IDUA Antibody

What is IDUA and what is its physiological function?

Alpha-L-iduronidase (IDUA) is a lysosomal enzyme that catalyzes the hydrolysis of unsulfated alpha-L-iduronosidic linkages in dermatan sulfate. This enzyme plays a critical role in the degradation of glycosaminoglycans (GAGs) such as dermatan sulfate and heparan sulfate by specifically hydrolyzing their terminal alpha-L-iduronic acid residues . IDUA belongs to the glycosyl hydrolase 39 family and is essential for proper lysosomal degradation of these glycosaminoglycans . The enzymatic function is particularly important in maintaining cellular homeostasis, as mutations in the IDUA gene resulting in enzymatic deficiency lead to mucopolysaccharidosis type I (MPS I), an autosomal recessive disease characterized by GAG accumulation in tissues and organs .

What types of IDUA antibodies are currently available for research applications?

Several validated IDUA antibodies are available for research purposes, each with distinct characteristics:

These antibodies vary in their specificity, host species, and validated applications, allowing researchers to select the most appropriate antibody for their specific experimental needs .

What is the expected molecular weight for IDUA detection and why is this important?

The calculated molecular weight of IDUA protein is approximately 71-73 kDa, with observed molecular weights in experimental studies typically around 73-74 kDa . In Western blot analysis, for example, specific bands for alpha-L-iduronidase/IDUA are detected at approximately 74 kDa under reducing conditions . This molecular weight information is critical for researchers to:

• Verify antibody specificity by confirming detection at the expected size

• Differentiate between full-length IDUA and potential degradation products

• Identify post-translational modifications that may alter apparent molecular weight

• Ensure proper experimental controls when validating novel antibodies

• Interpret Western blot results accurately when studying IDUA expression

What dilutions should be used for IDUA antibodies in different applications?

Optimal dilutions vary by antibody and application type. Based on manufacturer recommendations:

It is important to note that optimal dilutions should be determined by each laboratory for each application and sample type . Titration experiments are recommended to find the balance between specific signal and background.

What cell lines and tissues show confirmed IDUA expression for use as positive controls?

Based on validation data from antibody manufacturers, IDUA has been detected in:

Cell lines:

• IMR-90 human lung fibroblast cell line

• HepG2 human hepatocellular carcinoma cell line

• A549 cells

• HEK-293 cells

• LNCaP cells

Tissues:

• Human kidney tissue

• Mouse brain tissue

• Rat brain tissue

• Mouse kidney tissue

• Human prostate cancer tissue

These validated positive samples provide researchers with appropriate controls for experimental design and antibody validation .

What are the optimal storage conditions for maintaining IDUA antibody efficacy?

For maximum stability and retention of antibody activity:

• Short-term storage (up to 1 week): Store at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Storage buffer composition: Most IDUA antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) or similar buffers

• Avoid repeated freeze-thaw cycles as they can lead to denaturation and loss of antibody activity

• Shelf life: Typically one year from date of dispatch when stored properly

Proper storage is essential for maintaining antibody specificity and sensitivity throughout the experimental timeline .

How can non-specific binding be minimized when using IDUA antibodies?

To improve specificity and reduce background in IDUA detection:

• Optimize blocking conditions using appropriate blocking agents (BSA, serum, or commercial blockers)

• Titrate antibody concentrations to find the optimal dilution that minimizes background while maintaining specific signal

• Increase washing stringency and duration between antibody incubations

• For IHC applications, optimized antigen retrieval is critical - use TE buffer pH 9.0 or citrate buffer pH 6.0 as suggested by manufacturers

• Include appropriate negative controls (secondary antibody only, isotype controls)

• Consider sample-specific optimizations based on tissue or cell type being analyzed

• For Western blot applications, use the recommended buffer systems (e.g., Immunoblot Buffer Group 1)

What factors affect the detection sensitivity of IDUA in Western blotting?

Several experimental factors influence IDUA detection sensitivity:

• Sample preparation: Use proper lysis buffers with protease inhibitors to prevent degradation

• Protein loading: Optimize protein concentration to achieve adequate signal without overloading

• Membrane selection: PVDF membranes are recommended for IDUA detection

• Transfer efficiency: Optimize transfer conditions for high molecular weight proteins (~73 kDa)

• Blocking conditions: Over-blocking can reduce antibody binding

• Antibody quality and concentration: Verify activity and titrate to optimal concentration

• Incubation conditions: Temperature and duration affect binding kinetics

• Detection system: Enhanced chemiluminescence systems improve sensitivity

• Reducing conditions: All validated protocols used reducing conditions for IDUA detection

How can researchers verify IDUA antibody specificity in their experimental systems?

To validate antibody specificity:

• Confirm detection at the expected molecular weight (73-74 kDa for IDUA)

• Use validated positive controls (e.g., human kidney tissue, IMR-90 cells)

• Verify cellular localization patterns (IDUA should show primarily cytoplasmic/lysosomal localization)

• Compare results from multiple antibodies targeting different IDUA epitopes

• Consider using IDUA knockdown/knockout samples as specificity controls

• For western blot applications, test under reducing conditions as specified in validation data

• For immunohistochemistry, compare staining patterns with literature-reported IDUA expression

How can IDUA antibodies be utilized in mucopolysaccharidosis type I (MPS I) research?

IDUA antibodies offer valuable tools for MPS I research:

• Analyze IDUA protein levels in patient-derived samples compared to controls

• Study IDUA localization patterns in affected versus unaffected cells

• Monitor IDUA levels during enzyme replacement therapy (ERT)

• Evaluate therapeutic efficacy by measuring IDUA restoration in treated tissues

• Investigate protein-protein interactions through co-immunoprecipitation studies

• Detect recombinant IDUA distribution following therapeutic administration

• Study cellular pathways affected by IDUA deficiency

• Track enzyme uptake in cellular models of MPS I

These applications help advance understanding of disease mechanisms and therapeutic approaches for MPS I .

What considerations should be made when using IDUA antibodies in enzyme replacement therapy (ERT) studies?

ERT studies using IDUA antibodies require special considerations:

• Almost all ERT-treated patients develop anti-IDUA antibodies, which may affect therapeutic efficacy

• Differentiate between endogenous and recombinant IDUA when studying tissue distribution

• Consider antibody specificity when monitoring recombinant IDUA (laronidase) in treated patients

• Research shows that patients with low anti-IDUA antibody levels may exhibit more robust decreases in GAG body load

• Patients may develop immune tolerance after approximately 2 years of treatment with IDUA

• Most IDUA-directed antibodies appear to be non-neutralizing, not affecting the enzyme's active site

• The long-term impact of anti-IDUA antibodies on treatment efficacy remains incompletely understood

What techniques are optimal for studying IDUA localization in cellular compartments?

To investigate IDUA's subcellular distribution:

• Immunofluorescence microscopy: IDUA has been successfully detected in fixed HepG2 cells using specific antibodies at 15 μg/mL concentration

• Co-localization studies with lysosomal markers (e.g., LAMP1, LAMP2)

• Cell fractionation followed by Western blot analysis of different fractions

• Fluorescent ICC staining of cells on coverslips, as validated for IDUA detection

• Counterstaining with DAPI for nuclear visualization helps establish cellular context

• Specific staining has been localized to the cytoplasm in validated cell models

• Use appropriate fluorophore-conjugated secondary antibodies (e.g., NorthernLights 557-conjugated Anti-Sheep IgG)

How can researchers interpret conflicting IDUA antibody results in different experimental systems?

When encountering discrepancies in IDUA detection:

• Compare antibody epitopes - different antibodies target different regions of IDUA

• Consider species-specific variations in IDUA sequence and expression

• Evaluate tissue-specific post-translational modifications that may affect antibody binding

• Assess fixation and sample preparation effects on epitope accessibility

• Review antibody validation data for application-specific recommendations

• Verify experimental conditions align with manufacturer-validated protocols

• Consider antibody cross-reactivity with related glycosidase family members

• Test multiple antibodies targeting different epitopes to confirm results

How are IDUA antibodies being used to investigate the relationship between enzyme structure and function?

Current research applications include:

• Studying how specific IDUA mutations affect protein stability and expression

• Investigating structure-function relationships through epitope-specific antibodies

• Analyzing how post-translational modifications influence IDUA activity

• Examining conformational changes in IDUA during substrate binding

• Evaluating how therapeutic approaches affect IDUA folding and stability

• Studying IDUA interactions with other lysosomal proteins and degradation machinery

• Investigating differences between recombinant and native IDUA isoforms

What are the considerations for using IDUA antibodies in combination with other analytical techniques?

For multi-modal analytical approaches:

• When combining with enzymatic activity assays, consider whether antibodies might interfere with active sites

• For mass spectrometry studies, verify antibody compatibility with sample preparation methods

• In live-cell imaging studies, validate that antibody binding doesn't alter normal protein function

• For high-throughput screening, optimize antibody-based detection for reproducibility

• When using with genomic techniques, correlate protein expression data with transcriptomic profiles

• For flow cytometry applications, optimize fixation and permeabilization protocols for IDUA detection

• In animal model studies, verify cross-reactivity with the species-specific IDUA variants