SPOPL Antibody

What is SPOPL and why is it significant in cellular research?

SPOPL (Speckle-type POZ protein-like) is a member of the MATH-BTB protein family with approximately 82.6% sequence homology to Speckle-type POZ protein (SPOP). It functions as a component of a cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex that mediates the ubiquitination and subsequent proteasomal degradation of target proteins, albeit with relatively low efficiency compared to complexes containing only SPOP .

SPOPL's significance in cellular research stems from its crucial role in several biological processes:

• It acts as a regulator of protein degradation pathways, influencing protein turnover and cellular signaling.

• It has been identified as a potential prognostic biomarker for glioblastoma multiforme, promoting proliferation and stemness of glioma stem cells through activation of the Notch signaling pathway .

• It participates in endocytic trafficking regulation, with depletion causing changes in late endocytic markers, particularly MVB markers such as EPS15, HRS, STAM, and TSG101 .

• It forms part of protein complexes that may act as either homodimers or heterodimers with SPOP, with differential effects on ubiquitin ligase activity .

Understanding SPOPL function provides insights into fundamental cellular processes relating to protein degradation and signaling pathways relevant to both normal cell function and disease states.

What are the validated applications for SPOPL antibodies?

SPOPL antibodies have been validated for several research applications, with Western blot (WB) and ELISA being the most commonly confirmed. Based on current data, researchers can reliably use these antibodies for the following applications:

For Western blot applications, SPOPL antibodies have successfully detected bands at the expected molecular weight of approximately 39-45 kDa in various cell lines, including HeLa cells, PC-3 cells, and A549 cells . The predicted molecular weight based on amino acid sequence is 45 kDa, which aligns well with experimental observations.

When performing Western blot analysis, researchers should consider:

• Using proper positive controls (such as A549, HeLa, or PC-3 cell lysates)

• Following recommended secondary antibody systems (e.g., Goat polyclonal to rabbit IgG)

• Optimizing antibody concentration for each specific cell type or tissue being analyzed

While not extensively validated in the provided search results, researchers may also explore using SPOPL antibodies for immunoprecipitation, immunohistochemistry, or immunofluorescence applications after performing their own validation studies.

How should researchers select an appropriate SPOPL antibody for their experiments?

Selecting the appropriate SPOPL antibody requires careful consideration of several factors to ensure experimental success and reliable results:

1. Experimental Application: First determine your primary application (WB, ELISA, etc.) and select antibodies specifically validated for that technique . For instance, PACO57988 and 17740-1-AP are both validated for Western blot applications.

2. Species Reactivity: Ensure the antibody recognizes SPOPL in your species of interest. Available antibodies show reactivity with:

• Human SPOPL (PACO57988, 17740-1-AP)

• Mouse SPOPL (17740-1-AP)

• Rat SPOPL (17740-1-AP)

3. Clonality and Host: Consider whether a polyclonal or monoclonal antibody better suits your needs:

• Polyclonal antibodies (like the rabbit polyclonals described) recognize multiple epitopes, potentially providing stronger signals but with increased risk of cross-reactivity

• Both PACO57988 and 17740-1-AP are rabbit polyclonal antibodies

4. Epitope Information: When available, consider the immunogen used to generate the antibody:

• PACO57988: Recombinant Human Speckle-type POZ protein-like protein (1-120AA)

• 17740-1-AP: SPOPL fusion protein Ag11951

5. Validation Data: Review provided validation data, including:

• Western blot images showing the expected band size (39-45 kDa)

• Positive control samples (e.g., A549, HeLa, PC-3 cell lysates)

• Specificity tests (absence of non-specific bands)

6. Antibody Format: Consider the antibody's format and storage conditions:

• Both PACO57988 and 17740-1-AP are provided in liquid form with specific buffer compositions

• Storage requirements typically include -20°C with glycerol to prevent freeze-thaw damage

For researchers studying SPOPL's role in cancer or endocytic trafficking, antibodies with demonstrated efficacy in detecting endogenous SPOPL in relevant cell types (such as cancer cell lines) would be particularly valuable.

What protocol should researchers follow for SPOPL antibody Western blot applications?

For optimal Western blot results with SPOPL antibodies, researchers should follow this methodological approach:

Sample Preparation:

• Harvest cells (A549, HeLa, or PC-3 cells work well as positive controls)

• Lyse cells in a suitable lysis buffer containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare samples containing 20-50 μg of total protein with reducing sample buffer

• Heat samples at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Load prepared samples onto 10-12% SDS-PAGE gels (appropriate for the 39-45 kDa SPOPL protein)

• Run electrophoresis at 100-120V until adequate separation

• Transfer proteins to PVDF or nitrocellulose membrane (PVDF recommended for higher protein binding capacity)

Immunoblotting:

• Block membrane in 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Dilute primary SPOPL antibody:

  • For PACO57988: Use 1:500-1:5000 dilution

  • For 17740-1-AP: Use 1:500-1:2000 dilution

• Incubate with primary antibody overnight at 4°C with gentle agitation

• Wash membrane 3-5 times with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody (e.g., Goat Anti-Rabbit IgG) at recommended dilution (typically 1:5000-1:50000) for 1 hour at room temperature

• Wash membrane 3-5 times with TBST, 5 minutes each

• Develop using chemiluminescent substrate and image

Expected Results:

• Band size: 39-45 kDa (observed molecular weight)

• Predicted band size: 45 kDa (calculated from amino acid sequence)

Troubleshooting Tips:

• If signal is weak, try increasing antibody concentration or extending incubation time

• For high background, increase washing steps or reduce antibody concentration

• If multiple bands appear, optimize blocking conditions or antibody dilution

• Consider fractionation protocols if studying SPOPL in specific cellular compartments, as demonstrated in endosomal fractionation studies

This protocol can be optimized based on specific experimental conditions and sample types.

How does SPOPL function in the ubiquitin-proteasome pathway?

SPOPL functions as a critical component within the ubiquitin-proteasome pathway through its role in cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complexes. The mechanism and functional characteristics of SPOPL in this pathway reveal several important aspects:

• E3 ligase complex formation: SPOPL can form either homodimeric complexes or heterodimeric complexes with SPOP within the BCR E3 ubiquitin-protein ligase machinery .

• Efficiency differences: Notably, cullin-RING-based BCR E3 ubiquitin-protein ligase complexes containing homodimeric SPOPL or SPOP-SPOPL heterodimers show lower efficiency in ubiquitination compared to complexes containing only SPOP .

• Regulatory role: SPOPL may function to down-regulate the activity of cullin-RING-based BCR E3 ubiquitin-protein ligase complexes that contain SPOP, suggesting a potential modulatory role in controlling the rate of substrate ubiquitination .

• Functional domains: Like other MATH-BTB proteins, SPOPL likely contains:

  • A MATH domain responsible for substrate recognition

  • A BTB/POZ domain that enables interaction with Cullin-3

  • Regions that facilitate dimerization with itself or SPOP

• Biological processes: SPOPL participates in several ubiquitin-related processes including:

  • Negative regulation of protein ubiquitination

  • Proteasomal ubiquitin-dependent protein catabolic processes

Researchers investigating SPOPL's function in the ubiquitin-proteasome pathway should consider that its activity may be context-dependent, potentially varying based on its dimerization state and the presence of SPOP. Additionally, SPOPL's modulation of E3 ligase activity suggests it may serve as a fine-tuning mechanism for protein degradation within cells.

What is the relationship between SPOPL and SPOP proteins, and how can researchers distinguish between them?

SPOPL and SPOP share significant structural and functional similarities, but also exhibit important differences that researchers should understand when studying these proteins:

Structural Relationship:

• SPOPL shows 82.6% sequence homology with SPOP, making them close paralogs within the MATH-BTB protein family

• Both contain characteristic MATH domains (for substrate binding) and BTB/POZ domains (for Cullin-3 interaction)

• Their high sequence similarity can create challenges for antibody specificity and experimental design

Functional Relationship:

• Both function as substrate adaptors in cullin-RING-based BCR E3 ubiquitin-protein ligase complexes

• SPOPL can form heterodimers with SPOP, creating complexes with intermediate ubiquitination efficiency

• SPOPL may negatively regulate SPOP-containing E3 ligase complexes, suggesting a counterbalancing role

• SPOP depletion can influence mRNA levels of SPOPL, but SPOPL depletion does not affect SPOP expression, indicating potential regulatory asymmetry

Distinguishing Features:

• Cellular localization differences: While both may be present in endosomal fractions, they might show distinct distribution patterns

• Molecular weight: Both have similar predicted molecular weights (~45 kDa)

• Functional impact: Depletion studies show that SPOPL specifically affects late endocytic markers, while SPOP may have different targets

Methods to Distinguish SPOPL from SPOP:

Experimental Considerations:

• When examining depletion phenotypes, researchers should verify the specificity of knockdown effects, as SPOP depletion can affect SPOPL levels

• For antibody-based detection, ensure validation against recombinant versions of both proteins

• Consider the possibility of functional overlap, particularly in cells expressing both proteins

• When studying ubiquitination processes, account for the potential formation of heterodimers with altered activity compared to homodimers

Understanding these relationships is essential for correctly interpreting experimental results and developing specific interventions targeting either protein individually.

How can researchers validate the specificity of SPOPL antibodies?

Validating antibody specificity is crucial for obtaining reliable research results, especially when studying proteins like SPOPL that have high homology with other proteins (such as SPOP). Here is a comprehensive methodological approach to validate SPOPL antibody specificity:

1. Knockout/Knockdown Controls:

• Perform siRNA/shRNA knockdown of SPOPL and verify signal reduction in Western blot

• Use CRISPR-Cas9 to generate SPOPL knockout cell lines as negative controls

• Important: Validate that SPOP knockdown doesn't affect your SPOPL antibody signal to confirm specificity

2. Recombinant Protein Testing:

• Test antibody against purified recombinant SPOPL protein (positive control)

• Test cross-reactivity against recombinant SPOP protein

• Create a dilution series to determine detection limits and linear range

3. Peptide Competition Assay:

• Pre-incubate antibody with excess immunizing peptide/protein

• Perform parallel Western blots with blocked and unblocked antibody

• Specific signals should disappear in the blocked antibody sample

4. Multiple Antibody Validation:

• Compare results using different antibodies targeting distinct SPOPL epitopes

• Consistent results with antibodies recognizing different regions strongly support specificity

• For example, compare PACO57988 (immunogen: 1-120AA region) with antibodies targeting other regions

5. Mass Spectrometry Validation:

• Perform immunoprecipitation using the SPOPL antibody

• Analyze precipitated proteins via mass spectrometry

• Confirm presence of SPOPL peptides and absence/minimal presence of SPOP peptides

6. Cellular Localization Analysis:

• Perform subcellular fractionation and analyze distribution patterns

• Compare with known localization data (SPOPL has been detected in endosomal fractions)

• Verify distribution patterns match those from orthogonal detection methods

7. Expected Results Table:

Through these rigorous validation approaches, researchers can establish high confidence in their SPOPL antibody specificity, which is essential for accurate interpretation of experimental results, especially when studying the relationship between SPOPL and its close homolog SPOP.

What cellular compartments is SPOPL typically found in, and how can this inform experimental design?

SPOPL exhibits specific subcellular localization patterns that are important for researchers to consider when designing experiments. Based on cellular fractionation and gradient separation studies, SPOPL has been characterized with the following localization profile:

Primary Cellular Locations:

• Endosomal Compartments: SPOPL has been detected in endosomal organelles, particularly in late endocytic compartments

• Association with Multivesicular Bodies (MVBs): SPOPL is found in fractions containing MVB markers such as EPS15, HRS, STAM, and TSG101

• Co-distribution with CUL3: SPOPL shows co-distribution with its binding partner CUL3 in certain cellular fractions

Experimental Evidence of Localization:
Differential centrifugation and OptiPrep gradient fractionation studies have revealed SPOPL's presence in specific cellular compartments:

• In differential centrifugation experiments, SPOPL has been detected in fractions corresponding to endosomal organelles

• Further fractionation on 5-20% OptiPrep gradients showed SPOPL distribution across multiple fractions containing endosomal markers

• SPOPL depletion specifically affects late endocytic markers, further supporting its functional role in these compartments

Implications for Experimental Design:

Methodological Recommendations:

• For Biochemical Isolation: Follow protocols that effectively separate endosomal compartments:

  • Initial centrifugation at 1000g to remove nuclei

  • Subsequent centrifugation at 13,000g and 100,000g

  • Further separation on density gradients (5-20% OptiPrep) for refined fractionation

• For Functional Studies: Consider that SPOPL depletion affects:

  • Late endocytic marker levels (particularly MVB markers)

  • Certain receptors (EGFR, MET) but not others (VEGFR, IGF1R, HER2)

Understanding SPOPL's precise cellular localization helps researchers design more targeted experiments to investigate its functional roles in endocytic trafficking and receptor regulation, and informs the choice of appropriate cell lysis methods, fractionation approaches, and co-immunoprecipitation conditions.

What is SPOPL's role in cancer biology, and how can SPOPL antibodies advance cancer research?

SPOPL has emerged as a significant player in cancer biology, with particular relevance to glioblastoma multiforme (GBM). Understanding its role offers important insights for cancer researchers utilizing SPOPL antibodies:

SPOPL's Cancer-Related Functions:

• Prognostic Biomarker: SPOPL has been identified as a potential prognostic biomarker for glioblastoma multiforme, suggesting its expression levels may correlate with clinical outcomes .

• Promotion of Cancer Stemness: Research indicates SPOPL promotes the proliferation and stemness properties of glioma stem cells specifically through activation of the Notch signaling pathway . This connection to cancer stem cell maintenance has significant implications for tumor recurrence and treatment resistance.

• E3 Ubiquitin Ligase Activity: As a component of cullin-RING-based E3 ubiquitin ligase complexes, SPOPL likely influences the stability of proteins involved in oncogenic or tumor suppressor pathways .

• Receptor Regulation: SPOPL depletion studies have shown specific effects on receptor tyrosine kinases relevant to cancer, including EGFR and MET, while not affecting others like VEGFR, IGF1R and HER2 . This selectivity suggests SPOPL may regulate specific oncogenic signaling pathways.

Applications of SPOPL Antibodies in Cancer Research:

Experimental Considerations for Cancer Research:

• Cell Line Selection: Use cancer cell lines with known SPOPL expression levels; A549 and HeLa cells have been validated for SPOPL detection .

• Control Considerations: When studying SPOPL's cancer-specific functions:

  • Compare malignant versus non-malignant cells of the same tissue origin

  • Consider potential variability in SPOPL expression across different cancer subtypes

  • Account for potential differences in SPOPL function between primary tumors and metastatic lesions

• Pathway Analysis: Given SPOPL's connection to Notch signaling in glioma stem cells , researchers should:

  • Monitor Notch pathway components alongside SPOPL

  • Investigate potential cross-talk with other cancer-relevant pathways

  • Consider SPOPL's differential effects on specific receptors like EGFR and MET

Researchers utilizing SPOPL antibodies for cancer studies should design experiments that not only detect expression levels but also investigate functional interactions with cancer-relevant pathways, particularly focusing on receptor trafficking, Notch signaling, and cancer stem cell maintenance.

What are the challenges in detecting low-abundance SPOPL in different cell types and how can they be overcome?

Detecting low-abundance proteins like SPOPL presents several technical challenges, particularly when expression levels vary across cell types or experimental conditions. Researchers can implement the following advanced strategies to optimize SPOPL detection:

Common Challenges and Solutions:

• Low Expression Levels

  • Challenge: SPOPL may be expressed at low levels in certain cell types, making detection difficult.

  • Solutions:

    • Enrich for SPOPL-containing compartments through subcellular fractionation before analysis

    • Use OptiPrep gradient fractionation (5-20%) to concentrate endosomal fractions where SPOPL is enriched

    • Increase protein loading (50-100 μg per lane) for Western blot analysis

    • Employ signal amplification techniques such as biotin-streptavidin systems for immunodetection

• High Background/Non-specific Binding

  • Challenge: When using high antibody concentrations to detect low-abundance SPOPL, background signal may increase.

  • Solutions:

    • Optimize blocking conditions (consider 5% BSA instead of milk for phosphorylated protein studies)

    • Use more stringent washing protocols (increased number of washes, higher salt concentration)

    • Employ monoclonal antibodies when available for higher specificity

    • Consider using signal enhancers specifically designed for Western blot applications

• Signal Interference from SPOP

  • Challenge: The high homology between SPOPL and SPOP (82.6%) may create cross-reactivity issues .

  • Solutions:

    • Validate antibody specificity using SPOPL and SPOP recombinant proteins

    • Perform parallel experiments with SPOPL-specific and SPOP-specific knockdowns

    • Use epitope-specific antibodies targeting regions with lowest homology between SPOPL and SPOP

Advanced Detection Methods for Low-Abundance SPOPL:

Cell Type-Specific Considerations:

• Cancer Cell Lines vs. Normal Cells

  • SPOPL may be differentially expressed in cancer cells compared to normal cells, particularly in glioblastoma models

  • Adjust protein loading accordingly when comparing different cell types

• Cells with High Endosomal Activity

  • Given SPOPL's presence in endosomal compartments, cells with active endocytic pathways may show better detection

  • A549, HeLa, and PC-3 cells have been validated for SPOPL detection

• Optimization Protocol Flowchart:

  • Start with validated cell lines (A549, HeLa, PC-3) as positive controls

  • Perform subcellular fractionation to enrich endosomal compartments

  • Test increasing antibody concentrations (1:500 → 1:250 → 1:100) with extended incubation times

  • Implement signal enhancement strategies if needed

  • Validate all signals with appropriate controls (knockdown, peptide competition)

By implementing these advanced techniques, researchers can overcome the challenges associated with detecting low-abundance SPOPL across different cell types and experimental conditions.

How can SPOPL antibodies be utilized in studying endocytic trafficking pathways?

SPOPL has been identified as a critical regulator of endocytic trafficking, making SPOPL antibodies valuable tools for investigating these pathways. Advanced methodological approaches can help researchers leverage these antibodies to gain deeper insights into endocytic processes:

SPOPL's Role in Endocytic Trafficking:
SPOPL functions as part of a SPOPL/Cullin-3 ubiquitin ligase complex that regulates endocytic trafficking . Depletion studies have revealed that SPOPL specifically affects late endocytic markers, particularly multivesicular body (MVB) components such as EPS15, HRS, STAM, and TSG101, while having minimal impact on early endocytic and recycling markers .

Advanced Methodological Approaches:

• Subcellular Co-localization Studies

  • Method: Combined immunofluorescence microscopy using SPOPL antibodies with markers of different endocytic compartments

  • Technical implementation:

    • Fix cells using 4% paraformaldehyde to preserve membrane structures

    • Use optimized permeabilization (0.1% saponin better preserves endosomal membranes than Triton X-100)

    • Co-stain with markers for early endosomes (EEA1, Rab5), late endosomes/MVBs (Rab7, CD63), and recycling endosomes (Rab11)

    • Employ super-resolution microscopy (STED, STORM) for precise localization

• Endosomal Protein Complex Analysis

  • Method: Immunoprecipitation coupled with mass spectrometry to identify SPOPL-interacting proteins in endosomal compartments

  • Protocol refinements:

    • Isolate endosomal fractions using differential centrifugation and OptiPrep gradients

    • Cross-link proteins in live cells before lysis to preserve transient interactions

    • Use SPOPL antibodies for immunoprecipitation followed by mass spectrometry

    • Validate interactions using reciprocal co-immunoprecipitation

• Endocytic Cargo Tracking

  • Method: Pulse-chase experiments with labeled cargo proteins and SPOPL immunodetection

  • Experimental design:

    • Label endocytic cargo (e.g., fluorescent EGF for EGFR trafficking)

    • Fix cells at different time points after internalization

    • Perform SPOPL immunostaining to assess co-localization with cargo at various stages

    • Quantify co-localization coefficients at each time point

• Ubiquitination Profiling in Endocytic Compartments

  • Method: Combined SPOPL and ubiquitin detection in isolated endosomal fractions

  • Technical approach:

    • Fractionate endosomal compartments using established protocols

    • Detect SPOPL, CUL3, and ubiquitinated proteins in each fraction

    • Compare ubiquitination profiles between control and SPOPL-depleted cells

Research Applications with Data Tables:

Advanced Analytical Considerations:

• Quantitative Analysis of SPOPL Distribution:

  • When analyzing OptiPrep gradient fractions, quantify SPOPL signal intensity across fractions and correlate with specific endosomal markers

  • Create distribution profiles comparing SPOPL localization with CUL3 and endosomal markers to identify functional complexes

• Temporal Dynamics:

  • Study SPOPL localization changes under stimulated conditions (e.g., growth factor stimulation, starvation)

  • Track temporal changes in SPOPL association with endocytic compartments during cargo processing

• Comparative Analysis:

  • Compare SPOPL vs. SPOP distribution in endocytic compartments to determine functional specialization

  • Analyze differences in ubiquitination patterns mediated by SPOPL vs. SPOP in endosomal fractions

These methodological approaches enable researchers to precisely determine SPOPL's function in endocytic trafficking, particularly its role in regulating receptor fate decisions and MVB formation, with implications for both normal cellular function and disease states.

What are the considerations for using SPOPL antibodies in analyzing protein-protein interactions within ubiquitin ligase complexes?

Investigating protein-protein interactions (PPIs) within SPOPL-containing ubiquitin ligase complexes requires sophisticated methodological approaches. The following considerations and techniques will help researchers design robust experiments to study these complex interactions:

Structural and Functional Considerations:

• Complex Architecture: SPOPL functions within cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complexes, where it can form:

  • Homodimeric SPOPL complexes

  • Heterodimeric complexes with SPOP

  • Each with different efficiency in substrate ubiquitination

• Domain-Specific Interactions: SPOPL contains:

  • BTB/POZ domain for CUL3 interaction

  • MATH domain for substrate recognition

  • Regions mediating dimerization with itself or SPOP

• Dynamic Associations: SPOPL interactions may be:

  • Transient during ubiquitination processes

  • Compartment-specific (particularly in endosomal fractions)

  • Regulated by post-translational modifications

Advanced Methodological Approaches:

Antibody-Specific Strategic Approaches:

• Epitope Accessibility Considerations:

  • Ensure selected SPOPL antibodies do not interfere with interaction domains

  • For Co-IP applications, validate that the antibody does not disrupt native protein complexes

  • Consider using antibodies targeting different SPOPL epitopes to confirm interactions

• Validation Controls for Interaction Studies:

  • Perform reciprocal Co-IPs when possible (pull-down with SPOPL antibody and with antibodies against interacting partners)

  • Include SPOPL knockdown controls to confirm specificity of interactions

  • Compare SPOPL and SPOP interaction networks to identify unique vs. shared partners

• Distinguishing Homodimers vs. Heterodimers:

  • Design sequential immunoprecipitation approaches (first SPOPL, then SPOP antibodies)

  • Use tagged versions of SPOPL or SPOP in combination with antibody-based detection of the endogenous counterpart

  • Quantify relative abundance of complex types through quantitative proteomics

• Substrate Identification Strategy:

  • Combine SPOPL antibody immunoprecipitation with ubiquitination site profiling

  • Compare ubiquitinated proteins in control vs. SPOPL-depleted cells

  • Analyze differences between substrates preferentially ubiquitinated by SPOPL homodimers vs. SPOP/SPOPL heterodimers

Functional Validation Approaches:

• Reconstitution Studies:

  • Express recombinant SPOPL with mutations in key interaction domains

  • Use SPOPL antibodies to confirm complex formation (or lack thereof) with CUL3 and other partners

  • Correlate complex formation with ubiquitination activity

• Compartment-Specific Analysis:

  • Isolate endosomal fractions where SPOPL is enriched

  • Use SPOPL antibodies to identify compartment-specific interaction partners

  • Compare with interactions in other cellular compartments

• Dynamics of Complex Formation:

  • Study temporal changes in SPOPL interactions following cellular stimulation

  • Use SPOPL antibodies to track redistribution of interaction partners

  • Correlate with changes in ubiquitination patterns of target proteins

By implementing these advanced approaches, researchers can gain detailed insights into the composition, regulation, and function of SPOPL-containing ubiquitin ligase complexes, furthering our understanding of its role in protein degradation pathways and endocytic trafficking.