TMPO Antibody

What is TMPO and what is its role in cellular biology?

TMPO (Thymopoietin), also known as LAP2 (Lamina-associated polypeptide 2), is a nuclear protein involved in the structural organization of the nucleus and post-mitotic nuclear assembly. It plays an important role, together with LMNA, in the nuclear anchorage of RB1 . TMPO/LAP2 functions in directing the assembly of the nuclear lamina and helps maintain the structural organization of the nuclear envelope . It serves as a possible receptor for the attachment of lamin filaments to the inner nuclear membrane and may be involved in controlling the initiation of DNA replication through its interaction with NAKAP95 .

What are the different isoforms of TMPO and how do they differ?

TMPO exists in several isoforms with varying molecular weights:

These isoforms are generated through alternative splicing and have distinct functions in the nuclear envelope. The alpha isoform is predominantly involved in nuclear architecture organization, while beta/gamma isoforms play roles in chromatin interaction and nuclear assembly .

What are the recommended storage conditions for TMPO antibodies?

Most TMPO antibodies should be stored at -20°C for long-term preservation . For frequent use, short-term storage at 4°C for up to one month is acceptable . The antibodies are typically supplied in a buffer containing glycerol (e.g., 50%) and a preservative such as sodium azide (0.02%) . It is critical to avoid repeated freeze-thaw cycles as this can degrade antibody quality and reduce reactivity .

What epitope retrieval methods are recommended for TMPO detection in FFPE tissues?

For immunohistochemistry applications using formalin-fixed, paraffin-embedded (FFPE) tissue sections, the following epitope retrieval methods are recommended:

• Citrate buffer (pH 6.0) epitope retrieval is specifically recommended for FFPE tissue sections when using certain antibodies .

• TE buffer (pH 9.0) has been validated with several TMPO antibodies for optimal detection .

Selection of the appropriate retrieval method may be antibody-specific, and optimization might be necessary for different tissue types or fixation conditions .

How can I validate the specificity of a TMPO antibody?

Validating antibody specificity is crucial for reliable experimental results. For TMPO antibodies, several validation methods can be employed:

• Knockout validation: Use TMPO knockout cell lines to confirm absence of signal with your antibody. Some commercial antibodies are already KO-validated .

• Multiple detection methods: Compare results across different applications (WB, IHC, IF) to ensure consistent detection patterns .

• Positive controls: Use tissues or cell lines known to express TMPO at high levels, such as HeLa, HepG2, or MCF-7 cells .

• Molecular weight verification: Confirm that the detected bands match the expected molecular weights for TMPO isoforms (75 kDa, 51 kDa, and 39 kDa) .

• Blocking peptides: When available, use immunizing peptides to compete for antibody binding in parallel experiments .

Why might I observe multiple bands in TMPO Western blots?

Multiple bands in TMPO Western blots are common and could reflect:

• Multiple isoforms: TMPO exists in several splice variants with molecular weights of approximately 75 kDa, 51 kDa, and 39 kDa .

• Post-translational modifications: Phosphorylation during mitosis regulates TMPO interactions with chromosomes and lamin B1 , potentially causing band shifts.

• Degradation products: Sample preparation methods can influence protein integrity, leading to partial degradation products.

• Cross-reactivity: Some antibodies may cross-react with structurally similar proteins, particularly those containing LEM domains.

To distinguish between these possibilities, consider including positive controls with known TMPO expression patterns and using phosphatase treatments to identify modification-dependent shifts .

What cell types and tissues are optimal for studying TMPO expression?

Based on validation data from commercial antibodies, the following cell lines and tissues show strong TMPO expression and can serve as good experimental systems:

Western blot analysis has confirmed TMPO expression in these systems, making them suitable positive controls for antibody validation and experimental studies .

How does TMPO relate to disease pathologies?

TMPO/LAP2 has been implicated in several disease contexts, making it an important research target:

• Cardiomyopathy: The gene symbol CMD1T (Cardiomyopathy, Dilated, 1T) appears as an alias for TMPO, suggesting a link to cardiac disorders .

• Cancer research: TMPO antibodies have been validated on various cancer tissues, including liver cancer and cervical cancer, indicating potential roles in malignancy .

• Nuclear envelope disorders: As a nuclear lamina protein, TMPO may be involved in laminopathies and related nuclear envelope diseases.

• Cell cycle regulation: Through its interaction with RB1 and potential role in DNA replication control, TMPO may influence cell proliferation pathways relevant to cancer research .

What factors might affect subcellular localization patterns of TMPO in immunofluorescence?

When performing immunofluorescence experiments with TMPO antibodies, several factors can influence the observed localization pattern:

• Cell cycle stage: TMPO's interaction with chromosomes is regulated by phosphorylation during mitosis , potentially altering its distribution pattern.

• Fixation method: Different fixation protocols (paraformaldehyde vs. methanol) can affect nuclear envelope preservation and epitope accessibility.

• Permeabilization conditions: Overly harsh permeabilization may disrupt nuclear envelope structure, while insufficient permeabilization limits antibody access.

• Isoform-specific detection: Different antibodies may preferentially detect certain TMPO isoforms, which can have distinct subcellular distributions.

• Image acquisition parameters: Confocal vs. wide-field microscopy and exposure settings can significantly impact the apparent localization pattern.

For optimal immunofluorescence results, use dilutions of 1:200-1:800 and include appropriate nuclear counterstains to properly contextualize TMPO localization .

How can I optimize immunoprecipitation experiments with TMPO antibodies?

For successful immunoprecipitation (IP) of TMPO, consider these optimization strategies:

• Antibody amount: Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate as a starting point .

• Lysis conditions: Since TMPO is a nuclear membrane protein, ensure your lysis buffer effectively solubilizes nuclear envelope components (consider NP-40 or RIPA buffers with appropriate salt concentrations).

• Cross-linking: For studying TMPO protein interactions, consider mild cross-linking before lysis to preserve protein complexes.

• Pre-clearing lysates: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Controls: Always include an isotype control antibody IP to identify non-specific interactions.

Positive IP detection has been validated in HeLa cells, making this cell line a good starting point for TMPO immunoprecipitation experiments .

How can high-throughput biophysical assays be applied to TMPO antibody research?

Recent developments in antibody evaluation can be applied to TMPO research using the high-throughput developability workflow described in the literature :

• Antibody engineering: When TMPO antibodies show suboptimal properties, sequence engineering can be performed to remove post-translational modification sites or disrupt hydrophobic patches that lead to aggregation without affecting binding specificity .

• Biophysical characterization: TMPO antibodies can be evaluated for critical parameters such as colloidal properties (aggregation, self-interaction, hydrophobicity), fragmentation/clipping, and thermostability using small amounts of material (100s μgs) .

• Predictive tools: Computational methods for predicting aggregation risk can be applied to TMPO antibodies, such as SAP scores or machine learning methods to predict hydrophobic chromatography retention time .

• Iterative optimization: The analytical characterization process can be repeated during sequence engineering to ensure improved biophysical properties while maintaining target specificity .

What emerging applications exist for TMPO antibodies in disease research?

TMPO antibodies are finding new applications in several disease-related research areas:

• Cancer biomarker studies: TMPO expression patterns may serve as diagnostic or prognostic indicators in various cancers, as evidenced by validated detection in breast cancer and cervical cancer tissues .

• Nuclear envelope dynamics in pathology: As nuclear architecture disruption is implicated in aging and disease, TMPO antibodies can help track these changes in patient-derived samples.

• Cell cycle control mechanisms: Through its interaction with RB1 and potential role in DNA replication, TMPO studies may reveal new insights into cell proliferation control relevant to cancer and developmental disorders .

• Cardiovascular research: The association of TMPO with cardiomyopathy (CMD1T) suggests applications in heart disease research .

By combining TMPO antibodies with other nuclear envelope markers in multi-parameter analyses, researchers can develop more comprehensive understanding of nuclear structure alterations in disease states.