DUSP13 Antibody, HRP conjugated

What is DUSP13 and what are its primary functions in cellular signaling?

DUSP13 (Dual Specificity Phosphatase 13) is a member of the highly conserved protein-tyrosine phosphatase superfamily that plays vital roles in cell cycle regulation. It exhibits intrinsic phosphatase activity towards both phospho-seryl/threonyl and phospho-tyrosyl residues with similar specific activities in vitro. Research has established that DUSP13 specifically dephosphorylates MAPK8/JNK and MAPK14/p38, but not MAPK1/ERK2 .

DUSP13 exists in two major isoforms:

• DUSP13A (MDSP): Encoded by exons 1-3, known as muscle-restricted dual-specificity phosphatase

• DUSP13B (TMDP): Encoded by exons 7-9, known as testis and skeletal-muscle-specific dual-specificity phosphatase

These isoforms are expressed from alternative open reading frames (ORFs) of the same gene, which is extremely rare in eukaryotes . DUSP13 is primarily expressed in skeletal muscle and testis, suggesting tissue-specific regulatory functions .

How do HRP-conjugated antibodies work in laboratory applications?

HRP (Horseradish Peroxidase)-conjugated antibodies function as detection reagents by catalyzing colorimetric, chemiluminescent, or fluorescent reactions. When using DUSP13 antibodies conjugated to HRP, the enzyme enables direct detection without requiring secondary antibodies, simplifying experimental workflows and potentially reducing background noise.

For DUSP13 detection, researchers typically employ HRP-conjugated antibodies in methods such as:

• ELISA: The antibody-HRP conjugate binds to DUSP13 and catalyzes the conversion of substrates like TMB (3,3',5,5'-Tetramethylbenzidine) to produce a measurable signal proportional to DUSP13 concentration .

• Western blotting: Following protein transfer to membranes, HRP-conjugated DUSP13 antibodies enable protein detection through chemiluminescent substrates.

• Immunohistochemistry: HRP-conjugated antibodies allow for visualization of DUSP13 expression patterns in tissue sections.

The conjugation process preserves both the antibody's specificity for DUSP13 and the enzymatic activity of HRP, offering sensitive detection capabilities down to nanogram levels of target protein .

What are the optimal experimental conditions for using DUSP13 antibody, HRP conjugated in ELISA?

For optimal ELISA performance with HRP-conjugated DUSP13 antibodies, researchers should implement the following protocol:

Recommended ELISA Protocol:

• Coating: Immobilize capture antibody (non-conjugated anti-DUSP13) at 1-2 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Use 1-5% BSA or non-fat milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Sample incubation: Apply tissue lysates or recombinant DUSP13 diluted in blocking buffer for 1-2 hours at room temperature or overnight at 4°C.

• Detection: Apply HRP-conjugated DUSP13 antibody at optimal dilution (typically 1:1000 to 1:5000) in blocking buffer for 1-2 hours at room temperature.

• Substrate reaction: Use TMB substrate and measure absorbance at 450 nm after adding stop solution.

Critical Parameters:

• Temperature stability is essential for reproducibility

• Thorough washing between steps (3-5 times with PBS-T)

• Proper controls must be included (positive, negative, and background controls)

• Signal development should be monitored to prevent oversaturation

Researchers should validate antibody performance with known positive and negative samples before experimental use. Previous studies suggest anti-Human Kappa HRP shows lower background binding compared to polyclonal Rabbit Anti-Human IgG/HRP antibody for sandwich ELISA formats .

How can researchers differentiate between DUSP13A and DUSP13B isoforms in their experiments?

Differentiating between DUSP13A (MDSP) and DUSP13B (TMDP) isoforms requires careful experimental design due to their shared gene locus. Researchers can employ the following strategies:

1. Transcript-specific detection:

• Utilize RNAscope™ Multiplex Fluorescent V2 Assay with probe Mn-Dusp13a-O1-C1 for DUSP13A-specific detection

• Perform RT-PCR with isoform-specific primers that span unique exon junctions

2. Protein-specific detection:

• Select antibodies that target different molecular weights:

  • DUSP13A: 22 kDa

  • DUSP13B: 36 kDa

• Use antibodies targeting unique epitopes (e.g., antibodies directed against AA 81-198 region)

3. Functional discrimination:

• DUSP13B shows stronger anti-apoptotic ability than DUSP13A in H₂O₂-treated cardiomyocytes (54.7% vs. 23.7% reduction in apoptosis)

• DUSP13B, but not DUSP13A, demonstrates MAPK phosphatase activity in COS-7 and HEK 293 cells

For definitive isoform identification, researchers should combine multiple approaches rather than relying on a single detection method. Western blotting with careful molecular weight discrimination remains the most reliable technique for distinguishing the isoforms at the protein level.

How can DUSP13 antibodies be employed to study ASK1-mediated apoptosis pathways?

DUSP13 antibodies provide valuable tools for investigating the regulatory mechanisms of ASK1-mediated apoptosis, particularly because DUSP13A functions as a novel regulator of ASK1. When designing experiments to study this pathway, researchers should consider the following experimental approaches:

1. Protein-Protein Interaction Studies:

• Co-immunoprecipitation using anti-ASK1 antibody followed by DUSP13A detection can confirm interactions in various cell types beyond HEK 293 cells

• Use DUSP13A antibodies to perform pull-down assays to identify the N-terminal domain of ASK1 (residues 1-666) as the binding region for DUSP13A

2. Activation State Analysis:

• Utilize DUSP13 and phospho-ASK1 (Ser-83) antibodies to monitor how DUSP13A enhances ASK1 autophosphorylation in a dose-dependent manner

• Track kinase activity through immunoblotting for downstream targets JNK and p38 MAPK

3. Apoptosis Pathway Monitoring:

• Detect caspase-3 and caspase-9 cleavage following ASK1 activation by DUSP13A

• Use cytochrome c release assays in conjunction with DUSP13 antibodies to measure mitochondrial apoptotic events

4. Competitive Binding Assays:

• Employ DUSP13 antibodies to demonstrate how DUSP13A competes with Akt1 (a negative regulator) for binding to ASK1

• Gradually increase DUSP13A while monitoring Akt1 displacement from ASK1 complexes

Research has shown that DUSP13A enhances ASK1 kinase activity independent of its phosphatase activity. This activation leads to increased caspase-3 activity and caspase-9 cleavage, ultimately enhancing ASK1-mediated cell death .

What strategies can be used to validate the specificity of DUSP13 antibody, HRP conjugated?

Validating antibody specificity is crucial for ensuring experimental integrity. For HRP-conjugated DUSP13 antibodies, researchers should implement a comprehensive validation strategy:

1. Genetic Controls:

• Perform small interfering RNA (siRNA) knockdown of DUSP13 expression and verify reduced signal in Western blot or IHC

• Use DUSP13 knockout cell lines or tissues as negative controls

• Employ overexpression systems with tagged DUSP13 constructs as positive controls

2. Peptide Competition Assays:

• Pre-incubate the HRP-conjugated DUSP13 antibody with purified recombinant DUSP13 protein

• Observe signal reduction or elimination in subsequent applications

• Demonstrate dose-dependent competition with increasing concentrations of blocking peptide

3. Cross-Reactivity Assessment:

• Test against related phosphatases, particularly other DUSP family members

• Examine reactivity across multiple species to confirm expected conservation patterns

• Verify molecular weight specificity (22 kDa for DUSP13A and 36 kDa for DUSP13B)

4. Multi-method Concordance:

• Compare results across multiple applications (Western blot, IHC, ELISA)

• Verify localization patterns match known distribution (testis and skeletal muscle enrichment)

• Use alternative antibodies targeting different epitopes of DUSP13

5. Phosphatase Activity Correlation:

• Connect antibody staining intensity to functional phosphatase activity using OMFP (3-O-Methylfluorescein Phosphate) assays

• Correlate expression levels detected by the antibody with functional outcomes

The most robust validation combines multiple approaches to establish confidence in antibody specificity before proceeding with critical experiments or publishing results.

How does DUSP13 regulation of p53 impact experimental design when using DUSP13 antibodies?

Recent research has revealed a complex relationship between p53 and DUSP13 expression that researchers must consider when designing experiments using DUSP13 antibodies:

1. Alternative Promoter Activation:

• p53 strongly activates DUSP13 expression from an alternative promoter in the intron

• This promoter is activated by both endogenous and ectopically expressed p53

• The resulting isoform (TMDP-L1) localizes to the perinuclear region

Experimental Implications:

• When studying DUSP13 in p53 wild-type cells, researchers must account for potential p53-dependent expression variations

• Treatment with p53 activators (actinomycin D + nutlin-3a) dramatically increases DUSP13 expression (up to 55-fold)

• Use p53-null cell lines as controls to distinguish p53-dependent vs. independent DUSP13 regulation

2. Synergistic Regulation:

Methodological Considerations:

• Include appropriate controls for p53 status in cell lines

• Monitor p53 activation status alongside DUSP13 detection

• When using cancer cell lines with varying p53 status, anticipate differential DUSP13 expression patterns

3. Subcellular Localization:

• p53-induced DUSP13 isoform exhibits distinct perinuclear localization

• This pattern differs from the typical distribution of other DUSP13 isoforms

This regulatory relationship suggests that DUSP13 may modulate stress responses through inactivation of stress-activated MAPKs, representing a novel feedback mechanism in the p53 pathway . Researchers should carefully control for p53 status and activation when interpreting DUSP13 antibody results across different experimental systems.

What are the most common challenges when using DUSP13 antibody, HRP conjugated, and how can they be addressed?

Researchers frequently encounter several technical challenges when working with HRP-conjugated DUSP13 antibodies. Here are the most common issues and evidence-based solutions:

1. High Background Signal:

• Cause: Non-specific binding or excessive antibody concentration

• Solution: Optimize blocking (use 5% BSA rather than milk proteins) and increase washing steps (5× with 0.1% Tween-20 in PBS)

• Evidence: Studies show that anti-Human Kappa HRP exhibits lower background binding compared to polyclonal Rabbit Anti-Human IgG/HRP antibody in sandwich ELISA formats

2. Weak or Absent Signal:

• Cause: Insufficient antigen, denatured antibody, or suboptimal detection conditions

• Solution: Verify DUSP13 expression in your sample (highest in testis and skeletal muscle); use antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) ; ensure proper HRP substrate handling

• Evidence: DUSP13 has tissue-specific expression patterns, and some cell lines may express minimal levels

3. Multiple Bands in Western Blots:

• Cause: Detection of both DUSP13A (22 kDa) and DUSP13B (36 kDa) isoforms, degradation products, or cross-reactivity

• Solution: Use peptide competition to confirm specificity; optimize SDS-PAGE conditions; employ recombinant proteins as size controls

• Evidence: Both 22 kDa and 36 kDa bands represent legitimate DUSP13 isoforms, not non-specific binding

4. Variable Results Across Experiments:

• Cause: Antibody degradation from improper storage or handling

• Solution: Aliquot antibody upon receipt; avoid repeated freeze-thaw cycles; store in recommended buffer (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Evidence: Commercial recommendations based on stability testing suggest single-use aliquots stored at -20°C maintain optimal activity

5. Cross-Reactivity in Multi-Protein Assays:

• Cause: Antibody binding to homologous phosphatase domains

• Solution: Validate antibody specificity using DUSP13 knockdown controls; use epitope-mapped antibodies targeting unique regions

• Evidence: Antibodies targeting AA 81-198 or AA 147-173 regions show improved specificity

Implementing these evidence-based solutions will significantly improve experimental outcomes when working with HRP-conjugated DUSP13 antibodies.

How can researchers optimize signal-to-noise ratio when using DUSP13 antibody, HRP conjugated in different tissue types?

Optimizing signal-to-noise ratio is crucial for obtaining reliable results with HRP-conjugated DUSP13 antibodies, particularly across diverse tissue types. The following tissue-specific approaches are based on published methodologies:

1. Skeletal Muscle Tissue (High DUSP13 Expression):

• Optimal Dilution: Use higher dilution ranges (1:1000-1:2000 for WB; 1:100-1:200 for IHC)

• Fixation Method: 4% paraformaldehyde for 10-15 minutes provides optimal epitope preservation

• Background Reduction: Pre-adsorb antibody with acetone powder from mouse liver to reduce cross-reactivity

• Buffer Optimization: Add 0.1% Triton X-100 to improve antibody penetration

2. Testicular Tissue (High DUSP13 Expression):

• Antigen Retrieval: Use TE buffer pH 9.0 for optimal epitope exposure

• Detection System: Employ polymer-based HRP detection systems rather than avidin-biotin methods

• Blocking Strategy: 10% normal goat serum with 1% BSA significantly reduces background

• Washing Protocol: Extended washing steps (5× for 5 minutes each)

3. Neuronal Tissue (Low DUSP13 Expression):

• Signal Amplification: Use tyramide signal amplification (TSA) system to enhance detection sensitivity

• Antibody Concentration: Higher concentration (1:20-1:50) combined with longer incubation (overnight at 4°C)

• Control Strategy: Include human neuroblastoma SK-N-SH cells as positive control

• Alternative Approach: RNAscope™ for transcript detection followed by protein validation

4. Cardiac Tissue (Variable DUSP13 Expression):

• Pretreatment: Hydrogen peroxide (3%, 10 minutes) to block endogenous peroxidase activity

• Sensitivity Enhancement: Use DUSP13B-specific detection for stronger signal in cardiomyocytes

• Validation Control: Compare H₂O₂-treated versus untreated samples (DUSP13B has strong anti-apoptotic effects)

• Multiplexing Strategy: Co-stain with annexin V and 7-AAD to correlate DUSP13 expression with apoptotic status

Universal Optimization Techniques:

• Titrate antibody concentrations for each tissue type

• Include absorption controls with recombinant DUSP13 protein

• Adjust incubation times based on tissue thickness and fixation method

• Optimize substrate development time for each application

These tissue-specific approaches significantly improve signal-to-noise ratios and enhance the reliability of experimental results when using HRP-conjugated DUSP13 antibodies.

What controls are essential when using DUSP13 antibody, HRP conjugated for studying apoptotic pathways?

When investigating apoptotic pathways using HRP-conjugated DUSP13 antibodies, a comprehensive control strategy is essential for accurate data interpretation:

1. Expression Controls:

2. Apoptosis Pathway Controls:

• ASK1 Activity Correlation: Monitor ASK1 autophosphorylation alongside DUSP13 detection to establish functional relationships

• Caspase Activation Verification: Confirm caspase-3 and caspase-9 cleavage in cells with enhanced DUSP13 expression

• Stimulus-Specific Controls: Compare H₂O₂-treated cells (where DUSP13B exhibits anti-apoptotic activity) with untreated samples

• Phosphatase Activity Control: Include DUSP13 DACS mutant (catalytically inactive) to differentiate between phosphatase-dependent and independent functions

3. Technical Controls:

• Primary Antibody Omission: Evaluate background from detection system

• Isotype Control: Use irrelevant HRP-conjugated antibody of same isotype

• Absorption Control: Pre-incubate antibody with recombinant DUSP13 protein

• Substrate-Only Control: Assess endogenous peroxidase activity

4. Biological Pathway Controls:

• p53 Status Control: Use p53-null and p53-wild-type cells to account for p53-dependent DUSP13 expression

• Akt1 Competition Analysis: Monitor Akt1-ASK1 binding in relation to DUSP13A levels

• Downstream MAPK Monitoring: Track phosphorylation status of JNK and p38 to verify DUSP13 functional effects

Research has demonstrated that DUSP13A enhances ASK1-mediated apoptosis while DUSP13B exhibits anti-apoptotic effects in H₂O₂-treated cardiomyocytes . Including these essential controls will ensure proper interpretation of DUSP13's complex, context-dependent roles in apoptotic pathways.

What emerging applications of DUSP13 antibody, HRP conjugated could advance our understanding of muscle development and regeneration?

Recent research suggests several promising applications of HRP-conjugated DUSP13 antibodies for investigating muscle development and regeneration:

1. Muscle Stem Cell Fate Transition:

• DUSP13, along with DUSP27, appears to function as a key switch in muscle stem cell (MuSC) transition from proliferation to differentiation during myogenesis

• HRP-conjugated DUSP13 antibodies could enable high-resolution temporal mapping of this transition

• Potential Application: Tracking DUSP13 expression during different phases of muscle regeneration following injury

2. Myogenin-Mediated Regulation:

• Recent evidence shows that myogenin (MYOG) regulates DUSP13 to inhibit apoptosis induced by hydrogen peroxide

• HRP-conjugated antibodies could help characterize this regulatory circuit in detail

• The DUSP13 promoter region shows strong luciferase activity (40.5-fold) when activated by MYOG, with core binding sites located within 500bp upstream of the 5′UTR

3. Dual-Track Analysis of DUSP13 Isoforms:

• DUSP13B demonstrates stronger anti-apoptotic effects than DUSP13A in cardiomyocytes (54.7% vs. 23.7% reduction in apoptosis)

• Isoform-specific detection could reveal differential roles in muscle development

• Integration with RNAscope™ for simultaneous detection of transcripts and proteins

4. Stress Response in Muscle Fibers:

• DUSP13 modulates stress responses by deactivating MAPKs , suggesting a protective role in muscle adaptation

• HRP-conjugated antibodies could map stress-responsive DUSP13 expression patterns in different muscle fiber types

• Potential for identifying therapeutic targets for muscle wasting conditions

5. Methodological Advancements:

• Combining HRP-conjugated DUSP13 antibodies with specific fluorescent markers for muscle progenitor states

• Sequential chromogenic detection of multiple proteins in muscle tissue sections

• Adapting time-resolved multiplexed detection systems for studying DUSP13 dynamics in living muscle tissue

These emerging applications could significantly advance our understanding of DUSP13's role in muscle biology, potentially leading to new therapeutic approaches for muscle injuries, dystrophies, and age-related sarcopenia.