TPPP3 Antibody

What is TPPP3 and what cellular functions does it perform?

TPPP3, also known as TPPP/p20, is a 19 kDa protein member of the tubulin polymerization-promoting protein family. It functions primarily as a regulator of microtubule dynamics with demonstrated microtubule bundling activity . TPPP3 is predominantly localized in the cytoplasm where it interacts with the microtubule network. Multiple studies have shown that TPPP3 plays essential roles in various biological processes including:

• Embryo implantation through regulation of beta-catenin signaling

• Cellular decidualization processes

• Modulation of microtubule organization similar to its family member TPPP1

In neuronal contexts, TPPP3 has been identified as a novel marker for retinal ganglion cells (RGCs) and appears to play a significant role in axon regeneration, as demonstrated in mouse models .

What are the common experimental applications for TPPP3 antibodies?

TPPP3 antibodies are utilized across multiple experimental techniques, with application-specific optimization requirements. Based on current research practices, the primary applications include:

Most commercially available TPPP3 antibodies demonstrate reactivity with human, mouse, and rat samples, though some exhibit broader species cross-reactivity .

How is TPPP3 expression distributed across different tissues and developmental stages?

TPPP3 exhibits tissue-specific and developmentally regulated expression patterns. Research findings indicate:

• In retinal development: TPPP3 expression is detected in the inner retinal layer at embryonic day 12 (E12) and becomes specifically localized to the ganglion cell layer by postnatal day 0 (P0)

• Expression peaks at E14.5, coinciding with the peak of retinal ganglion cell differentiation

• Approximately 75% of cells in the retinal ganglion cell layer co-express the RGC marker Brn3a and TPPP3

• In adult tissues: TPPP3 has been identified as a specific marker for connective tissue

• In endometrium: TPPP3 is expressed during the mid-secretory phase (LH + 7)

Single-cell RNA sequencing analysis has revealed that TPPP3 expression is widespread across all retinal ganglion cell sub-clusters in mice and is also present within RGC clusters of macaque and human samples .

What approaches should be used to validate TPPP3 antibody specificity?

Antibody validation is critical when working with TPPP3 due to the presence of related family members (TPPP1, TPPP3) and potential cross-reactivity. Recommended validation approaches include:

A. Western Blot Validation:

• Confirm band size at the expected molecular weight (19-20 kDa)

• Include positive control samples with known TPPP3 expression (e.g., PC-3 cells, HEK-293 cells)

• Test reactivity in TPPP3 overexpression systems compared to vector controls

• Perform antibody validation in TPPP3 knockdown or knockout models

B. Immunohistochemistry/Immunofluorescence Validation:

• Compare staining patterns with mRNA expression (e.g., RNAscope in situ hybridization)

• Perform co-localization studies with established TPPP3 markers

• Use both positive control tissues (e.g., retinal ganglion cell layer) and negative control tissues

• Validate using peptide competition assays where pre-incubation with the immunizing peptide should abolish specific staining

C. ELISA-Based Validation:

• Competitive ELISA can be used to assess specificity between TPPP family members

• In one study, ELISA plates were coated with 5 μg/mL TPPP1 or TPPP3, and a monoclonal antibody was tested at serial dilutions to confirm specificity

The most comprehensive validation combines multiple approaches to establish antibody specificity across different experimental conditions.

How should researchers address contradictory findings regarding TPPP3 expression in different cancer types?

The literature presents conflicting data regarding TPPP3 expression patterns in various cancers, requiring careful experimental design and interpretation:

Contradictory Expression Patterns:

• Elevated expression: TPPP3 shows significantly elevated expression in colorectal carcinoma and invasive ductal breast carcinoma according to Oncomine database analysis

• Reduced expression: TPPP3 expression is significantly downregulated in nasopharyngeal carcinoma (NPC) tissues and cells compared to normal controls

Methodological Approaches to Address Contradictions:

• Multi-method validation: Employ multiple detection methods (qRT-PCR, immunohistochemistry, and western blotting) in the same samples

• Database integration: Analyze multiple public datasets (e.g., GEO datasets GSE12452, GSE53819, and GSE61218 for NPC)

• Cell line validation: Compare expression across both normal and cancer cell lines (e.g., NP69 vs. CNE2, HK1, 5-8F, and HONE1 for NPC)

• Biological functional validation: Perform overexpression or knockdown experiments to correlate expression with functional outcomes

  • In NPC cells, TPPP3 overexpression significantly inhibited proliferation and attenuated invasion ability

  • TPPP3 overexpression diminished the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) mRNA in NPC cells

When reporting results, researchers should clearly specify cancer type, detection methods, and functional validation approaches to help resolve discrepancies in the literature.

What are the optimal fixation and antigen retrieval protocols for TPPP3 detection in tissue samples?

Successful immunohistochemical detection of TPPP3 requires careful attention to fixation and antigen retrieval conditions:

Fixation Protocols:

• For frozen sections: 12 μm OCT-frozen tissue sections have been successfully used for TPPP3 detection using RNAscope in situ hybridization

• For paraffin-embedded sections: Standard formalin fixation has been used successfully for TPPP3 immunohistochemistry

Antigen Retrieval Methods:

• The primary recommended method is TE buffer at pH 9.0

• Alternative approach: Citrate buffer at pH 6.0 has also been reported effective

• For RNAscope detection of TPPP3 mRNA, sections should be pretreated with hydrogen peroxide, followed by antigen retrieval and protease application before hybridization

Optimization Recommendations:

• Test multiple antibody dilutions, typically starting with 1:50-1:500 range for IHC

• Include both positive control tissues (e.g., stomach cancer tissue has been used successfully)

• For neuronal tissue, particularly retinal samples, longer fixation times may require more aggressive antigen retrieval

The specific protocol should be optimized based on the tissue type, fixation method, and the particular TPPP3 antibody being used.

How can researchers effectively distinguish between TPPP family members in experimental systems?

The TPPP protein family contains multiple members with structural similarities, necessitating specific approaches to distinguish between them:

Antibody-Based Discrimination:

• Select antibodies raised against non-conserved regions of TPPP3

• Many commercial TPPP3 antibodies target the C-terminal region (AA 117-146) or specific epitopes (AA 50-150)

• Validate antibody specificity using competitive ELISA: In one study, plates were coated with 5 μg/mL TPPP1 or TPPP3, and the monoclonal antibody was tested at serial dilutions to confirm specificity

Development of Monoclonal Antibodies:

• For maximum specificity, custom monoclonal antibodies can be developed using TPPP3-specific peptides

• Example method: Female Balb/c mice were immunized with TPPP3 peptide-thyreoglobulin conjugate, followed by cell fusion and ELISA-based screening

Expression Analysis Approaches:

• qRT-PCR using primers specifically designed for non-conserved regions

• Western blotting with careful attention to molecular weight differences (TPPP3 is ~19-20 kDa)

• Immunoprecipitation followed by mass spectrometry for definitive identification

Experimental Controls:

• Include recombinant TPPP1 and TPPP3 as positive and negative controls

• Utilize TPPP3 knockout or knockdown systems as negative controls

• When studying protein-protein interactions, use purified recombinant proteins to verify specificity

These approaches should be used in combination for conclusive identification of specific TPPP family members.

What are the recommended protocols for studying TPPP3's role in regulating microtubule dynamics?

TPPP3 functions as a regulator of microtubule dynamics with demonstrated bundling activity. To effectively study this function:

In Vitro Microtubule Polymerization Assays:

• Purify recombinant TPPP3 protein using established protocols

• Monitor tubulin polymerization in the presence of varying concentrations of TPPP3

• Use turbidity measurements (absorbance at 350 nm) to track polymerization kinetics

• Compare with known microtubule-stabilizing agents (e.g., Taxol) as positive controls

Cellular Imaging Approaches:

• Perform co-localization studies using anti-TPPP3 and anti-tubulin antibodies

• Example: Co-labeling experiments using neuronal marker anti-β-III-tubulin antibody-E7 have confirmed co-localization of TPPP3 with β-III-tubulin in primary retinal ganglion cells

• Use live-cell imaging with fluorescently tagged TPPP3 to monitor dynamic associations with microtubules

• Implement super-resolution microscopy techniques for detailed structural analysis

Functional Perturbation Studies:

• Generate TPPP3 mutants with altered microtubule binding domains

• Perform overexpression and knockdown experiments to assess effects on microtubule stability

• Measure parameters such as microtubule growth rates, catastrophe frequency, and rescue events

Biochemical Interaction Studies:

• Use co-immunoprecipitation to identify TPPP3 binding partners within the microtubule network

• Employ chemical crosslinking mass spectrometry to identify specific interaction sites

• Perform in vitro binding assays with purified components to determine direct interactions

These methodologies provide complementary approaches to characterize TPPP3's role in microtubule regulation.

How can researchers effectively use TPPP3 as a marker for retinal ganglion cells (RGCs)?

Recent research has identified TPPP3 as a novel marker for retinal ganglion cells, with specific methodological considerations:

Validation of TPPP3 as an RGC Marker:

• Approximately 75% of cells co-express the established RGC marker Brn3a and TPPP3 in the ganglion cell layer

• TPPP3 expression is localized within the RGC layer and the retinal nerve fiber layer (RNFL)

• Expression has been validated using multiple techniques: immunofluorescence, western blot, and RNAscope in situ hybridization

Optimal Detection Protocols:

• For immunofluorescence: TPPP3 is primarily expressed in RGC soma, with lower expression in neurites

• For developmental studies: RNAscope in situ hybridization can trace TPPP3 expression from E12 through P0

• Western blot analysis can be used to quantify expression levels in retinal tissue samples

Applications in RGC Research:

• TPPP3 can be used to identify and isolate RGCs from mixed retinal cell populations

• In RGC culture systems, TPPP3 markers can confirm cellular identity alongside established markers (BRN3A, RBPMS, THY1)

• For axon regeneration studies, TPPP3 overexpression has been shown to enhance axonal regeneration and improve RGC survival following optic nerve crush (ONC)

Comparative Analysis with Other RGC Markers:

• Single-cell RNA sequencing analysis shows TPPP3 expression across all RGC sub-clusters in mice

• TPPP3 expression is also detected within RGC clusters of macaque and human samples

• Consider using TPPP3 in conjunction with other established RGC markers for comprehensive identification

These approaches enable effective utilization of TPPP3 as a valuable tool in retinal ganglion cell research.

What are the methodological approaches for studying TPPP3's role in epithelial-mesenchymal transition (EMT) in cancer research?

Recent evidence suggests TPPP3 may play a role in epithelial-mesenchymal transition in certain cancer types, particularly through interactions with Snail1 in glioblastomas. Effective study of this relationship requires:

Expression Analysis Approaches:

• Immunohistochemical analysis of patient tumor samples to assess correlation between TPPP3 and EMT markers

• Western blot analysis using validated antibodies against TPPP3 and EMT markers (E-cadherin, N-cadherin, Vimentin, Snail1, Slug, Twist1, ZEB1)

• qRT-PCR to quantify mRNA expression levels of TPPP3 and EMT-related genes

Functional Studies:

• Generate stable TPPP3 overexpression and knockdown cell lines using lentiviral transduction

• Example: After lentivirus transfection and puromycin selection, GFP expression can be used to monitor transfection efficiency

• Confirm altered expression by qRT-PCR and western blot before proceeding with functional assays

EMT Phenotype Assessment:

• Cell morphology examination using phase-contrast microscopy

• Migration assays (wound healing, transwell migration)

• Invasion assays (Matrigel-coated transwell chambers)

• Analysis has shown that TPPP3 overexpression can attenuate invasion ability of cancer cells and diminish expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)

Molecular Mechanism Investigation:

• Co-immunoprecipitation to study TPPP3 interaction with EMT transcription factors

• Chromatin immunoprecipitation (ChIP) to investigate transcriptional regulation

• Analysis of clinical data for correlation between TPPP3 and Snail1 protein expression levels

These methodological approaches provide a comprehensive framework for investigating TPPP3's role in epithelial-mesenchymal transition in cancer.