TPD52L1 Antibody

What is TPD52L1 and what are its key biological functions?

TPD52L1, also known as hD53, is a member of the tumor protein D52 (TPD52) family. This protein contains a coiled-coil domain that enables it to form homo- or hetero-dimers with other TPD52 family members . TPD52L1 plays significant roles in:

• Cell proliferation pathways

• Calcium signaling mechanisms

• Apoptotic regulation through interaction with mitogen-activated protein kinase kinase kinase 5 (MAP3K5/ASK1)

Unlike its family member TPD52, TPD52L1 does not appear to significantly participate in lipid storage or lipid droplet formation in cells . Multiple alternatively spliced transcript variants of TPD52L1 have been identified, though the complete characterization of some variants remains ongoing .

What applications are TPD52L1 antibodies validated for?

TPD52L1 antibodies have been validated for multiple experimental applications with specific recommended parameters:

The specific application determines the optimal working dilution, and researchers should conduct preliminary experiments to establish the ideal conditions for their specific experimental system .

What is the molecular weight pattern of TPD52L1 protein?

TPD52L1 exhibits a notable discrepancy between its calculated and observed molecular weights:

• Calculated molecular weight: 22 kDa (based on amino acid sequence)

• Observed molecular weight in experiments: 25 kDa (in Western blot analyses)

This difference is likely attributable to post-translational modifications. Additionally, different isoforms resulting from alternative splicing demonstrate varying molecular weights:

• Isoform 1: 22.5 kDa

• Isoform 3: 16.2 kDa

• Isoform 4: 14.7 kDa

When analyzing Western blot results, researchers should anticipate these variations and optimize gel resolution accordingly.

How should antigen retrieval be performed for TPD52L1 immunohistochemistry?

For optimal TPD52L1 detection in formalin-fixed, paraffin-embedded tissues, appropriate antigen retrieval is critical. The following methods are recommended:

• Primary method: Heat-induced epitope retrieval using TE buffer pH 9.0

  • Immerse slides in TE buffer and heat to 95-98°C for 15-20 minutes

  • Allow gradual cooling before proceeding with IHC protocol

• Alternative method: Citrate buffer pH 6.0

  • May be employed if the primary method yields suboptimal results

  • Follow similar heating parameters as above

When working with human prostate cancer tissue (a positive control for TPD52L1), TE buffer at pH 9.0 has been demonstrated to produce reliable results . The method selection may depend on the specific antibody clone and tissue type being examined.

What positive controls are recommended for TPD52L1 antibody validation?

Based on extensive validation data, the following positive controls are recommended:

• Cell lines:

  • MCF-7 cells (human breast adenocarcinoma cell line)

    • Validated for Western Blot applications

    • Validated for Immunoprecipitation

    • Validated for Immunofluorescence/ICC applications

• Tissue samples:

  • Human prostate cancer tissue has been verified as a positive control for IHC applications

When establishing a new experimental system, these samples serve as appropriate positive controls to verify antibody performance. For negative controls, researchers should consider using cell lines with confirmed low TPD52L1 expression or employing siRNA knockdown in positive cell lines.

How should TPD52L1 antibodies be stored and handled?

Proper storage and handling are essential for maintaining antibody performance over time:

• Storage temperature: -20°C is optimal for long-term stability

• Typical formulation: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Stability: One year from shipment date when stored properly

For aliquoting practices:

Always ensure proper temperature maintenance during shipping and handling to preserve antibody functionality.

How does TPD52L1 differ functionally from TPD52 in lipid metabolism research?

Despite belonging to the same protein family, TPD52L1 and TPD52 demonstrate distinct functional roles in lipid metabolism:

• TPD52 expression substantially increases neutral lipid storage and lipid droplet formation in cultured cells (approximately 4-10 fold increase in lipid droplet numbers compared to control cells)

• In contrast, TPD52L1-expressing cell lines show lipid droplet staining patterns comparable to control cells, suggesting minimal involvement in enhanced lipid storage

This functional distinction is further supported by protein interaction studies:

• TPD52 directly interacts with ADRP (also known as PLIN2), a lipid droplet-associated protein

• No direct interaction has been detected between TPD52L1 and ADRP in yeast two-hybrid system analyses

When designing comparative studies of TPD52 family proteins, researchers should account for these isoform-specific functional differences and employ standardized lipid quantification methods.

What is known about TPD52L1's role in MAP3K5-mediated apoptosis?

TPD52L1 has been identified as an interactor with mitogen-activated protein kinase kinase kinase 5 (MAP3K5/ASK1), a central regulator of stress-induced apoptosis. Current research indicates that:

• TPD52L1 positively regulates MAP3K5-induced apoptotic pathways

• The interaction likely involves the coiled-coil domain of TPD52L1

• This function represents a distinct role from other TPD52 family members

For researchers investigating these apoptotic mechanisms, several experimental approaches should be considered:

• Co-immunoprecipitation assays to confirm the interaction in specific cell types

• Knockdown/knockout studies to evaluate effects on MAP3K5 signaling

• Phosphorylation analysis to determine if TPD52L1 is a substrate of MAP3K5

• Apoptosis assays (caspase activation, PARP cleavage, Annexin V staining) with and without TPD52L1 expression

The precise mechanisms underlying TPD52L1's enhancement of MAP3K5-induced apoptosis represent an important area for ongoing investigation.

How can alternative splicing variants of TPD52L1 be detected and distinguished?

TPD52L1 undergoes alternative splicing, producing multiple transcript variants with distinct functional properties. To effectively detect and distinguish these variants, researchers can employ several complementary approaches:

• RT-PCR with isoform-specific primers:

  • Design primers spanning exon-exon junctions unique to each variant

  • Use nested PCR for low-abundance transcripts

  • Quantitative real-time PCR provides relative expression levels

• Western blotting:

  • Different isoforms exhibit distinct molecular weights:

    • Isoform 1: 22.5 kDa

    • Isoform 3: 16.2 kDa

    • Isoform 4: 14.7 kDa

  • High-resolution gel systems (12-15% acrylamide) are recommended to separate closely sized isoforms

• Isoform-specific antibodies:

  • Some antibodies recognize specific isoforms

  • For example, certain goat polyclonal antibodies recognize isoforms 1, 3, and 4 (NP_003278.1, NP_001003396.1, NP_001003397.1)

When reporting research findings, clear specification of which isoforms were detected and the methods used for distinction is essential, as different isoforms may possess distinct functional properties.

Why might the observed molecular weight of TPD52L1 (25 kDa) differ from the calculated weight (22 kDa)?

The discrepancy between calculated molecular weight (22 kDa) and observed migration pattern (25 kDa) can be attributed to several factors:

• Post-translational modifications:

  • Phosphorylation: TPD52 family proteins undergo phosphorylation, adding approximately 80 Da per phosphate group

  • Potential glycosylation, though not extensively reported for TPD52L1

• Protein structure and composition:

  • Proportion of acidic amino acids can cause anomalous migration on SDS-PAGE

  • The coiled-coil domain may affect SDS binding efficiency

• Technical considerations:

  • Gel percentage and buffer system influence apparent molecular weight

  • Molecular weight standards may not behave identically to the protein of interest

To address this discrepancy experimentally:

• Use multiple molecular weight markers

• Consider running dephosphorylation controls (treat lysates with phosphatase)

• Compare migration patterns under reducing and non-reducing conditions

Understanding the basis for this difference may provide insights into TPD52L1's post-translational regulation in different cellular contexts.

What approaches can resolve weak TPD52L1 signal in Western blots?

Researchers encountering weak signal when detecting TPD52L1 by Western blot can implement several optimization strategies:

• Sample preparation enhancement:

  • Enrich for TPD52L1 using subcellular fractionation

  • Use comprehensive protease inhibitor cocktails during lysis

  • Consider phosphatase inhibitors if phosphorylated forms are of interest

• Protein loading optimization:

  • Increase total protein loaded (30-50 μg per lane)

  • Use cell lines with known higher expression (e.g., MCF-7)

• Detection system improvements:

  • Employ more sensitive detection methods (enhanced chemiluminescence systems)

  • Consider fluorescent secondary antibodies for quantitative analysis

  • Try biotin-streptavidin amplification systems

• Antibody optimization:

  • Adjust antibody concentration (use the more concentrated end of the recommended range: 1:500)

  • Extend primary antibody incubation (overnight at 4°C)

  • Test different antibody clones if available

When implementing these approaches, modify one parameter at a time to identify the specific factor limiting signal intensity. Document successful optimization strategies for reproducibility.

How can specificity of TPD52L1 antibody be validated?

Thorough validation of antibody specificity is essential for reliable experimental outcomes. For TPD52L1 antibody, several complementary approaches are recommended:

• Genetic manipulation approaches:

  • siRNA or shRNA knockdown of TPD52L1 should proportionally reduce signal intensity

  • CRISPR/Cas9 knockout provides a definitive negative control

  • Overexpression systems can confirm expected molecular weight patterns

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Should abolish specific signal while non-specific binding remains

• Multiple antibody validation:

  • Utilize antibodies raised against different epitopes of TPD52L1

  • Consistent results across different antibodies increase confidence in specificity

• Cross-reactivity assessment:

  • Test against recombinant TPD52, TPD52L1, and TPD52L2 proteins

  • Ensures the antibody discriminates between family members

• Orthogonal methods:

  • Compare protein expression with mRNA levels

  • Concordance between protein and mRNA suggests specific detection

Comprehensive documentation of validation experiments should be maintained to support the reliability of research findings involving TPD52L1 antibody.

What approaches are recommended for studying TPD52L1 protein-protein interactions?

Given TPD52L1's roles in various cellular processes through protein-protein interactions, several complementary methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4.0 μg of TPD52L1 antibody per 1.0-3.0 mg of total protein lysate

  • Include appropriate controls (IgG control, reverse IP)

  • Western blot to detect interacting partners

• Yeast two-hybrid (Y2H) screening:

  • Has been successfully used to identify TPD52 family interactions

  • Use full-length TPD52L1 or specific domains (e.g., coiled-coil region) as bait

  • Confirmatory assays required to validate initial interactions

• GST pulldown assays:

  • Express TPD52L1 as a GST fusion protein

  • Incubate with cell lysates or purified potential interacting partners

  • Detect binding by Western blot analysis

• Proximity-ligation assays (PLA):

  • Allows visualization of protein interactions in situ

  • Provides spatial information about interaction sites within cells

When studying TPD52L1 interactions, consider that the protein may form both homo- and hetero-dimers with TPD52 family members, potentially complicating interpretation of results. Multiple approaches should be employed to confirm interactions.

Future directions in TPD52L1 antibody-based research

As TPD52L1 continues to be investigated in various cellular processes, several emerging research directions warrant consideration:

• Development of isoform-specific antibodies to better distinguish between alternatively spliced variants

• Exploration of TPD52L1's potential roles in cancer progression through comparative studies of normal and malignant tissues

• Investigation of post-translational modifications of TPD52L1 and their functional significance

• Further characterization of the regulatory mechanisms controlling TPD52L1 expression and activity