UMODL1 Antibody

What is UMODL1 and where is it primarily expressed?

UMODL1 (Uromodulin-like 1) encodes Ca²⁺-dependent EGF-like membrane-bound proteins that are notably expressed in both the immune and female reproductive systems. Research has demonstrated its expression in the mouse olfactory region along the migratory route of GnRH neurons, suggesting roles in olfactory development and reproductive function . In the immune system, UMODL1 shows a prompt and robust response in proliferating CD4+ T cells when stimulated by CD3/CD28 antibodies, indicating potential involvement in immune defense against pathogens . Additionally, UMODL1 expression in the ovary is regulated by gonadotropins, highlighting its importance in reproductive processes .

What structural domains characterize UMODL1 protein?

UMODL1 protein shares a distinctive combination of WAP (whey acidic protein) and FNIII (fibronectin type III) repeats with anosmin-1 (the product of the ANOS1 gene). These domains are expressed in complementary patterns throughout the protein structure . In silico analysis reveals significant similarity between UMODL1 and anosmin-1 at the N-terminal region, particularly in the arrangement of WAP and FNIII domains . The WAP domains of both proteins show marked similarities in their binding modes of interaction with the FGFR1-FGF2 complex, which may explain their functional overlap . This structural organization contributes to UMODL1's roles in cellular signaling and migration processes.

How does UMODL1 function in reproductive biology?

In reproductive biology, UMODL1 plays a critical role in ovarian follicle development and maintenance. Mice overexpressing Umodl1 develop premature infertility by 6 months of age despite appearing normal when young . Histological analyses reveal that ovaries from these mice contain very few discernible follicles in the cortical region and lack distinguishable corpus lutea (CL) . The multilayered preantral follicles show elevated apoptosis in both oocytes and surrounding granulosa cells (GCs) . Furthermore, high levels of PPARγ in these ovaries indicate abnormal adipogenesis, resulting in the conversion of granulosa cells into adipocytes . By 6 months, all mutant mice become anovulatory with degenerated ovarian tissues including CL, follicles of various stages, and associated stromal cells .

What are the validated applications for UMODL1 antibody?

The commercially available UMODL1 polyclonal antibody (targeting mouse UMODL1) has been validated for multiple applications in laboratory research . These validated applications include Western Blotting (WB) for protein detection and quantification, Immunohistochemistry (IHC) for localization studies in tissue sections, Immunocytochemistry (ICC) for cellular localization, and Immunoprecipitation (IP) for protein isolation and interaction studies . These diverse applications make the antibody versatile for researchers studying UMODL1 expression, localization, and interactions across different experimental contexts. Researchers studying the olfactory system and reproductive biology have successfully employed these techniques to characterize UMODL1 function in multiple model systems.

What protein regions does the UMODL1 antibody recognize?

The UMODL1 polyclonal antibody described in the search results was developed using an immunogen spanning from Ser34 to Pro306 of the mouse UMODL1 protein (Accession Number: Q5DID3, Gene ID: 52020) . This N-terminal region is particularly significant as experimental evidence suggests that the N-terminal portion of UMODL1 (olfactorin) is released into the culture medium, potentially through post-translational processing . Western blot analyses of UMODL1-transfected COS-7 cells revealed both a full-length intracellular protein (approximately 148 kDa) and a shorter secreted form (approximately 60-70 kDa) that was detectable using antibodies recognizing the N-terminal region . This information is crucial for researchers designing experiments to detect either the full-length protein or its processed forms.

How is UMODL1 antibody reactivity confirmed in experimental systems?

Confirming UMODL1 antibody reactivity typically involves multiple complementary approaches. In studies cited in the search results, researchers employed both overexpression systems and immunodetection techniques . For instance, recombinant UMODL1 was expressed in COS-7 cells using expression vectors (pCMV-Sport6.1-FLAG-BQ88765), and immunofluorescence of transfected cells showed clear immunoreactivity with anti-FLAG monoclonal antibodies . Western blot analysis of whole-cell extracts confirmed the overexpression of a protein corresponding to the predicted size of intracellular full-length UMODL1 (approximately 148 kDa) . Additionally, antibody specificity can be confirmed through immunoneutralization experiments, where pre-incubation with the antibody blocks the biological activity of the protein, as demonstrated in chemotaxis assays with GnRH neurons .

How should researchers design Western blot protocols for UMODL1 detection?

For optimal Western blot detection of UMODL1, researchers should consider both intracellular and secreted forms of the protein. Based on the provided research, the following protocol framework is recommended:

• Sample preparation:

  • For cell lysates: Extract proteins using standard lysis buffers containing protease inhibitors

  • For secreted proteins: Collect conditioned medium (CM) in the presence of protease inhibitors (e.g., aprotinin at 0.5 mg/ml), centrifuge at 3,000g for 5 min at 4°C

• Gel electrophoresis:

  • Load 30 μg of protein extracts or 20 μl of concentrated CM

  • Use SDS-PAGE gels with appropriate percentage (7-10% for full-length UMODL1)

• Transfer and detection:

  • Transfer to PVDF membrane

  • Block with appropriate blocking buffer

  • Incubate overnight with anti-UMODL1 antibody (1:1,000 dilution)

  • Use peroxidase-conjugated secondary antibodies (1 hour at room temperature)

  • Detect using chemiluminescence (e.g., Cyanagen Ultra kit)

  • Include tubulin expression (1:2,000 dilution) as loading control

Note that UMODL1 appears at approximately 148 kDa (full-length form) in cell lysates and 60-70 kDa (processed form) in conditioned medium .

What methodologies are effective for studying UMODL1 function in migration assays?

To study UMODL1's function in cell migration, researchers can employ the Boyden's microchemotaxis chamber assay as demonstrated in prior research . This methodology allows for quantitative assessment of UMODL1's chemoattractant properties:

• Chamber preparation:

  • Use a 48-well Boyden's microchemotaxis chamber

  • Coat porous membranes with extracellular matrix proteins if needed

• Cell preparation:

  • Prepare responsive cells (e.g., GN11 immortalized GnRH neurons)

  • Place cells in the upper compartment of the chamber

• Chemoattractant preparation:

  • Use conditioned medium from cells expressing UMODL1 (e.g., COS-7 cells transfected with UMODL1 expression vectors)

  • Include appropriate controls (e.g., CM from untransfected cells, 0.1% FBS as positive control)

• Migration assessment:

  • Allow migration to proceed for an appropriate time period (e.g., 3 hours)

  • Fix and stain migrated cells

  • Count cells per square mm of porous membrane

  • Express results as relative response vs. control stimuli

• Validation of specificity:

  • Perform immunoneutralization using specific antibodies against UMODL1 to confirm that the observed effect is due to this protein

This assay effectively demonstrated that UMODL1-enriched CM induced a 44% higher chemotactic response than control CM in GnRH neurons, comparable to the effect of anosmin-1 .

How can researchers perform immunofluorescence studies with UMODL1 antibody?

For effective immunofluorescence detection of UMODL1, researchers should follow these methodological guidelines based on previously successful studies:

• Cell preparation:

  • Culture cells on appropriate coverslips

  • For transfection studies, transfect cells with UMODL1 expression vectors (e.g., pCMV-Sport6.1-FLAG-BQ88765)

  • Allow 24-48 hours for protein expression

• Fixation and permeabilization:

  • Fix cells with paraformaldehyde (typically 4%)

  • Permeabilize with appropriate detergent (e.g., Triton X-100) if detecting intracellular protein

• Antibody incubation:

  • Block with appropriate serum or BSA solution

  • For detection of tagged constructs, use anti-tag antibodies (e.g., anti-FLAG M2 antibody)

  • For endogenous UMODL1, use specific anti-UMODL1 antibodies

  • Use fluorophore-conjugated secondary antibodies

• Mounting and imaging:

  • Mount slides with appropriate medium containing DAPI for nuclear counterstaining

  • Examine using confocal or fluorescence microscopy

• Controls:

  • Include untransfected cells as negative controls

  • Use cells expressing known markers as positive controls

  • Consider co-staining with organelle markers to determine subcellular localization

This approach has successfully demonstrated clear immunoreactivity for UMODL1 in transfected COS-7 cells, revealing its cellular distribution pattern .

How can UMODL1 antibody be utilized to study its role in reproductive aging?

Investigating UMODL1's role in reproductive aging represents an advanced research application that builds on findings that UMODL1 overexpression accelerates follicle depletion . Researchers can employ the following methodological approach:

• Experimental design:

  • Utilize age-matched cohorts of control and UMODL1-overexpressing mice

  • Collect ovarian samples at multiple time points (e.g., 2, 4, and 6 months) to track progression

• Histological analysis:

  • Process ovarian tissues with standard fixation and sectioning techniques

  • Perform UMODL1 immunohistochemistry to localize protein expression

  • Quantify follicle numbers at different developmental stages

  • Assess corpus luteum formation

• Apoptosis assessment:

  • Perform TUNEL assays to detect apoptotic cells in follicles

  • Use anti-cleaved caspase-3 immunostaining as a complementary apoptosis marker

  • Quantify apoptotic indices in oocytes and granulosa cells

• Molecular analysis:

  • Extract RNA from ovarian tissues

  • Perform qRT-PCR to assess expression of:

    • UMODL1 itself

    • PPARγ (indicator of abnormal adipogenesis)

    • Ovary-specific markers (AMH, Gdf-9, Rnf35, NOHLH, Gcx-1)

    • Follicle-stimulating hormone receptors

• Hormone analysis:

  • Measure serum levels of reproductive hormones (FSH, LH, estrogen, progesterone)

  • Correlate hormonal changes with follicular depletion

This comprehensive approach will illuminate UMODL1's role in premature ovarian failure or early ovarian aging, providing insights into potential therapeutic targets for age-related fertility decline .

What methods can be employed to study UMODL1's interaction with FGFR signaling pathways?

Given UMODL1's demonstrated involvement in FGFR and MAPK pathways , studying these interactions requires sophisticated methodological approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Use anti-UMODL1 antibody for immunoprecipitation

  • Probe for FGFR1 and downstream signaling molecules in the precipitated complex

  • Perform reciprocal Co-IP with anti-FGFR1 antibodies

• Pathway inhibition experiments:

  • Treat cells with specific inhibitors of FGFR (e.g., SU5402) or MAPK pathway components (e.g., PD98059 for MEK inhibition)

  • Assess the effect on UMODL1-induced cellular responses (e.g., chemotaxis, proliferation)

  • Measure phosphorylation of downstream targets (ERK1/2, AKT) by Western blotting

• Binding assays:

  • Produce recombinant UMODL1 domains (particularly WAP and FNIII domains)

  • Perform surface plasmon resonance (SPR) to measure binding kinetics with FGFR1 and FGF2

  • Conduct competitive binding assays with anosmin-1, which shares similar domain structure

• Structural studies:

  • Use computational modeling to predict interaction interfaces

  • Perform site-directed mutagenesis of key residues

  • Validate the functional importance of these residues in cellular assays

• In vivo pathway analysis:

  • Develop zebrafish models with fluorescent reporters for MAPK pathway activation

  • Down-regulate z-umodl1 using morpholino technology

  • Assess changes in pathway activity using confocal microscopy

This multifaceted approach would provide detailed mechanistic insights into how UMODL1 engages with and modulates FGFR signaling, potentially revealing new therapeutic targets for conditions involving dysregulated FGFR signaling .

How can researchers investigate the comparative functions of UMODL1 and anosmin-1?

Given the structural and functional similarities between UMODL1 and anosmin-1 , comparative studies can provide valuable insights into their respective roles and potential redundancies:

• Domain-swapping experiments:

  • Generate chimeric constructs exchanging WAP and FNIII domains between UMODL1 and anosmin-1

  • Express these constructs in relevant cell systems

  • Assess functional outcomes (migration, proliferation, differentiation)

• Comparative expression analysis:

  • Perform dual immunofluorescence with antibodies against both proteins

  • Map their spatial and temporal expression patterns during development

  • Identify regions of overlap versus distinct expression

• Dual knockdown/knockout studies:

  • Design experiments with single and combined knockdown/knockout of both genes

  • Utilize morpholino technology in zebrafish models as demonstrated for z-umodl1

  • Compare phenotypes to identify synergistic versus additive effects

• Rescue experiments:

  • In models with UMODL1 deficiency, attempt rescue with anosmin-1 and vice versa

  • Identify domains required for functional complementation

• Receptor competition assays:

  • Determine if UMODL1 and anosmin-1 compete for the same receptors (e.g., FGFR1)

  • Measure binding affinity and downstream signaling under competitive conditions

This comprehensive approach would clarify the extent of functional overlap between these proteins and potentially explain why mutations in ANOS1 lead to Kallmann syndrome despite the presence of UMODL1 .

What are common issues in UMODL1 antibody applications and how can they be resolved?

Researchers working with UMODL1 antibodies may encounter several technical challenges. Here are common issues and recommended solutions:

When troubleshooting, always include appropriate positive and negative controls and consider the specific characteristics of UMODL1, such as its calcium dependency and potential for post-translational processing .

How should researchers interpret conflicting data between UMODL1 expression and function?

When researchers encounter apparent contradictions between UMODL1 expression data and functional outcomes, methodical approaches are needed:

• Consider post-translational processing:

  • UMODL1 undergoes processing that results in a 60-70 kDa secreted form versus the 148 kDa full-length protein

  • Different antibodies may detect different forms, leading to seemingly conflicting results

  • Verify which protein form is being detected in each assay

• Examine temporal dynamics:

  • UMODL1 effects may be age-dependent (e.g., mice overexpressing UMODL1 appear normal when young but develop premature infertility at 6 months)

  • Design time-course experiments to capture developmental changes

• Assess tissue-specific effects:

  • UMODL1 functions differently in different tissues (immune system, reproductive system, olfactory system)

  • Context-dependent effects may explain apparent contradictions

• Analyze signaling pathway interactions:

  • UMODL1 activates FGFR and MAPK pathways

  • Different cell types may have varying levels of pathway components

  • Measure pathway activation directly rather than assuming consistent downstream effects

• Methodological reconciliation:

  • Use multiple complementary techniques (IHC, WB, qPCR)

  • When differences persist, molecular methods (RNA-seq, proteomics) may reveal underlying mechanisms

  • Consider genetic background effects in animal models

This systematic approach helps distinguish true biological complexity from technical artifacts, providing a more complete understanding of UMODL1 biology.

How can researchers quantitatively assess UMODL1 expression in developmental studies?

For rigorous quantification of UMODL1 expression across developmental stages, researchers should implement these methodological approaches:

• RNA quantification:

  • Perform RT-PCR analysis to measure UMODL1 mRNA levels as demonstrated in zebrafish studies

  • Use qRT-PCR with validated primer sets spanning exon-exon junctions

  • Include appropriate reference genes (GAPDH, β-actin) for normalization

  • Consider RNA-seq for genome-wide expression context

• Protein quantification:

  • Use quantitative Western blotting with:

    • Standard curves of recombinant protein

    • Digital imaging and analysis software

    • Normalization to loading controls

  • Perform ELISA if available for UMODL1

• Spatial localization quantification:

  • Use immunofluorescence with standardized image acquisition settings

  • Perform quantitative image analysis:

    • Measure mean fluorescence intensity

    • Quantify percentage of positive cells

    • Assess subcellular distribution

  • Use automated high-content imaging systems for larger datasets

• Statistical analysis:

  • Apply appropriate statistical tests (ANOVA, t-tests)

  • Include biological replicates (n≥3) and technical replicates

  • Present data with measures of central tendency and dispersion

  • Use developmental time as a continuous variable where appropriate

• Validation approaches:

  • Confirm specificity with genetic models (knockdown/knockout)

  • Use multiple antibodies targeting different epitopes

  • Include morpholino controls as demonstrated in zebrafish studies

This comprehensive quantitative approach enables robust assessment of UMODL1 developmental expression patterns, facilitating correlation with functional outcomes in olfactory and reproductive system development .