DUSP13 Antibody, Biotin conjugated

What is DUSP13 and why is it a significant research target?

DUSP13 (Dual Specificity Phosphatase 13) is an important phosphatase enzyme that dephosphorylates both phospho-seryl/threonyl and phospho-tyrosyl residues. It plays critical roles in cellular signaling pathways, particularly in muscle development and apoptosis regulation. DUSP13 specifically dephosphorylates MAPK8/JNK and MAPK14/p38, but not MAPK1/ERK2, in vitro . Recent studies have identified DUSP13 as a direct target for MYOD, making it a key switch in muscle development . Additionally, DUSP13A functions as a novel regulator of Apoptosis Signal-regulating Kinase 1 (ASK1), enhancing ASK1 kinase activity and thus inducing ASK1-mediated apoptosis through caspase-3 activation . These dual functions in muscle development and apoptosis regulation make DUSP13 a significant target for research in developmental biology, cancer research, and neurodegenerative diseases.

How does a biotin-conjugated DUSP13 antibody differ functionally from other conjugated versions?

Biotin-conjugated DUSP13 antibodies utilize the exceptional biotin-streptavidin binding system, which offers several functional advantages over other conjugations:

The biotin-conjugated antibodies provide exceptional versatility due to their ability to interact with various streptavidin-linked molecules, allowing for signal amplification and modular experimental design. The biotin-streptavidin interaction is 10^3 to 10^6 times stronger than typical antigen-antibody interactions, making it highly suited for isolation and amplification of weak signals . Additionally, biotin's small size (240 Da) and flexible valeric side chain facilitate protein labeling without interfering with the antibody's natural binding properties .

What are the optimal applications for biotin-conjugated DUSP13 antibodies in molecular research?

Biotin-conjugated DUSP13 antibodies excel in multiple research applications, with optimization parameters for each:

For achieving optimal results in protein-protein interaction studies, biotin-conjugated DUSP13 antibodies are particularly effective when combined with streptavidin-saporin systems, which allow for targeted intracellular delivery and functional studies of DUSP13's role in cellular pathways . The modular nature of biotin-streptavidin binding enables researchers to efficiently screen multiple experimental conditions without having to create multiple direct conjugates .

How can I optimize the detection of DUSP13 in different tissue types using biotin-conjugated antibodies?

Tissue-specific optimization for DUSP13 detection requires adjustment of several experimental parameters:

• Tissue-specific considerations:

  • For skeletal muscle tissue: Use 1:500 dilution with extended incubation (overnight at 4°C) to account for dense tissue architecture

  • For testis tissue: Use 1:1000 dilution with standard processing due to high endogenous expression

  • For neuronal tissues: Consider 1:800 dilution with specialized permeabilization steps

• Antigen retrieval optimization:

  • Primary option: TE buffer pH 9.0 with heat-induced retrieval (95°C for 20 minutes)

  • Alternative option: Citrate buffer pH 6.0 when studying phosphorylation-dependent interactions

• Signal amplification strategy:

  • For low expression tissues: Implement streptavidin-biotin amplification system with tyramide signal enhancement

  • For co-localization studies: Use streptavidin conjugated to distinct fluorophores for multiplex detection

• Background reduction techniques:

  • Pre-block endogenous biotin with avidin/biotin blocking kit

  • Include carrier proteins (BSA 0.1-1%) in diluents to reduce non-specific binding

  • Use 0.02% sodium azide in storage buffers to maintain antibody integrity

What are the optimal methodologies for validating the specificity of biotin-conjugated DUSP13 antibodies?

A multi-step validation approach ensures specificity of biotin-conjugated DUSP13 antibodies:

• Expression system validation:

  • Test antibodies against recombinant DUSP13 protein expressed in E. coli

  • Compare against DUSP13 overexpressed in mammalian cells (HEK 293 recommended)

  • Include DUSP13 mutants (e.g., DUSP13_D97A and DUSP13_C128S) as negative controls

• Knockout/knockdown validation:

  • Verify signal reduction in DUSP13 siRNA-treated cells

  • Confirm specificity with CRISPR/Cas9 knockout models where available

• Cross-reactivity assessment:

  • Test against related DUSP family members to assess potential cross-reactivity

  • Verify tissue-specific expression patterns (high in testis tissue and skeletal muscle)

• Biotin conjugation-specific validation:

  • Confirm that biotin conjugation doesn't interfere with epitope recognition

  • Compare detection efficiency between biotin-conjugated and unconjugated antibodies

  • Test for biotin interference from endogenous sources in biological samples

For phosphatase activity studies, a validation approach using 3-O-Methylfluorescein Phosphate (OMFP) substrate can determine if antibody binding affects DUSP13 enzymatic function. The assay should be performed at 30°C in reaction buffer containing 100 mM Tris-HCl pH 8.2, 40 mM NaCl, 1 mM DTT, 20% glycerol, and 500 μM OMFP, with fluorescence measured at excitation 485 nm and emission 525 nm .

How can I mitigate biotin interference issues in experimental systems using biotin-conjugated DUSP13 antibodies?

Biotin interference is a significant concern in research and diagnostic applications. Implement these strategies to minimize interference:

• Sample preparation modifications:

  • Pre-treat samples with streptavidin-agarose beads to deplete endogenous biotin

  • Use non-biotin containing culture media when growing cells for experiments

  • Consider using serum alternatives if working with samples from subjects taking biotin supplements

• Assay design considerations:

  • Include biotin-blocking steps before adding biotin-conjugated antibodies

  • Implement stringent washing steps with detergent-containing buffers

  • Use dilution series to determine the minimum effective antibody concentration

• Alternative detection approaches:

  • Consider two-step detection methods where streptavidin reagent is added after washing

  • Implement competitive binding controls to assess endogenous biotin levels

  • For critical applications, validate findings using non-biotin conjugated antibodies

• Controls to include:

  • Samples with added free biotin at varying concentrations (10-1000 ng/mL)

  • Streptavidin-only controls without biotin-conjugated antibody

  • Parallel assay with non-biotin conjugated detection system

The FDA has issued safety recommendations regarding biotin interference in clinical diagnostics, which can also inform research practices. For research applications, verify that laboratory personnel are aware of potential biotin sources and implement appropriate controls .

How can biotin-conjugated DUSP13 antibodies be utilized in protein-protein interaction studies to investigate DUSP13's role in cell signaling pathways?

Biotin-conjugated DUSP13 antibodies offer sophisticated approaches for mapping protein interaction networks:

• In vivo competition assay methodology:

  • Transfect cells with FLAG-ASK1 (1 μg), HA-DUSP13A (0-2 μg), and HA-Akt1 (1 μg)

  • After 48 hours, lyse cells in PTP buffer (4°C for 30 min)

  • Immunoprecipitate with anti-FLAG agarose (4°C for 4 hours)

  • Analyze bound proteins by SDS-PAGE and immunoblotting

  • This approach revealed that DUSP13A competes with Akt1 for binding to ASK1

• ASK1 oligomerization assay:

  • Express FLAG-ASK1 and HA-ASK1 in separate cell populations

  • Mix lysates and add recombinant DUSP13 (wild-type or mutant)

  • Immunoprecipitate with anti-FLAG agarose

  • Detect co-precipitated HA-ASK1 by immunoblotting

  • This method demonstrated DUSP13's effect on ASK1 oligomerization

• Streptavidin-saporin approach:

  • Conjugate biotin-DUSP13 antibody with streptavidin-linked saporin

  • Apply to cells at concentrations starting at 200 nM

  • Monitor internalization and cell killing

  • This strategy helps evaluate DUSP13's functional role in cellular pathways

• Proteomic analysis integration:

  • Use biotin-conjugated DUSP13 antibodies to purify DUSP13 complexes

  • Perform mass spectrometry to identify associated proteins

  • Validate key interactions with reciprocal co-immunoprecipitation

  • Map interaction networks to specific cellular pathways and functions

These approaches have revealed that DUSP13A functions as a positive regulator of ASK1, enhancing its kinase activity independently of DUSP13A's phosphatase activity, suggesting a scaffolding role in signaling complexes .

What are the current methodological approaches for studying DUSP13's dual roles in muscle development and apoptosis using biotinylated antibodies?

Investigating DUSP13's dual functions requires specialized experimental designs:

• Muscle development analysis:

  • RNAscope Multiplex Fluorescent V2 Assay for simultaneous detection of DUSP13 with muscle markers

  • Use probes like Mn-Dusp13a-O1-C1 and Mn-Styxl2 (Dusp27)-C1

  • Visualize with TSA Vivid Fluorophore Kit 520

  • Counter-stain with antibodies against PAX7, MYOD, and MF20

  • This approach revealed DUSP13 as a direct target for MYOD in muscle development

• Apoptosis pathway investigation:

  • Analyze cytochrome c release from mitochondria using cellular fractionation

  • Detect cleaved caspase-3 (Asp-175) and caspase-9 as markers of apoptosis

  • Monitor phosphorylation status of ASK1 at Ser-83 using phospho-specific antibodies

  • These methods demonstrated DUSP13A's pro-apoptotic function through ASK1 activation

• Structure-function analysis:

  • Generate DUSP13 mutants lacking phosphatase activity (DUSP13_D97A and DUSP13_C128S)

  • Express mutants in cellular models using adenoviral vectors

  • Compare phenotypes with wild-type DUSP13 expression

  • This revealed that DUSP13's regulation of ASK1 is independent of its phosphatase activity

• In vivo functional studies:

  • Use streptavidin-linked saporin conjugated to biotinylated antibodies for selective targeting

  • Apply concentration gradients (starting at 200 nM) to evaluate dose-dependent effects

  • Monitor both muscle differentiation markers and apoptosis indicators

  • Integrate findings to understand tissue-specific functions

These methodologies have demonstrated that DUSP13 operates through different mechanisms in different contexts: as a phosphatase in some pathways and as a scaffolding protein in others, highlighting its versatility in cellular regulation .

What are common technical challenges when using biotin-conjugated DUSP13 antibodies in multi-step detection protocols?

Researchers frequently encounter several challenges with biotin-conjugated antibodies in complex protocols:

• High background signal issues:

  • Problem: Non-specific streptavidin binding or endogenous biotin

  • Solution: Include avidin/biotin blocking steps before antibody application

  • Validation: Run parallel detection with non-biotinylated antibody to compare background

• Signal masking in tissues with high endogenous biotin:

  • Problem: Particularly significant in kidney, liver, and brain tissues

  • Solution: Implement specialized blocking with free streptavidin pre-treatment

  • Validation: Include tissue-matched biotin quantification controls

• Inconsistent results between experiments:

  • Problem: Variable biotin conjugation efficiency between antibody lots

  • Solution: Standardize protocols using conjugation ratio assessments

  • Validation: Maintain reference standards across experiments

• Cross-reactivity with related DUSP family members:

  • Problem: DUSP family shares structural similarities

  • Solution: Validate with recombinant DUSP proteins and knockout models

  • Validation: Compare reactivity profiles against DUSP5, DUSP7, and DUSP10

• Diminished antibody performance after storage:

  • Problem: Biotin conjugates can lose activity over time

  • Solution: Store at -20°C with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Validation: Include positive controls with each experiment to verify activity

For optimizing signal-to-noise ratio in immunofluorescence applications, a systematic approach comparing various amplification methods revealed that tyramide signal amplification (TSA) with biotin-conjugated antibodies provided 4-8 fold signal enhancement compared to direct detection methods .

How can I design controlled experiments to distinguish between DUSP13's phosphatase-dependent and phosphatase-independent functions?

Distinguishing between DUSP13's dual functional modes requires carefully designed experiments:

• Phosphatase activity mutation strategy:

  • Generate DUSP13 mutants with substitutions at catalytic sites:

    • DUSP13_D97A (aspartic acid to alanine at position 97)

    • DUSP13_C128S (cysteine to serine at position 128)

  • Express these mutants alongside wild-type DUSP13

  • Compare phenotypic outcomes across experimental conditions

• Substrate-specific phosphatase assay:

  • Use 3-O-Methylfluorescein Phosphate (OMFP) as substrate

  • Measure activity at 30°C in reaction buffer (100 mM Tris-HCl pH 8.2, 40 mM NaCl, 1 mM DTT, 20% glycerol)

  • Monitor fluorescence at excitation 485 nm and emission 525 nm

  • Compare wild-type and mutant DUSP13 activity profiles

• Protein interaction analysis:

  • Perform co-immunoprecipitation with wild-type and mutant DUSP13

  • Identify differential binding partners using mass spectrometry

  • Validate key interactions with reciprocal co-immunoprecipitation

  • Map interactions to specific signaling pathways

• Functional outcome assessment:

  • Monitor ASK1 kinase activity in the presence of wild-type or mutant DUSP13

  • Assess downstream effects on JNK and p38 MAPK activation

  • Evaluate cellular outcomes (apoptosis, differentiation)

  • This approach demonstrated that DUSP13A enhances ASK1 activity independently of phosphatase function

Research using these approaches revealed that DUSP13A competes with Akt1 (a negative regulator) for binding to ASK1, thus promoting ASK1 activation through a scaffolding mechanism rather than enzymatic function . This exemplifies how DUSP13 can regulate signaling pathways through both catalytic and non-catalytic mechanisms.

How are biotin-conjugated DUSP13 antibodies being utilized in developing targeted therapeutic approaches for diseases involving dysregulated MAPK signaling?

The development of targeted therapeutics leveraging DUSP13 biology is an emerging research area:

• Antibody-drug conjugate (ADC) development:

  • Streptavidin-biotin conjugation allows rapid screening of antibody-toxin combinations

  • This platform enables assessment of both antitumor activity and pre-clinical safety

  • Example: Trastuzumab-SB-DM1 created via streptavidin-biotin showed comparable efficacy to clinically approved T-DM1

• Selective cellular targeting:

  • Streptavidin-ZAP (saporin) conjugation to biotinylated DUSP13 antibodies

  • Application at concentrations starting at 200 nM demonstrates selective internalization

  • This approach allows evaluation of DUSP13 inhibition in specific cell populations

• Therapeutic target validation:

  • DUSP13's role in ASK1-mediated apoptosis provides potential intervention points

  • Assessment of competing interactions between DUSP13A and Akt1 for ASK1 binding

  • This competition mechanism offers possibilities for modulating apoptotic pathways

• Tissue-specific therapeutic strategies:

  • DUSP13's role as a key switch in muscle development suggests applications for muscular disorders

  • Direct targeting of MYOD-DUSP13 pathways could modulate muscle regeneration

  • This approach builds on findings of DUSP13 as a direct MYOD target

Research has demonstrated that the streptavidin-biotin platform facilitates efficient generation of functionally active antibody-drug conjugates, providing a rapid and cost-effective screening method to identify promising therapeutic candidates . This approach has particular relevance for DUSP13-targeted therapies given its roles in both muscle development and apoptosis regulation.

What are the current methodological limitations in studying DUSP13 isoform-specific functions with available antibodies?

Current research faces several methodological challenges when investigating DUSP13 isoform specificity:

• Isoform discrimination limitations:

  • DUSP13 gene produces multiple protein isoforms (including DUSP13A and DUSP13B)

  • Many commercial antibodies react with multiple isoforms

  • Solution: Develop epitope-specific antibodies targeting unique regions of each isoform

• Cross-reactivity with related DUSP family members:

  • The catalytic domains of DUSP family proteins share structural similarities

  • This can lead to false positive signals in certain applications

  • Solution: Validate specificity with recombinant proteins and knockout controls

• Post-translational modification detection:

  • DUSP13 function is regulated by phosphorylation and other modifications

  • Most antibodies do not distinguish modified forms

  • Solution: Develop modification-specific antibodies for studying regulation mechanisms

• Tissue-specific expression challenges:

  • DUSP13 shows differential expression across tissues (high in testis and skeletal muscle)

  • Low expression in some tissues requires enhanced detection methods

  • Solution: Implement signal amplification strategies with biotin-streptavidin systems

• Temporal dynamics limitations:

  • Current methods provide static snapshots rather than dynamic information

  • Understanding temporal regulation requires new approaches

  • Solution: Develop biosensor systems using antibody-derived binding domains

Research has shown that DUSP13A and potentially other isoforms have distinct functions: DUSP13A regulates ASK1 in apoptosis pathways, while other forms may have tissue-specific roles in muscle development. Developing methods to distinguish these isoforms will be crucial for advancing therapeutic applications targeting specific pathways .