.4 μg/mL for some polyclonal)

  • Increasing blocking time/concentration

  • Adding 0.05-0.1% Tween-20 to washing buffers

• Weak or absent signal:

  • Ensure sample contains sufficient RDH14 (consider using positive controls like HEK293T cells transfected with RDH14)

  • Decrease antibody dilution

  • Increase exposure time

  • Verify transfer efficiency

  • Check if denaturing conditions affect epitope recognition

• Multiple bands:

  • May represent different isoforms, post-translational modifications, or degradation products

  • Run alongside a known positive control

  • Consider using more specific monoclonal antibodies like clone 3E1

• High background:

  • Increase washing duration/frequency

  • Dilute primary and secondary antibodies

  • Use fresher blocking reagents

How can researchers optimize flow cytometry protocols for RDH14 antibodies?

For optimal flow cytometry results with RDH14 antibodies:

• Cell preparation:

  • Fix and permeabilize cells appropriately since RDH14 is primarily intracellular (nucleoplasm/ER/mitochondria)

  • Use gentle permeabilization to preserve epitope integrity

• Antibody concentration:

  • Start with recommended dilution (approximately 1:100 for most RDH14 monoclonal antibodies)

  • Titrate to determine optimal concentration for your specific cell type

• Controls:

  • Include isotype controls matched to your RDH14 antibody (IgG1 for most monoclonal versions)

  • Use cells with known RDH14 expression levels (e.g., HEK293T transfected with RDH14 versus empty vector)

• Signal amplification:

  • For unconjugated antibodies, select secondary antibodies with appropriate brightness

  • For directly conjugated antibodies (like DyLight 755), ensure your cytometer has appropriate lasers/filters

• Data analysis:

  • Gate appropriately based on controls

  • Consider dual-parameter analysis when examining co-expression with other markers

What is the evidence linking RDH14 to neurological disorders?

Recent research has identified RDH14 as a candidate gene involved in neurological disorders:

• A 2021 study published in Scientific Reports identified a biallelic frameshifting variant in RDH14 in individuals with intellectual disability and cerebellar atrophy from a Pakistani family .

• Magnetic resonance imaging of individuals with the RDH14 mutation showed cerebellar atrophy without signs of polymicrogyria .

• Functional studies demonstrated that:

  • RDH14 is expressed in brain tissue but not in retina

  • RDH14 localizes primarily to the nucleoplasm in HEK293 cells

  • The frameshifting mutation greatly diminished RDH14's binding to proton-activated chloride channel 1 (PACC1/TMEM206)

• The study proposed two potential disease mechanisms:

  • Disrupted retinoic acid signaling

  • Disrupted chloride ion homeostasis in the brain through altered interaction with PACC1

How can researchers design experiments to further investigate RDH14's role in retinoid metabolism?

To advance understanding of RDH14's function in retinoid metabolism:

• Enzyme activity assays:

  • Use purified RDH14 protein with various retinoid substrates to measure kinetic parameters

  • Compare activity with different cofactors (NADPH vs. NADH)

  • Validate findings in cellular models using RDH14 antibodies to confirm protein expression

• Gene modification approaches:

  • Generate RDH14 knockdown/knockout models to examine effects on retinoid levels

  • Create point mutations mimicking those found in human disorders

  • Use RDH14 antibodies to confirm protein reduction/absence

• Metabolomics analysis:

  • Compare retinoid profiles in tissues/cells with normal versus altered RDH14 expression

  • Combine with RDH14 antibody-based techniques to correlate protein levels with metabolite changes

• Tissue-specific expression studies:

  • Use RDH14 antibodies for immunohistochemistry across multiple tissues

  • Correlate expression patterns with tissue-specific retinoid metabolism

• Structural biology approaches:

  • Use insights from antibody epitope mapping to inform structural studies

  • Investigate how mutations affect protein structure and function

What experimental designs would help clarify RDH14's interaction with PACC1 and its implications for neurological function?

To further investigate the RDH14-PACC1 interaction and its role in neurological disorders:

• Detailed mapping of interaction domains:

  • Use truncated versions of RDH14 in co-immunoprecipitation experiments with RDH14 antibodies

  • Identify specific residues required for interaction through site-directed mutagenesis

• Functional consequence analysis:

  • Measure chloride channel activity in the presence of wild-type versus mutated RDH14

  • Assess neuronal chloride homeostasis in cellular and animal models with altered RDH14

• Developmental studies:

  • Examine RDH14 and PACC1 expression during brain development using specific antibodies

  • Investigate effects of RDH14 mutations on neuronal differentiation and cerebellar development

• In vivo models:

  • Generate conditional knockout models of RDH14 in specific brain regions

  • Examine behavioral and cognitive outcomes alongside cerebellar morphology

  • Validate models with RDH14 antibodies to confirm tissue-specific knockdown

• Therapeutic exploration:

  • Test compounds that might restore RDH14-PACC1 interaction or compensate for lost function

  • Use RDH14 antibodies to monitor protein levels and localization during therapeutic interventions