UOX Antibody

What is UOX and why are UOX antibodies important in research?

UOX (Urate Oxidase) is an enzyme that catalyzes the conversion of uric acid to allantoin. Knockout of this gene can lead to severe hyperuricemia, making it a crucial target for research on gout and related conditions . UOX antibodies are essential tools for detecting and studying this enzyme in experimental models, particularly in validating gene knockout strategies and investigating uric acid metabolism disorders.

In humans, the UOX gene is naturally inactivated during evolution, which makes humans susceptible to hyperuricemia and gout. Mouse models with UOX gene deletion have been developed to create "human-like" hyperuricemia models with serum uric acid levels approximately 5.5-fold higher than wild-type mice (1351.04±276.58μmol/L vs. 248.19±100.59μmol/L) .

What types of UOX antibodies are available for scientific applications?

Several types of UOX antibodies are available for research applications, varying in host species, target epitopes, and conjugations:

Antibodies targeting different epitopes of UOX are valuable for confirming experimental results through multiple detection methods. The choice of conjugation depends on the specific application, with fluorescent conjugates preferred for microscopy and HRP conjugates for Western blotting and ELISA .

How should researchers validate UOX antibody specificity?

Proper validation of UOX antibodies is essential for ensuring experimental reliability:

• Genetic controls: Use UOX knockout tissues or cells as negative controls to confirm antibody specificity

• Peptide competition: Pre-incubate the antibody with purified antigen or immunizing peptide to block specific binding sites

• Multiple epitope targeting: Compare results using antibodies targeting different regions of UOX

• Cross-reactivity testing: Verify reactivity against predicted species (mouse and rat UOX show high homology)

• Orthogonal validation: Correlate antibody detection with UOX mRNA expression or enzyme activity measurements

For Western blot validation, researchers should observe a band at approximately 35 kDa for mouse UOX, with normalization against housekeeping proteins such as β-tubulin .

How can UOX antibodies be utilized to characterize CRISPR/Cas9-generated UOX knockout models?

CRISPR/Cas9-mediated deletion of UOX represents a powerful approach for generating hyperuricemia models. UOX antibodies play critical roles in validating and characterizing these models:

• Knockout confirmation: Western blot analysis using UOX-specific antibodies at 1:500 dilution against UOX with β-tubulin (1:10000) as loading control can verify complete protein absence

• Off-target effect assessment: Immunohistochemistry with UOX antibodies can detect unexpected UOX expression patterns in non-targeted tissues

• Phenotype correlation: Comparing UOX protein levels with serum uric acid measurements:

  Model Type	UOX Protein Expression	Serum Uric Acid Level (μmol/L)	Reference
  Wild-type	Normal (100%)	248.19±100.59
  UOX KO (-/-)	Undetectable (0%)	1351.04±276.58
  UOX KO + Allopurinol	Undetectable (0%)	612.55±146.98

• Tissue-specific expression analysis: Immunofluorescence using fluorophore-conjugated UOX antibodies can map UOX expression across tissue types before and after gene modification

When analyzing CRISPR/Cas9-generated UOX knockout models, researchers should consider potential frameshift mutations and ensure that deletions cover critical exons (exons 2-4 account for about 46% of the entire UOX protein coding gene) .

What methodological considerations are crucial when using UOX antibodies for studying protein-protein interactions?

When investigating UOX interactions with other proteins (such as ubiquitin-mediated regulation), researchers should follow these methodological principles:

• Denaturing lysis conditions: Use denaturing buffers to remove non-covalently interacting proteins from UOX and inactivate cellular deubiquitylating enzymes (DUBs)

• Metal affinity purification: For His-tagged ubiquitin experiments:

  • Transfer Metal Affinity Resin in ~50 μL HEPES buffer to a PCR tube

  • Set aside ~10% of resin for pre-elution control

  • Elute ubiquitylated proteins with 300 mM imidazole

  • Rotate tubes for 4 hours at 4°C

  • Collect and pool eluates for analysis

• Stepwise renaturation: Dialyze purified proteins to gradually restore native conditions for functional studies

• Reducing conditions: Include DTT (dithiothreitol) to prevent oxidation of catalytic cysteine residues in interaction partner proteins

• Immunoblotting specificity: Detect UOX using specific antibodies, while ubiquitylated proteins can be detected with ubiquitin-specific or His-tag-specific antibodies

How can UOX antibodies contribute to the development of therapeutic UOX variants for hyperuricemia treatment?

UOX antibodies are vital tools in developing novel therapeutics for hyperuricemia treatment, particularly for recombinant UOX variants with extended half-lives:

• Therapeutic variant characterization: UOX antibodies can detect and quantify novel conjugates like PAT101 (recombinant human albumin (rHA)-conjugated Aspergillus flavus UOX)

• Pharmacokinetic profiling: Antibodies help measure circulating levels of therapeutic UOX:

  UOX Variant	Half-life Compared to Original	Activity Retention After 4 Weeks	Reference
  Rasburicase	Baseline	24%
  PAT101 (rHA-UOX)	>2x vs. pegloticase	86%

• Immunogenicity assessment: UOX antibodies can help detect anti-drug antibodies (ADAs) in samples, and in CD4+/CD8+ T-cell activation analysis, PAT101 showed lower immune response compared to rasburicase

• Site-specific conjugation optimization: When developing albumin-conjugated UOX variants, antibodies can verify successful incorporation of non-natural amino acids (NNAAs) and subsequent bioorthogonal conjugation through inverse electron demand Diels-Alder reaction (IEDDA)

• In vivo efficacy monitoring: UOX antibodies support tissue distribution studies and correlation with pharmacodynamic effects in UOX KO animal models

What are the optimal protocols for UOX detection by Western blotting?

For successful Western blot detection of UOX, researchers should follow these methodological guidelines:

• Sample preparation:

  • Homogenize tissues in RIPA buffer with protease inhibitors

  • Centrifuge lysates at 12,000g for 15 minutes at 4°C

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Separate 20-50 μg protein on 10-12% SDS-PAGE

  • Transfer to PVDF membrane at 100V for 1-2 hours or 30V overnight

• Antibody incubation:

  • Block with 5% non-fat milk for 1 hour at room temperature

  • Incubate with anti-UOX primary antibody (1:500-1:1000) overnight at 4°C

  • Wash 3x with TBST for 10 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:10000) for 1 hour at room temperature

• Detection and analysis:

  • Visualize using ECL substrate and appropriate imaging system

  • Normalize UOX signal to loading control (β-tubulin at 1:10000)

  • Perform densitometry analysis with ImageJ or similar software

• Troubleshooting:

  • For weak signals: Increase protein loading or primary antibody concentration

  • For multiple bands: Optimize lysis conditions or try different antibody

  • For high background: Increase washing time or decrease antibody concentration

Each measurement should be performed in triplicate, and all results should be normalized against β-tubulin to account for loading variations .

How should researchers design experiments using UOX antibodies for immunofluorescence?

For optimal immunofluorescence detection of UOX in tissues and cells:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde for 24 hours

  • Process and embed in paraffin or OCT for frozen sections

  • Cut sections at 4-6 μm thickness

• Antigen retrieval and blocking:

  • For paraffin sections: Perform heat-mediated antigen retrieval in citrate buffer (pH 6.0)

  • Block with 5% normal serum from secondary antibody host species for 1 hour

• Antibody incubation:

  • Incubate with primary UOX antibody (1:100-1:500) overnight at 4°C

  • Wash 3x with PBS for 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody (1:500) for 1 hour at room temperature

• Nuclear counterstaining and mounting:

  • Counterstain with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

• Imaging parameters:

  • Use appropriate excitation/emission filters for the fluorophore (e.g., AbBy Fluor® 350)

  • Capture multiple fields per sample (at least 5-10)

  • Include z-stack imaging for 3D analysis when relevant

It's essential to include both positive controls (wild-type mouse liver) and negative controls (UOX knockout tissues) to validate antibody specificity .

What are the critical considerations for sample preparation when studying UOX in knockout models?

When working with UOX knockout models, proper sample preparation is crucial for reliable results:

• Animal handling and sample collection:

  • For UOX KO mice, administer allopurinol (25 mg/kg daily) to pregnant female mice for survival of offspring

  • Collect blood by cardiac puncture into heparinized tubes for plasma separation

  • Harvest tissues rapidly and either flash-freeze or fix depending on downstream application

• Genotyping protocols:

  • Extract DNA from tail biopsies

  • Perform PCR using primers flanking the targeted exons (e.g., exons 2-4)

  • Verify deletion by gel electrophoresis and/or sequencing

• Tissue preservation for protein analysis:

  • For protein extraction: Snap-freeze tissues in liquid nitrogen and store at -80°C

  • For immunohistochemistry: Fix tissues in 4% paraformaldehyde for 24 hours, then process

• RNA analysis protocols:

  • Extract RNA using TRIzol or commercial kits

  • Perform RT-PCR with appropriate primers (e.g., 35 cycles with 30 seconds at 94°C, 30 seconds at 60°C, 1 minute at 72°C)

  • Calculate relative expression using the 2^-ΔΔCt method

• Experimental treatment groups:

  Group	Treatment	Route	Dosage (mg/kg)	Interval	Purpose
  G1	None (KO only)	-	-	-	Control
  G2	Allopurinol	Oral	25	Daily	Single treatment
  G3	Allopurinol + UOX therapy	Oral + I.P.	25 + 6	Daily + Weekly	Combination therapy
  G4	Allopurinol + pegloticase	Oral + I.P.	25 + 2	Daily + Weekly	Comparison therapy

  This experimental design allows for comprehensive evaluation of UOX replacement therapies compared to standard-of-care treatments .

How can UOX antibodies be utilized in antibody-oligonucleotide conjugate (AOC) research?

UOX antibodies can contribute to the emerging field of antibody-oligonucleotide conjugates (AOCs), which represent a promising approach for targeted delivery of nucleic acid therapeutics:

• Conjugation chemistry optimization:

  • UOX antibodies can be used as model systems for developing conjugation methods

  • Typical protocols use partial reduction of antibody interchain disulfide bonds with TCEP followed by conjugation with maleimide linker-oligonucleotides

  • Purification via ion exchange or hydrophobic interaction chromatography yields conjugates with controlled drug-to-antibody ratios (DAR)

• Linker chemistry selection:

  • MCC (maleimidocaproyl) linkers are commonly used for non-cleavable conjugation

  • Alternative linkers like MC offer simplified synthesis with potentially fewer steps

• Efficacy measurements:

  • Tissue pharmacokinetics showed >15-fold higher concentration in muscle with αTfR1 AOCs compared to unconjugated oligonucleotides

  • Binding affinity to target receptors must be preserved after conjugation (e.g., 42 pM for TfR1)

• Structure-activity relationship studies:

  • Evaluate impact of linker chemistry, oligonucleotide conjugation positions, and DARs on efficacy

  • For PMO conjugates, β-alanine conjugation handles provide flexible connection between the PMO and antibody

What emerging technologies are advancing UOX antibody research and applications?

Several cutting-edge technologies are enhancing UOX antibody research:

• Machine learning and active learning approaches:

  • Novel active learning strategies for antibody-antigen binding prediction have reduced the number of required experiments by up to 35%

  • The best algorithms can speed up the learning process by 28 steps compared to random baseline approaches

• Bioorthogonal conjugation chemistry:

  • Site-specific conjugation through non-natural amino acids (NNAAs) enables precise placement of conjugation sites

  • Inverse electron demand Diels-Alder reaction (IEDDA) offers faster reaction kinetics compared to strain-promoted azide-alkyne cycloaddition (SPAAC)

• Advanced immunogenicity prediction:

  • In vitro assays that assess T cell responses from healthy donors to specific biopharmaceuticals can predict potential immunogenicity

  • CD4+/CD8+ T-cell activation analysis has demonstrated that albumin-conjugated UOX (PAT101) shows lower immune response compared to unconjugated rasburicase

• Near-infrared-fluorescence amplification:

  • Nanostructured plasmonic gold substrates enhance detection sensitivity for antibodies

  • This technology enables simultaneous detection of multiple antibodies with quantification of immunoglobulin avidities

• People Also Ask (PAA) data mining:

  • Google's PAA feature appears in over 80% of English searches, generally within the first few results

  • This data provides valuable insights into researcher questions and needs, helping to guide experimental design and troubleshooting resources

How can researchers troubleshoot contradictory results when using UOX antibodies?

When researchers encounter contradictory results with UOX antibodies, systematic troubleshooting is essential:

• Antibody validation discrepancies:

  • Cross-validate using multiple antibodies targeting different epitopes

  • Verify antibody lot-to-lot consistency with standardized positive controls

  • Perform epitope mapping to ensure the recognized region is present in your samples

• Species-specific considerations:

  • Confirm antibody species reactivity matches your experimental model

  • For cross-species studies, select antibodies validated across multiple species or use species-specific antibodies in parallel

  • Consider evolutionary differences in UOX structure (human UOX gene is naturally inactivated)

• Experimental conditions optimization:

  • For Western blotting: Adjust reducing conditions, sample preparation method, and blocking agents

  • For immunohistochemistry: Compare different fixation methods and antigen retrieval protocols

  • For ELISA: Optimize coating concentration, blocking buffer, and detection system

• Resolving knockout model inconsistencies:

  • Different UOX knockout strategies may yield varying phenotypes:

  Knockout Strategy	Targeted Region	Genetic Background	Serum Uric Acid (μmol/L)	Phenotype	Reference
  CRISPR/Cas9	Exons 2-4	C57BL/6J	1351.04±276.58	Death by 4 weeks without allopurinol
  Neomycin cassette	Exon 3	C57BL/6J*129Sv hybrid	650	12× higher than WT
  TALEN	28bp of exon 3	C57BL/6J	420-520	2-3× higher than WT

• Combining orthogonal techniques:

  • Correlate antibody-based detection with functional assays (e.g., enzymatic activity)

  • Compare protein detection results with mRNA expression data

  • Verify protein-protein interactions using multiple methods (co-immunoprecipitation, proximity ligation, mass spectrometry)

When troubleshooting contradictory results, researchers should document all experimental conditions meticulously and contact antibody manufacturers for technical support with specific lot numbers and detailed protocols.