TPBG Antibody

What is TPBG and what epitopes do researchers target with antibodies?

TPBG is a leucine-rich repeat glycoprotein with an intracellular PDZ-binding motif. It functions in cellular processes including migration, adhesion, and cytoskeletal organization. Two primary epitope regions are commonly targeted by researchers: the N-terminal (extracellular) leucine-rich repeat domain and the C-terminal region containing the PDZ-interacting motif. Anti-TPBG-CT antibodies typically target a 15-amino acid synthetic peptide within amino acids 385-420 of human TPBG, while anti-TPBG-NT antibodies target the region from amino acids 30 to 361 of mouse TPBG . When selecting a TPBG antibody, researchers should consider whether they need to detect the extracellular or intracellular domains based on their experimental questions, as these antibodies may reveal different aspects of TPBG biology.

What expression pattern does TPBG show in normal tissues versus disease states?

In normal tissues, TPBG shows restricted expression patterns with notable presence in specific neural cell populations. In the retina, TPBG is localized to dendrites and synaptic terminals of rod bipolar cells and in cell bodies and dendrites of an uncharacterized amacrine cell type . Outside the nervous system, TPBG functions as an oncofetal antigen, showing low expression in normal adult tissues but significant upregulation in various human cancers . This differential expression pattern makes TPBG an attractive target for cancer therapeutics, with high TPBG expression typically associated with poor clinical outcomes . Researchers should be aware of these tissue-specific patterns when designing experiments, as detection protocols may need optimization depending on the tissue being studied.

What molecular characteristics of TPBG are important for antibody detection?

TPBG appears as a broad band around 72 kDa in immunoblots, reflecting its glycosylated nature . The protein contains multiple post-translational modifications, most notably glycosylation, which contributes to its apparent molecular weight being higher than predicted from the amino acid sequence alone. Manufacturers of TPBG antibodies have verified immunoreactivity via immunoblot of various cell lysates, typically detecting broad bands of glycosylated proteins at 70-80 kDa . When probing for TPBG, researchers should anticipate this glycosylation pattern rather than expecting a sharp single band. Deglycosylation experiments may be useful controls when characterizing TPBG expression in new experimental systems.

How should immunofluorescence protocols be optimized for TPBG detection in retinal tissue?

For optimal TPBG detection in retinal tissue, post-fixation in 4% paraformaldehyde for 10 minutes before blocking and permeabilization steps has been shown to significantly improve immunofluorescence signal with both anti-TPBG-CT and anti-TPBG-NT antibodies . Use antibody dilutions of approximately 1:500 for immunofluorescence applications, and include DAPI nuclear staining to identify different retinal layers . For retina sections, block and permeabilize by incubation at room temperature for 60 minutes in appropriate blocking solution, then incubate with primary antibodies for 1 hour at room temperature . After thorough washing with PBS, incubate with appropriate secondary antibodies and proceed with mounting. This approach will allow visualization of TPBG localization in rod bipolar cell dendrites and terminals, as well as in amacrine cells.

What are the recommended conditions for Western blot applications with TPBG antibodies?

For Western blot applications, anti-TPBG-CT antibodies have been successfully used at dilutions of 1:5000, while other TPBG antibodies may require different concentrations . When performing immunoblotting for TPBG, expect to detect a broad band around 72 kDa representing the glycosylated protein rather than a sharp band . Specificity can be confirmed through immunoprecipitation followed by immunoblotting from wild-type tissue lysates and TPBG-transfected cell lines as positive controls. Run appropriate negative controls such as lysates from TPBG knockout tissues or cells where available. Sample preparation should preserve glycosylated proteins, as TPBG contains significant post-translational modifications that contribute to its apparent molecular weight and potentially to epitope recognition.

How can researchers validate TPBG antibody specificity in their experimental systems?

To validate TPBG antibody specificity, researchers should employ multiple complementary approaches. Immunoprecipitation followed by immunoblotting from wild-type tissue lysates compared with TPBG-transfected cell lines has been used successfully to confirm specificity of both N-terminal and C-terminal targeting antibodies . Use multiple antibodies targeting different epitopes of TPBG and compare labeling patterns. Consistent results across antibodies provide increased confidence in specificity. Include appropriate controls in experiments, such as TPBG knockout tissues where available, or tissues known to have very low TPBG expression. For transfection experiments, compare TPBG-transfected cells with vector-only controls to identify specific labeling patterns.

How does neuronal activity affect TPBG antibody labeling in retinal tissue?

TPBG immunofluorescence in rod bipolar cells shows remarkable activity-dependent patterns, particularly when detected with C-terminal antibodies. Labeling is significantly reduced in dark-adapted retina compared to light-adapted retina, despite no change in total TPBG protein levels as determined by immunoblotting . Similar reductions in labeling occur in light-adapted PKCα knockout and TRPM1 knockout retinas compared to wild type . This suggests that the C-terminal epitope becomes masked or inaccessible in certain activity states, possibly through interaction with PDZ domain-containing proteins. Researchers should consider these activity-dependent changes when designing experiments and interpret immunofluorescence results in the context of the tissue's activity state. Use multiple antibodies (targeting different epitopes) and complementary techniques (such as Western blotting) to distinguish between changes in protein levels versus changes in epitope accessibility.

What developmental patterns of TPBG expression should researchers consider?

TPBG expression in the retina increases dramatically just prior to eye opening, with a time course closely correlated with TRPM1 expression . This developmental pattern suggests TPBG may play important roles in synaptic development and maturation. When studying TPBG in developmental contexts, researchers should carefully time their experiments relative to key developmental milestones relevant to their tissue of interest. In the retina, comparing pre- and post-eye opening timepoints may reveal important functional aspects of TPBG. Developmental studies should include appropriate age-matched controls and consider that antibody dilutions may need to be adjusted for developmental stages with lower TPBG expression. Correlating TPBG expression with other developmental markers can provide insights into its functional roles.

How does TPBG localization differ between cell types in the retina?

In the retina, TPBG shows distinct localization patterns in different cell types. In rod bipolar cells, TPBG is localized to dendrites and synaptic terminals, with punctate immunoreactivity in the outer plexiform layer (OPL) closely associated with dendritic tips . These puncta co-localize with or near GPR179 and CtBP2, suggesting localization to ribbon synapses. In the inner plexiform layer (IPL), TPBG labels the membrane and cytoplasm of rod bipolar cell synaptic terminals, overlapping with PKCα . In amacrine cells, TPBG is present in cell bodies with low-density distribution in the inner nuclear layer (INL) and dense dendritic stratification between the ON and OFF sublamina in the IPL . Researchers studying TPBG in retinal tissues should use co-labeling with cell-type specific markers to properly identify TPBG-expressing populations and consider that different fixation or permeabilization protocols might be necessary to optimize detection in different cellular compartments.

How can humanized mouse models advance TPBG-targeted therapeutic research?

Humanized mouse models where the murine Tpbg coding sequence is replaced with human TPBG under endogenous regulatory sequences provide valuable tools for therapeutic development. These models express human TPBG in patterns consistent with endogenous mouse expression, allowing for more translational pharmacology studies . When using such models, researchers should confirm that the gene replacement is functional by assessing whether the mice exhibit phenotypes associated with Tpbg deletion. In pharmacokinetic studies, researchers can use these models to evaluate target-related clearance of TPBG-targeted therapeutics, as demonstrated with the antibody-drug conjugate A1mcMMAF, which showed lower exposure in humanized TPBG knockin mice versus wild-type animals . This approach generates more precise pharmacokinetic and pharmacodynamic data for current and future TPBG-targeted therapies, particularly relevant for antibody-drug conjugates and other biotherapeutics in cancer research.

What approaches are effective for studying TPBG in signaling pathways?

To study TPBG's role in signaling pathways, researchers should employ multiple complementary techniques. In vascular research, TPBG has been shown to orchestrate migratory and angiogenic activities of pericytes through activation of the CXCL12/CXCR7/pERK axis . Techniques such as proximity ligation assays can reveal protein-protein interactions, while ELISA, Western blot, and immunocytochemistry can detect hypoxia-dependent regulation of TPBG expression . When investigating TPBG's interactions with PDZ domain-containing proteins, consider that epitope accessibility may change depending on binding status. This has been observed in retinal tissues where C-terminal epitope detection varies with neuronal activity state . Co-immunoprecipitation experiments followed by mass spectrometry can identify novel binding partners, while mutation of the PDZ-binding motif can confirm the specificity and functional significance of these interactions.

How can TPBG antibodies be used to study subcellular localization in transfected cell systems?

Transfected cell systems offer controlled environments to study TPBG trafficking and localization. In HEK293 cells, transfected TPBG localizes primarily to the plasma membrane and intracellular membranes, with enrichment at the tips of filopodia-like membrane projections . Researchers can use this system to study mutations in TPBG that might affect its trafficking or membrane localization. For optimal detection in transfected cells, use both N-terminal and C-terminal antibodies to gain comprehensive understanding of the protein's localization. Live-cell imaging with fluorescently tagged TPBG constructs, complemented by fixed-cell immunofluorescence with TPBG antibodies, can provide insights into dynamic trafficking processes. Co-transfection with markers for specific subcellular compartments can help identify the precise localization patterns of TPBG within the cell.

Why might N-terminal and C-terminal TPBG antibodies show different labeling patterns?

Different labeling patterns between N-terminal and C-terminal TPBG antibodies can result from several factors. C-terminal (intracellular) epitopes may be masked by protein-protein interactions, particularly with PDZ domain-containing proteins, resulting in activity-dependent accessibility . This has been observed in retinal tissue where C-terminal antibody labeling varies with light adaptation state. N-terminal (extracellular) antibodies target the leucine-rich repeat domain, which may have different accessibility constraints based on protein conformation or interactions with extracellular matrix components. When confronted with discrepancies between antibodies, researchers should consider that they may be revealing different aspects of TPBG biology rather than representing technical artifacts. Use both antibody types in parallel experiments and complement with additional approaches such as Western blotting to distinguish between changes in protein levels versus changes in epitope accessibility.

What fixation and permeabilization protocols optimize TPBG antibody applications?

For retinal tissue sections, post-fixation in 4% paraformaldehyde for 10 minutes before blocking and permeabilization has been shown to improve immunofluorescence with both anti-TPBG-CT and anti-TPBG-NT antibodies . Following fixation, block and permeabilize sections by incubation at room temperature for 60 minutes in appropriate blocking solution before proceeding with primary antibody incubation . For dissociated cell preparations, fixation protocols may need adjustment compared to tissue sections. The choice of detergent for permeabilization can significantly impact antibody access to intracellular epitopes, with Triton X-100 typically used for accessing cytoplasmic epitopes and milder detergents like saponin sometimes preferred for membrane-associated proteins. Researchers should systematically optimize fixation time, fixative concentration, and permeabilization conditions for their specific tissue and application.

How can researchers address epitope masking issues in TPBG detection?

Epitope masking is particularly relevant for TPBG C-terminal antibodies, where PDZ domain interactions may block antibody access. If epitope masking is suspected, several approaches can help: (1) Compare results from antibodies targeting different epitopes of TPBG; (2) Manipulate conditions that affect protein-protein interactions, such as phosphorylation state (using phosphatase inhibitors or activators); (3) Use denaturing conditions for techniques like Western blotting to disrupt protein interactions; (4) Consider using detergents or salt concentrations that may disrupt protein-protein interactions without affecting antibody binding; (5) For tissues with activity-dependent epitope masking (like retina), explicitly control and report the activity state of the tissue . The observation that TPBG immunofluorescence varies with light adaptation while total protein levels remain constant highlights the importance of considering epitope accessibility when interpreting results.

What are the optimal working dilutions for different TPBG antibodies across applications?

These recommended dilutions serve as starting points, and researchers should optimize conditions for their specific experimental systems. Factors affecting optimal dilution include tissue type, fixation method, detection system, and specific batch of antibody. Validation with appropriate positive and negative controls is essential when establishing working conditions for a new experimental system.

What cell types express TPBG across different tissues?

This information helps researchers select appropriate positive control tissues and understand the biological context of TPBG expression. When studying TPBG in a new tissue or cell type, co-labeling with established cell-type markers can help confirm the identity of TPBG-expressing cells.