DUSP9 Antibody, HRP conjugated

What is DUSP9 and what is its molecular function?

DUSP9 (Dual specificity phosphatase 9) is a member of the dual specificity phosphatase family that dephosphorylates threonine/serine and tyrosine residues of its substrates . It has a calculated molecular weight of 42 kDa and is typically observed at 42-44 kDa in experimental conditions . DUSP9 specifically inactivates MAP kinases with particular specificity for the ERK family . It plays critical roles in multiple biological processes, including lipid metabolism and inflammatory responses, particularly by blocking apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and subsequent activation of p38 and c-Jun NH2-terminal kinase signaling pathways .

What are the key applications for DUSP9 antibodies in laboratory research?

DUSP9 antibodies are utilized across multiple experimental applications:

For HRP-conjugated antibodies specifically, they provide direct detection capability without requiring secondary antibodies, which is particularly valuable in ELISA applications and can reduce background signals in some experimental contexts .

What sample types can be analyzed with DUSP9 antibodies?

Based on the available literature, DUSP9 antibodies have demonstrated reactivity with:

Researchers have successfully used DUSP9 antibodies in studying various tissue samples, including liver specimens from mice with specific Dusp9 genetic modifications (knockout and transgenic models) . For immunohistochemistry, DUSP9 expression has been visualized in amnion membranes from pregnant mice at different developmental stages (15.5 and 18.5 dpc) .

What is the recommended storage protocol for maintaining DUSP9 antibody activity?

Optimal storage conditions for DUSP9 antibodies include:

• Temperature: Store at -20°C

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Stability: Stable for one year after shipment when properly stored

• Aliquoting: For the unconjugated antibody, aliquoting is unnecessary for -20°C storage, but is generally recommended for HRP-conjugated versions to minimize freeze-thaw cycles

Note that smaller-sized preparations (20μl) may contain 0.1% BSA as a stabilizer . For HRP-conjugated antibodies specifically, avoiding repeated freeze-thaw cycles is critical for maintaining enzymatic activity.

How does DUSP9 function in metabolic disease models?

DUSP9 plays a crucial role in preventing nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Research using conditional liver-specific Dusp9-knockout (Dusp9-CKO) mice and Dusp9-transgenic (Dusp9-TG) mice has demonstrated that:

• Dusp9 expression is markedly decreased in liver tissue of mice fed a high-fat diet (HFD)

• Hepatocyte-specific Dusp9 prevents:

  • Lipid accumulation

  • Glucose metabolism disorders

  • Enhanced inflammation

  • Liver fibrosis

The molecular mechanism involves DUSP9 blocking apoptosis signal-regulating kinase 1 (ASK1) phosphorylation, which prevents subsequent activation of p38 and c-Jun NH2-terminal kinase signaling pathways . This suggests DUSP9 as a potential therapeutic target for treating NAFLD and NASH.

When studying metabolic disease models with DUSP9 antibodies, researchers should consider:

• Using both knockout and overexpression models for comprehensive functional analysis

• Examining temporal changes in DUSP9 expression during disease progression

• Correlating DUSP9 levels with specific metabolic parameters

What methodological considerations are important when using DUSP9 antibodies in ELISA?

When using DUSP9 antibodies in ELISA, particularly HRP-conjugated versions, researchers should consider:

• Assay principle: Most DUSP9 ELISAs utilize sandwich ELISA technology where:

  • Anti-DUSP9 capture antibody is pre-coated onto 96-well plates

  • Biotin-conjugated anti-DUSP9 detection antibody binds to the captured DUSP9

  • HRP-Streptavidin is added to bind with the biotin-conjugated antibody

  • TMB substrate visualizes the HRP enzymatic reaction

• Protocol optimization:

  • Incubation times and temperatures should be strictly followed

  • Washing steps must be thorough to remove unbound conjugates

  • For directly HRP-conjugated DUSP9 antibodies, the protocol can be simplified by eliminating the streptavidin-HRP step

• Quantification:

  • The concentration of DUSP9 in samples should be calculated by preparing a standard curve

  • The concentration is proportional to the OD450 value measured by a microplate reader

• Controls and validation:

  • Include positive and negative controls to ensure assay specificity

  • Consider spike-recovery experiments to validate antibody performance in complex samples

How can researchers validate DUSP9 antibody specificity?

Validating antibody specificity is crucial for ensuring experimental rigor. For DUSP9 antibodies, consider:

• Genetic models:

  • Use tissue from Dusp9-knockout mice as negative controls

  • Compare with samples from Dusp9-overexpressing models

  • When using CRISPR/Cas9-edited cell lines lacking DUSP9, ensure complete knockout by verifying at both mRNA and protein levels

• Molecular weight verification:

  • DUSP9 has a calculated molecular weight of 42 kDa

  • In experimental conditions, it is typically observed at 42-44 kDa

  • Check for additional bands that might indicate cross-reactivity

• Peptide competition assay:

  • Pre-incubate the antibody with specific peptide immunogens

  • This should eliminate or significantly reduce specific binding

• Multiple antibody comparison:

  • Use antibodies targeting different epitopes of DUSP9

  • Consistent results across antibodies increase confidence in specificity

• Correlation with mRNA expression:

  • Compare antibody-based protein detection with RT-qPCR results

  • Research has shown corresponding changes in both DUSP9 mRNA and protein levels

What is the relationship between DUSP9 and miRNA regulation?

Recent research has uncovered important connections between DUSP9 and microRNA regulation:

• miR-132-3p targeting:

  • DUSP9 has been identified as a direct target of miR-132-3p

  • The miR-132-3p binding site in DUSP9 3′UTR is evolutionarily conserved across species

  • Luciferase reporter assays confirm direct binding between miR-132-3p and DUSP9 3′UTR

• Expression correlation:

  • miR-132-3p overexpression leads to decreased DUSP9 expression

  • miR-132-3p inhibition results in increased DUSP9 expression

  • This has been verified at both mRNA and protein levels

• Functional significance:

  • miR-132-3p modulates DUSP9-dependent p38/JNK signaling

  • This regulatory mechanism may contribute to inflammation and PGE2 release

  • The pathway has been implicated in preterm birth mechanisms

When studying this relationship, researchers should:

• Consider the temporal dynamics of miRNA-mediated regulation

• Use both gain- and loss-of-function approaches for miR-132-3p

• Examine downstream effects on p38/JNK phosphorylation status

How can researchers optimize immunohistochemistry protocols for DUSP9 detection?

For effective IHC detection of DUSP9 in tissue sections:

• Tissue preparation:

  • Use paraffin-embedded sections for optimal morphology preservation

  • Ensure proper fixation to maintain antigen integrity while allowing antibody access

• Antibody selection and dilution:

  • For unconjugated antibodies, a working dilution of 1:200 has been successfully used in published studies

  • For HRP-conjugated antibodies, further dilution optimization may be required

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended

  • Optimization of retrieval time and temperature may be necessary for different tissue types

• Visualization system:

  • For unconjugated primary antibodies, use horseradish peroxidase-conjugated secondary antibodies

  • Visualization with 3,3′-diaminobenzidine (DAB) produces a brown precipitate

  • Counterstain nuclei with hematoxylin for structural context

• Controls and validation:

  • Include tissue sections known to express DUSP9 as positive controls

  • Use sections from Dusp9-knockout models as negative controls when available

  • Perform peptide neutralization tests to confirm specificity

Examples from published research include successful DUSP9 immunohistochemical staining in amnion membranes from pregnant mice at 15.5 and 18.5 dpc, with nuclei counterstained with hematoxylin .

What are common issues when using HRP-conjugated DUSP9 antibodies?

When working with HRP-conjugated DUSP9 antibodies, researchers may encounter several challenges:

• High background signal:

  • Possible causes: Insufficient blocking, cross-reactivity, or excessive antibody concentration

  • Solution: Optimize blocking conditions (duration, buffer composition), increase wash stringency, and titrate antibody concentration

• Low signal intensity:

  • Possible causes: Degraded HRP activity, insufficient antigen, suboptimal substrate reaction

  • Solution: Verify HRP activity with control experiments, ensure proper storage, and optimize substrate incubation time

• Non-specific bands in Western blot:

  • Possible causes: Cross-reactivity with similar phosphatases, degradation products

  • Solution: Increase blocking stringency, use gradient gels to better resolve proteins of similar size, verify with knockout controls

• Variable results between experiments:

  • Possible causes: Inconsistent sample preparation, antibody degradation

  • Solution: Standardize sample collection and processing protocols, aliquot antibodies to avoid repeated freeze-thaw cycles

How can DUSP9 antibodies be used to study its interaction with ASK1?

Given DUSP9's role in blocking ASK1 phosphorylation, researchers can investigate this interaction using:

• Co-immunoprecipitation (Co-IP):

  • Use DUSP9 antibodies to pull down protein complexes

  • Probe for ASK1 in the immunoprecipitates

  • Verify the interaction bidirectionally by using ASK1 antibodies for pull-down

• Proximity ligation assay (PLA):

  • Apply DUSP9 and ASK1 primary antibodies simultaneously

  • Use species-specific PLA probes to detect close proximity

  • Quantify interaction events through fluorescent signal analysis

• Domain mapping:

  • Research has created truncated versions of both DUSP9 (1-200 and 201-384 amino acids) and ASK1 (1-678, 679-936, and 937-1374 amino acids)

  • These constructs can be used to map the specific interaction domains

• Phosphorylation analysis:

  • Use phospho-specific antibodies to monitor ASK1 phosphorylation status

  • Compare wild-type conditions with DUSP9 knockdown or overexpression

  • This approach has been successfully used to demonstrate that DUSP9 blocks ASK1 phosphorylation

What considerations are important when using DUSP9 antibodies in multitissue or developmental studies?

When studying DUSP9 across different tissues or developmental stages:

• Expression patterns:

  • DUSP9 expression varies across insulin-sensitive tissues

  • Expression changes during development and in response to metabolic conditions

  • For example, DUSP9 shows different expression levels in amnion membranes at 15.5 versus 18.5 dpc in mice

• Antibody validation across tissues:

  • Verify antibody performance in each tissue type separately

  • Optimization may be required for different fixation methods and tissue processing protocols

  • Consider tissue-specific positive and negative controls

• Quantification methods:

  • For comparative studies, standardize quantification approaches

  • Use internal loading controls appropriate for each tissue type

  • Consider digital image analysis for objective quantification of IHC results

• Experimental design:

  • Include appropriate time points for developmental studies

  • Consider both spatial and temporal expression patterns

  • When studying disease models, include multiple disease stages to capture dynamic changes

How can DUSP9 antibodies be used to investigate inflammatory pathways?

Research has shown that DUSP9 plays a role in regulating inflammatory responses:

• Macrophage studies:

  • Use DUSP9 antibodies to monitor expression in different macrophage activation states

  • Correlate DUSP9 levels with inflammatory cytokine production

• Signaling pathway analysis:

  • DUSP9 inhibition promotes proinflammatory cytokines and COX2/PGE2 expression

  • Monitor p38 and JNK phosphorylation status in parallel with DUSP9 detection

  • This approach can reveal how DUSP9 modulates inflammatory signaling cascades

• In vivo inflammation models:

  • Use DUSP9 antibodies to track expression changes during inflammation progression

  • Compare with markers of inflammation severity

  • Correlate with therapeutic interventions targeting inflammatory pathways

• Combined approaches:

  • Implement multiplex assays to simultaneously detect DUSP9 and inflammatory mediators

  • Use flow cytometry with DUSP9 antibodies to analyze inflammatory cell populations

  • Combine with phospho-specific antibodies to monitor downstream signaling events

What are the considerations for quantifying DUSP9 in clinical samples?

When translating DUSP9 research to clinical applications:

• Sample collection and processing:

  • Standardize collection procedures to minimize pre-analytical variables

  • Optimize preservation methods to maintain protein integrity

  • Document sample handling times and conditions

• Assay selection:

  • For protein quantification, sandwich ELISA using validated DUSP9 antibodies provides reliable results

  • For localization studies in pathological specimens, IHC with appropriate controls is preferred

• Reference standards:

  • Develop reliable calibrators and quality controls

  • Consider recombinant DUSP9 protein as a reference standard

  • Implement internal quality control procedures

• Data interpretation:

  • Establish reference ranges for DUSP9 expression in relevant tissues

  • Consider demographic and clinical variables that might affect expression

  • Correlate DUSP9 levels with disease parameters and outcomes

• Validation strategies:

  • Use multiple detection methods when possible

  • Compare protein levels with mRNA expression data

  • Confirm findings in independent sample sets

How might new DUSP9 antibody technologies advance research in this field?

Emerging antibody technologies hold promise for advancing DUSP9 research:

• Single-domain antibodies:

  • Smaller size allows better tissue penetration

  • Potentially greater specificity for distinct DUSP9 epitopes

  • May enable novel in vivo imaging applications

• Multiplexed detection systems:

  • Simultaneous detection of DUSP9 and interacting partners

  • Co-localization studies with downstream targets

  • Integration with mass cytometry for highly multiparametric analysis

• Intrabodies and nanobodies:

  • Targeting DUSP9 in living cells

  • Monitoring real-time dynamics of DUSP9 localization

  • Potential for targeted modulation of DUSP9 activity

• Conformational-specific antibodies:

  • Detecting distinct activation states of DUSP9

  • Distinguishing between free and ASK1-bound DUSP9

  • Providing insights into regulatory mechanisms

What is known about DUSP9 post-translational modifications and how can antibodies help study them?

Research into DUSP9 post-translational modifications remains an emerging area:

• Phosphorylation status:

  • Develop phospho-specific DUSP9 antibodies

  • Map regulatory phosphorylation sites

  • Investigate how phosphorylation affects DUSP9 activity and interactions

• Ubiquitination and stability:

  • Use antibodies to monitor DUSP9 degradation

  • Investigate ubiquitination sites and machinery

  • Explore stability regulation in different cellular contexts

• Other modifications:

  • Investigate potential SUMOylation, acetylation, or methylation

  • Develop modification-specific antibodies

  • Correlate modifications with functional outcomes

The development of antibodies specifically recognizing modified forms of DUSP9 would significantly advance understanding of its regulation and function in both normal physiology and disease states.