ADPRHL2 Antibody

What is ADPRHL2 and why is it important to study?

ADPRHL2 encodes a member of the ADP-ribosylglycohydrolase family that catalyzes the removal of ADP-ribose from ADP-ribosylated proteins. This enzyme is particularly important because it functions in reversing post-translational modifications involved in key cellular processes including transcription, DNA repair, translation, and apoptosis . Mutations in ADPRHL2 have been linked to a progressive neurodegenerative disorder manifesting in childhood or adolescence characterized by developmental delay, seizures, ataxia, and axonal (sensori-)motor neuropathy . The enzyme localizes to mitochondria in addition to the nucleus and cytoplasm and plays a critical role in the maintenance of normal neuronal cell function .

What applications are ADPRHL2 antibodies typically used for?

ADPRHL2 antibodies are primarily utilized for Western blotting (WB), enzyme immunoassay (EIA), flow cytometry (FC), and immunohistochemistry (IHC) . These applications enable researchers to:

• Detect ADPRHL2 protein expression in different cell types and tissues

• Analyze subcellular localization patterns

• Evaluate changes in expression levels under different experimental conditions

• Study the effects of mutations on protein stability and function

How should ADPRHL2 antibodies be stored and handled?

For optimal performance, ADPRHL2 antibodies should be:

• Maintained refrigerated at 2-8°C for up to 2 weeks

• For long-term storage, keep at -20°C in small aliquots to prevent freeze-thaw cycles

• When diluting for applications, use recommended dilution ratios (typically 1:1000 for Western blotting and 1:10-50 for flow cytometry)

How can I validate the specificity of an ADPRHL2 antibody?

To validate ADPRHL2 antibody specificity:

• Conduct Western blot analysis using:

  • Positive controls: Cell lines known to express ADPRHL2

  • Negative controls: ADPRHL2 knockdown/knockout cells

  • Recombinant ADPRHL2 protein as a reference standard

• Perform peptide competition assays:

  • Pre-incubate the antibody with the immunogenic peptide (typically between amino acids 87-114 or 93-123 from the N-terminal region)

  • Compare immunoblotting signals with and without peptide blocking

  • A significant reduction in signal indicates specificity

• Cross-validate with multiple antibodies targeting different epitopes:

  • N-terminal antibodies (AA 87-114 or 93-123)

  • C-terminal antibodies

  • Compare staining/detection patterns

What cellular models are appropriate for studying ADPRHL2 function?

Based on published research, appropriate models include:

• Patient-derived fibroblasts:

  • From individuals with confirmed ADPRHL2 mutations

  • Useful for studying pathogenic mechanisms

• HeLa cells:

  • Well-characterized for ADP-ribosylation studies

  • Responsive to H₂O₂ treatment to induce stress conditions

• Neuronal cell lines:

  • Especially relevant given ADPRHL2's importance in neuronal function

  • Can be used to study the consequences of ADPRHL2 deficiency

• Cells grown in different media conditions:

  • High-glucose DMEM (4.5 g/L)

  • Low-glucose DMEM (1 g/L) to promote mitochondrial respiration

What is the optimal protocol for detecting ADPRHL2 in Western blotting?

For optimal Western blot detection of ADPRHL2:

• Sample preparation:

  • Extract proteins using a buffer containing protease inhibitors

  • Include subcellular fractionation if studying localization patterns

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels

  • Load 20-40 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST

• Antibody incubation:

  • Primary antibody: Use at 1:1000 dilution

  • Incubate overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG (typically 1:5000-1:10000)

• Signal detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Expected molecular weight of ADPRHL2 is approximately 39 kDa

How can I study ADPRHL2 subcellular localization?

To analyze ADPRHL2 subcellular localization:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1-0.5% Triton X-100

  • Use anti-ADPRHL2 antibody at appropriate dilution

  • Co-stain with organelle markers (mitochondria, nucleus)

  • Analyze using confocal microscopy

• Subcellular fractionation:

  • Separate mitochondrial, nuclear, and cytosolic fractions

  • Perform Western blotting on each fraction

  • Include fraction-specific markers as controls (e.g., VDAC for mitochondria, Lamin for nucleus)

• For studying mutant localization:

  • Express wild-type and mutant ADPRHL2 constructs (e.g., V335G mutant)

  • Compare localization patterns between wild-type and mutant proteins

How can I assess the functional impact of ADPRHL2 deficiency on ADP-ribosylation levels?

To evaluate the impact on ADP-ribosylation:

• Immunodetection of ADP-ribosylation:

  • Use recombinant antibody-like ADP-ribose binding reagents:

    • WWE(RNF146)–Fc for PAR chains

    • Macro(AF1521)–Fc, Macro(mH2A1.1)–Fc, or Macro2/3(PARP14)–Fc for different ADP-ribosylation patterns

  • Compare basal and stress-induced ADP-ribosylation levels

• Stress induction experiments:

  • Treat cells with H₂O₂ (typically 600 μM for 24 hours) to induce oxidative stress

  • Compare ADP-ribosylation patterns between control and ADPRHL2-deficient cells

  • Include time-course analysis (e.g., 0, 20 min, 2 hrs post-treatment)

• Validation experiments:

  • Hydroxylamine treatment to cleave ADPR from glutamate and aspartate residues

  • Treatment with ARH3 enzyme as a control

  • PARP inhibitor (e.g., DPQ) treatment to block new ADP-ribosylation

What approaches can be used to study the relationship between ADPRHL2 and cellular stress responses?

For investigating ADPRHL2's role in stress responses:

• Cell viability assays:

  • Culture cells in low-glucose DMEM (1 g/L) to promote mitochondrial respiration

  • Expose to H₂O₂ concentrations ranging from 0-1000 μM

  • Measure viability using alamarBlue Cell Viability Reagent

  • Compare wild-type, ADPRHL2-deficient, and rescued cells

• Rescue experiments:

  • Transduce ADPRHL2-deficient cells with wild-type ADPRHL2 cDNA

  • Confirm expression by immunoblotting

  • Test for restoration of normal cellular responses to stress

  • Compare with mutant ADPRHL2 constructs

• Pharmacological interventions:

  • Treat cells with PARP1 inhibitors (e.g., DPQ at 25 μM)

  • Assess whether inhibiting PAR formation rescues stress sensitivity

  • Determine downstream mechanisms affected by ADPRHL2 deficiency

How can I investigate the potential role of ADPRHL2 in neurodegeneration?

To explore ADPRHL2's role in neurodegeneration:

• Patient-derived cellular models:

  • Obtain fibroblasts from patients with ADPRHL2 mutations

  • Consider reprogramming to induced pluripotent stem cells (iPSCs)

  • Differentiate into neurons for disease-relevant studies

• Comparative analysis of different ADPRHL2 mutations:

  • C26F mutation (causes protein instability)

  • V335G mutation (affects subcellular localization)

  • Analyze differential effects on cellular phenotypes

• Mitochondrial function assessment:

  • Measure respiratory function in ADPRHL2-deficient cells

  • Analyze mitochondrial membrane potential

  • Assess changes in mitochondrial morphology

  • Investigate links to neuronal vulnerability

What are common issues when using ADPRHL2 antibodies and how can they be addressed?

Common challenges and solutions include:

• Weak or absent signal:

  • Increase antibody concentration

  • Extend incubation time

  • Use more sensitive detection methods

  • Check protein expression levels in your sample

  • Verify antibody reactivity with your species of interest

• High background:

  • Increase blocking time/concentration

  • Optimize antibody dilution

  • Include additional washing steps

  • Try alternative blocking agents (BSA vs. milk)

  • Consider using more specific antibodies

• Multiple bands:

  • Verify expected molecular weight (~39 kDa)

  • Check for known isoforms or degradation products

  • Include appropriate controls (recombinant protein, knockout samples)

  • Use fresh samples with protease inhibitors

How should I interpret changes in ADPRHL2 expression or localization in disease models?

When analyzing ADPRHL2 in disease contexts:

• Expression level changes:

  • Quantify relative to appropriate housekeeping proteins

  • Compare across multiple cell lines/patient samples

  • Consider tissue-specific expression patterns

  • Correlate with clinical severity if possible

• Localization alterations:

  • Compare nuclear vs. mitochondrial vs. cytoplasmic distribution

  • Analyze whether mutations affect specific compartments (e.g., V335G affects nuclear localization)

  • Consider functional consequences of mislocalization

• Functional implications:

  • Connect changes to specific cellular phenotypes (e.g., stress sensitivity)

  • Determine whether defects are reversible (through rescue experiments)

  • Investigate impact on tissue-specific functions, particularly in neurons