TPD52L3 Antibody

What is TPD52L3 and what is its functional significance in research?

TPD52L3 is a member of the tumor protein D52-like family of proteins characterized by an N-terminal coiled-coil motif that facilitates the formation of homo- and heteromeric complexes with other tumor protein D52-like proteins. The protein is primarily believed to play a significant role in spermatogenesis . TPD52L3 is encoded by a gene that undergoes alternative splicing, resulting in multiple transcript variants, which adds complexity to its functional characterization . Research interest in TPD52L3 stems from its potential involvement in cancer biology pathways, particularly as related proteins in the family have been identified as proto-oncogenes that are overexpressed in various cancers.

How does TPD52L3 differ from other members of the TPD52 family?

TPD52L3 (isoform 3) is structurally similar to other TPD52 family members but exhibits specific functional characteristics:

• Structural features: While all TPD52 family proteins contain the characteristic N-terminal coiled-coil motif, TPD52L3 has specific domains including PEST and D2-motifs that are critical for protein-protein interactions

• Tissue expression: Unlike TPD52 which is broadly expressed, TPD52L3 shows more restricted expression patterns with higher presence in testicular tissue

• Protein interactions: Recent molecular modeling and docking studies have revealed that TPD52L3 interacts with LKB1 (Liver kinase B1) through specific residues (E5, L8, E16, K147, and T151) that form polar contacts

• Cell signaling pathways: While TPD52 has been shown to inhibit AMPK activation through LKB1 interaction, the specific signaling pathways affected by TPD52L3 are still being investigated

What types of TPD52L3 antibodies are available for research applications?

Multiple types of TPD52L3 antibodies are available, each with specific characteristics suitable for different research applications:

Selecting the appropriate antibody depends on the specific experimental requirements, including the technique being employed, species of interest, and the specific epitope being targeted .

What critical factors should be considered when selecting a TPD52L3 antibody for specific experimental techniques?

When selecting a TPD52L3 antibody, researchers should consider several factors to ensure optimal experimental outcomes:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IF, IHC, ELISA)

• Species reactivity: Ensure cross-reactivity with your experimental model organism (human, rat, mouse)

• Epitope location: Consider whether the experimental design requires detection of specific domains or isoforms of TPD52L3

• Clonality: Polyclonal antibodies provide higher sensitivity but potentially lower specificity compared to monoclonal antibodies

• Validation data: Review available validation data including Western blot images, immunofluorescence patterns, and specificity tests

• Detection method: Consider whether the experiment requires conjugated or unconjugated antibodies based on the detection system

For specialized applications such as protein interaction studies, antibodies targeting specific domains (such as the N-terminal coiled-coil motif or PEST domain) may be more appropriate based on recent structural interaction data .

How should TPD52L3 antibodies be optimized for Western blot analysis?

Optimizing TPD52L3 antibodies for Western blot requires careful consideration of several experimental parameters:

• Sample preparation:

  • Extract proteins using RIPA buffer supplemented with protease inhibitor cocktail

  • For cell lines, harvest by scraping and lyse by passing through 29-gauge insulin syringes

  • Use 25μg of protein per lane for optimal detection

• Dilution optimization:

  • Start with 1:1000 dilution for primary TPD52L3 antibody in TBST with 3% non-fat dry milk

  • Use HRP-conjugated secondary antibodies (e.g., Goat Anti-Rabbit IgG) at 1:10000 dilution

• Detection conditions:

  • Block membranes with 3% non-fat dry milk in TBST to minimize non-specific binding

  • Use ECL Basic Kit for detection with approximately 10 seconds exposure time

  • Validate specificity with appropriate positive controls (testicular tissue) and negative controls

• Expected results:

  • TPD52L3 typically appears at approximately 20-25 kDa

  • Multiple bands may be observed due to alternative splicing variants or post-translational modifications

What are the optimal protocols for immunofluorescence studies using TPD52L3 antibodies?

Immunofluorescence studies using TPD52L3 antibodies should follow these methodological guidelines for optimal results:

• Cell preparation:

  • Seed cells (5 × 10^4) in 2-chambered glass slides and culture to 70% confluence

  • Fix cells with 4% PFA for 15 minutes followed by permeabilization with 0.25% Triton X-100 for 10 minutes

• Antibody incubation:

  • Block with 2% BSA to reduce non-specific binding

  • Dilute primary TPD52L3 antibody at 1:100 to 1:200 in 2% BSA and incubate overnight at 4°C

  • Wash thoroughly with TBST to remove excess primary antibody

  • Incubate with fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 555 for anti-mouse or Alexa Fluor 488 for anti-rabbit) at 1:1000 dilution for 2 hours in the dark

• Visualization and analysis:

  • Mount slides using Fluoroshield or similar mounting medium containing DAPI for nuclear counterstaining

  • Image using confocal microscopy with appropriate filter settings

  • For co-localization studies, calculate Pearson correlation coefficients using appropriate imaging software

• Expected pattern:

  • TPD52L3 typically shows cytoplasmic distribution with possible enrichment in specific subcellular compartments

  • In testicular tissue, strong immunoreactivity is observed

How can TPD52L3 antibodies be effectively used to study protein-protein interactions?

TPD52L3 antibodies can be valuable tools for investigating protein-protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Use TPD52L3 antibodies to pull down protein complexes from cell lysates

  • Analyze precipitated proteins by Western blot to identify interaction partners

  • For known interactions like TPD52L3-LKB1, use reciprocal Co-IP with antibodies against both proteins

• Proximity Ligation Assay (PLA):

  • Utilize a combination of TPD52L3 and partner protein antibodies (e.g., LKB1 antibodies)

  • Follow PLA protocols to visualize protein interactions in situ with subcellular resolution

  • Quantify interaction signals to assess the strength of protein-protein associations

• Double immunofluorescence:

  • Perform double immunofluorescence staining with TPD52L3 antibodies and antibodies against potential interaction partners

  • Calculate co-localization coefficients using imaging software

  • Based on existing research, TPD52L3 and LKB1 show significant co-localization that can be disrupted by specific mutations

• Validation using recombinant proteins:

  • Validate interactions identified in cellular systems using purified recombinant proteins

  • Consider the critical interaction residues (E5, L8, E16, K147, and T151) identified in TPD52L3 for designing interaction studies

How does TPD52L3 contribute to AMPK signaling pathways in cancer progression?

Recent research has uncovered an intriguing relationship between TPD52 family proteins, including TPD52L3, and the AMPK signaling pathway in cancer:

• Interaction with LKB1:

  • TPD52 family proteins interact with LKB1, an upstream kinase for AMPK

  • Molecular modeling and MD simulations indicate that this interaction may mask LKB1's auto-phosphorylation sites

  • TPD52 overexpression reduces phosphorylation of LKB1 (Ser428) and AMPK (Thr172)

• Regulation of AMPK activity:

  • The interaction between TPD52L3 and LKB1 may inhibit LKB1 kinase activity

  • This inhibition could prevent AMPK activation, promoting cancer cell growth and proliferation

  • AMPK activation with AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) has been shown to inhibit cancer cell growth by silencing TPD52 expression

• Downstream effects:

  • AMPK activation leads to downregulation of TPD52 via activation of GSK3β

  • This occurs through a decrease in inactive phosphorylation of GSK3β at Ser9

  • Inhibition of GSK3β with LiCl attenuates the downregulation of TPD52 by AICAR

• Implications for targeted therapy:

  • AMPK activators may be useful in suppressing cancer cell growth in tumors with TPD52 overexpression

  • Small molecules that disrupt the TPD52L3-LKB1 interaction could potentially restore AMPK signaling

What are the critical domains of TPD52L3 for protein-protein interactions and how can they be targeted experimentally?

Understanding the structural domains of TPD52L3 that mediate protein interactions is crucial for developing targeted experimental approaches:

• Critical interaction domains:

  • N-terminal PEST domain: Contains residues essential for protein-protein interactions

  • D2-motif: Works in conjunction with the PEST domain for binding partner proteins

  • Residues E5, L8, E16, K147, and T151 form polar contacts with interaction partners like LKB1

• Experimental targeting approaches:

  • Site-directed mutagenesis: Generate mutants (K147A, E16A, T151A) that disrupt critical interaction sites

  • Domain-specific antibodies: Use antibodies that recognize specific domains to block interactions

  • Peptide inhibitors: Design peptides that mimic interaction domains to competitively inhibit protein binding

  • In silico screening: Identify small molecules that bind to interaction interfaces using molecular docking

• Validation methods:

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

  • Use GST pull-down assays with recombinant proteins containing specific domains

  • Employ fluorescence resonance energy transfer (FRET) to measure interaction disruption

  • Analyze phenotypic changes in cellular models when interactions are disrupted

How do TPD52L3 expression patterns differ in normal versus cancerous tissues, and what techniques can be used to investigate these differences?

Understanding TPD52L3 expression patterns across normal and pathological tissues requires specialized experimental approaches:

• Expression patterns:

  • Normal tissues: Primarily expressed in testicular tissue with a potential role in spermatogenesis

  • Cancer tissues: Related family member TPD52 shows overexpression in prostate cancer due to gene amplification

  • Differential expression may contribute to cancer progression through modulation of AMPK signaling

• Investigative techniques:

  • Tissue microarrays (TMAs): Analyze TPD52L3 expression across multiple tissue samples simultaneously

  • Immunohistochemistry (IHC): Use TPD52L3 antibodies at 1:100 dilution for tissue section analysis

  • Quantitative RT-PCR: Measure TPD52L3 mRNA expression levels across tissue types

  • Single-cell RNA sequencing: Characterize expression at the single-cell level to identify cell type-specific patterns

  • Proteomic analysis: Use mass spectrometry to quantify protein expression and post-translational modifications

• Correlation with clinical parameters:

  • Analyze expression in relation to tumor grade, stage, and patient outcome

  • Investigate co-expression with signaling pathway components like LKB1 and AMPK

  • Examine associations with markers of cell proliferation, migration, and apoptosis

What are common challenges in TPD52L3 antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with TPD52L3 antibodies:

• Specificity issues:

  • Challenge: Cross-reactivity with other TPD52 family members

  • Solution: Use antibodies targeting unique epitopes in TPD52L3, validate with overexpression and knockdown controls, and perform peptide competition assays to confirm specificity

• Sensitivity limitations:

  • Challenge: Low detection in tissues with minimal expression

  • Solution: Employ signal amplification methods such as tyramide signal amplification for IHC/IF or use highly sensitive ECL substrates for Western blotting

• Isoform detection:

  • Challenge: Multiple transcript variants making specific isoform detection difficult

  • Solution: Use isoform-specific antibodies when available, or perform parallel detection with antibodies recognizing different epitopes

• Background signal:

  • Challenge: High background in immunofluorescence and IHC

  • Solution: Optimize blocking conditions (3% non-fat dry milk for Western blot, 2% BSA for IF), extend washing steps, and adjust antibody dilutions

How should experimental conditions be optimized when working with TPD52L3 antibodies across different model systems?

Optimizing experimental conditions for TPD52L3 antibodies requires system-specific considerations:

• Cell line models:

  • Validate antibody reactivity in each cell line before experimental use

  • For prostate cancer studies, LNCaP and VCaP cells have been successfully used

  • Consider doxycycline-inducible expression systems for controlled TPD52L3 expression

• Tissue samples:

  • For human samples: Optimize antigen retrieval methods for formalin-fixed paraffin-embedded tissues

  • For rodent samples: Adjust fixation times to preserve epitope accessibility

  • Consider using fresh frozen tissue sections for optimal antibody binding

• Species considerations:

  • Verify cross-reactivity when transitioning between human and rodent models

  • Be aware that some TPD52L3 antibodies are species-specific (human-only or human/rat/mouse)

  • Use species-appropriate positive controls to validate staining patterns

• Storage and handling:

  • Store antibodies at 4°C for short-term use

  • For long-term storage, aliquot and maintain at -20°C

  • Avoid freeze-thaw cycles that can degrade antibody quality

What controls are essential for validating TPD52L3 antibody specificity in research applications?

Rigorous validation of TPD52L3 antibody specificity requires implementation of several controls:

• Positive controls:

  • Use tissues or cell lines with confirmed TPD52L3 expression (testicular tissue is ideal)

  • Include recombinant TPD52L3 protein as a reference standard in Western blots

  • Consider using TPD52L3-overexpressing cells generated through transfection

• Negative controls:

  • Employ TPD52L3 knockdown/knockout cells or tissues

  • Use isotype control antibodies to assess non-specific binding

  • Include tissues known not to express TPD52L3 as negative controls

• Blocking peptide controls:

  • Pre-incubate antibody with blocking peptide containing the target epitope

  • Compare staining patterns with and without peptide blocking

  • Specific staining should be eliminated or significantly reduced in blocked samples

• Multiple antibody validation:

  • Use antibodies from different sources targeting distinct epitopes

  • Confirm that staining patterns are consistent across different antibodies

  • Consider orthogonal validation using mRNA detection methods (qPCR, RNA-seq)

How can TPD52L3 antibodies be utilized to investigate the protein's role in therapeutic resistance mechanisms?

Recent findings suggest potential applications of TPD52L3 antibodies in studying therapeutic resistance:

• Investigating AMPK pathway resistance:

  • Use TPD52L3 antibodies to monitor expression changes in response to AMPK-targeting therapies

  • Assess TPD52L3-LKB1 interaction status in treatment-resistant cells

  • Explore combination therapies targeting both TPD52L3 and AMPK signaling

• Biomarker development:

  • Employ TPD52L3 antibodies in tissue microarrays to correlate expression with treatment outcomes

  • Develop immunohistochemical scoring systems to quantify TPD52L3 levels in patient samples

  • Investigate potential for circulating TPD52L3 detection as a liquid biopsy approach

• Functional studies in resistant models:

  • Generate treatment-resistant cell lines and assess TPD52L3 expression and localization changes

  • Use TPD52L3 antibodies in combination with phospho-specific antibodies for LKB1 and AMPK to map signaling alterations

  • Perform immunoprecipitation to identify novel interaction partners in resistant contexts

What emerging techniques could enhance the utility of TPD52L3 antibodies in cancer research?

Several cutting-edge methodologies offer potential to maximize the research value of TPD52L3 antibodies:

• Proximity-based labeling approaches:

  • Combine TPD52L3 antibodies with BioID or APEX2 proximity labeling to identify the local interactome

  • Use spatially-resolved proteomics to map TPD52L3 interaction networks in specific cellular compartments

  • Employ TPD52L3 antibodies in conjunction with mass spectrometry to identify post-translational modifications

• Advanced imaging techniques:

  • Implement super-resolution microscopy (STORM, PALM) for nanoscale localization of TPD52L3

  • Apply live-cell imaging with fluorescently-tagged nanobodies derived from TPD52L3 antibodies

  • Utilize correlative light and electron microscopy to precisely localize TPD52L3 at the ultrastructural level

• Single-cell analysis:

  • Integrate TPD52L3 antibodies into mass cytometry (CyTOF) panels for high-dimensional analysis

  • Apply multiplexed immunofluorescence techniques to assess heterogeneity in TPD52L3 expression

  • Combine with single-cell transcriptomics to correlate protein and mRNA expression patterns

• Therapeutic development:

  • Assess potential for developing TPD52L3 antibody-drug conjugates

  • Explore TPD52L3 antibody fragments for therapeutic targeting of cancer cells

  • Investigate antibody-based disruption of the TPD52L3-LKB1 interaction as a therapeutic strategy