TMEM240 Antibody, Biotin conjugated

What is TMEM240 and what cellular functions has it been associated with?

TMEM240 (Transmembrane protein 240, also known as C1orf70) is a protein associated with synaptic membrane function . Recent research has revealed that TMEM240 exhibits tumor suppressor potential, affecting cancer cell growth and migration. Studies show that TMEM240 can lead to G1 cell cycle arrest, repress cancer cell proliferation, and inhibit cancer cell migration in colorectal cancer cells . When TMEM240 was overexpressed in DLD-1 cancer cells, their growth slowed by 66.6% compared to control groups, while knockdown of TMEM240 increased cell growth by 2.0-fold, demonstrating its inhibitory effects on cancer progression .

What are the properties of commercially available TMEM240 antibodies?

The TMEM240 antibody (biotin conjugated or FITC conjugated) is typically a polyclonal antibody raised in rabbits against human TMEM240. The immunogen used is recombinant human Transmembrane protein 240 protein (specifically amino acids 42-87). These antibodies demonstrate human species reactivity and have been tested for ELISA applications, though additional applications may require validation by individual researchers . The antibodies are typically >95% pure, processed through Protein G purification, and are provided in liquid form with specific storage buffers (typically containing preservatives like 0.03% Proclin 300 and 50% Glycerol in PBS at pH 7.4) .

What is the significance of biotin conjugation for TMEM240 antibodies?

Biotin conjugation of antibodies enables detection through avidin or streptavidin coupled to reporter molecules, providing a versatile detection system for immunohistochemistry and other protein detection methods . The biotin-streptavidin interaction is one of the strongest non-covalent biological interactions known, making it highly stable in experiments. For TMEM240 research, biotin-conjugated antibodies can be particularly valuable for detecting this protein in tissues where its expression may be altered in pathological conditions, such as various cancer types . The conjugation method significantly impacts the specificity of staining and experimental outcomes, with methods specifically targeting the Fc portion of antibodies (like ZBPA conjugation) providing superior results compared to non-specific conjugation methods .

How is TMEM240 expression altered in colorectal cancer and other malignancies?

TMEM240 expression demonstrates significant alterations across multiple cancer types, with hypermethylation of its promoter region being a dominant feature. Research data reveals:

The decreased expression of TMEM240 appears to be functionally significant, as experimental evidence demonstrates that it can suppress cancer cell proliferation and migration. In colorectal cancer specifically, hypermethylation of TMEM240 has been detected in 87.8% of tumor tissues compared to matched normal colorectal tissues, suggesting its potential use as a biomarker .

What methodologies can be employed to study TMEM240 in cancer cell lines?

Several methodologies have proven effective in studying TMEM240 in cancer research:

• DNA Methylation Analysis: Quantitative methylation-specific real-time PCR (QMSP) can be used to assess TMEM240 promoter methylation status in tumor tissues compared to normal tissues .

• Expression Studies: Transient transfection to overexpress TMEM240 or siRNA-mediated knockdown can be used to manipulate TMEM240 levels in cancer cell lines. For example, transfection of TMEM240 plasmid into DLD-1 cells demonstrates the protein's effect on cell growth, while si-TMEM240 (s50536 and s50534) can be used for knockdown studies in HCT116 colon cancer cells .

• Cell Proliferation Assays: Sulforhodamine B (SRB) assays can quantify changes in cell proliferation following TMEM240 manipulation .

• Migration Assays: Transwell assays effectively demonstrate TMEM240's influence on cancer cell motility. Research shows that increased TMEM240 expression suppressed DLD-1 cell migration by 39.7% .

• Cell Cycle Analysis: Flow cytometry can determine how TMEM240 affects cell cycle progression. Studies show TMEM240 expression increases the percentage of cells in G1 phase by 4.28% while decreasing the proportion in G2M phase by 4.53% .

What are the optimal methods for biotinylation of TMEM240 antibodies?

Based on comparative research, the Z-domain of protein A (ZBPA) conjugation method demonstrates superior performance compared to commercial kits like Lightning-Link for antibody biotinylation. The ZBPA method specifically targets the Fc portion of antibodies, resulting in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer .

For optimal biotinylation of TMEM240 antibodies:

• Choose the appropriate biotinylation method: ZBPA conjugation is recommended for its specificity in targeting the Fc region, which helps maintain antibody function and reduces non-specific binding .

• Consider antibody concentration and purity: Higher purity antibodies (>95%, as with commercially available TMEM240 antibodies) yield better biotinylation results .

• Optimize biotinylation ratio: The ideal biotin-to-antibody ratio should be determined empirically, but typically ranges from 4:1 to 8:1 for most applications.

• Buffer considerations: The presence of preservatives like 0.03% Proclin 300 in TMEM240 antibody preparations should be accounted for in the biotinylation protocol .

• Validation: Following biotinylation, antibodies should be validated through ELISA or Western blot to confirm retained specificity and functionality.

What are the recommended protocols for using biotin-conjugated TMEM240 antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) using biotin-conjugated TMEM240 antibodies:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin. Sections should be cut at 4-6μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Test both to determine optimal conditions for TMEM240 detection.

• Blocking: Block endogenous biotin using a commercial biotin blocking kit before applying the biotin-conjugated primary antibody to prevent non-specific binding.

• Primary antibody incubation: Dilute biotin-conjugated TMEM240 antibody (typically 1:100 to 1:500) and incubate at 4°C overnight or at room temperature for 1-2 hours. Optimal dilution should be determined empirically.

• Detection: Apply streptavidin-conjugated reporter (HRP, AP, or fluorophore) followed by appropriate substrate or fluorescence visualization.

• Controls: Include positive controls (tissues known to express TMEM240) and negative controls (primary antibody omitted) in each experiment.

• Signal amplification: For weak signals, consider using tyramide signal amplification (TSA) to enhance detection sensitivity while maintaining specificity.

These recommendations are based on general immunohistochemistry principles, as specific protocols for TMEM240 may need optimization based on tissue type and experimental conditions.

How can researchers address non-specific binding when using biotin-conjugated TMEM240 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies. To minimize this issue:

• Optimize blocking: Use protein blocking solutions containing BSA or serum from the same species as the secondary reagent. For tissues with high endogenous biotin (liver, kidney), use specific biotin/avidin blocking kits.

• Consider conjugation method: Research demonstrates that ZBPA biotinylation results in distinct immunoreactivity without off-target staining, whereas commercial kits like Lightning-Link may produce characteristic patterns of nonspecific staining .

• Titrate antibody concentration: Perform a dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

• Modify washing steps: Increase the number and duration of washing steps using buffers containing 0.1-0.3% Tween-20 to remove unbound antibodies.

• Consider alternative detection methods: For tissues with high endogenous biotin, consider using alternative conjugates like FITC or enzyme-labeled antibodies instead of biotin-streptavidin systems .

• Pre-adsorb antibodies: If cross-reactivity is suspected, pre-adsorb the antibody with the immunogen or related proteins to improve specificity.

What are the optimal storage conditions for maintaining the activity of biotin-conjugated TMEM240 antibodies?

To maintain optimal activity of biotin-conjugated TMEM240 antibodies:

• Storage temperature: Store at -20°C or -80°C for long-term stability. The antibody is typically shipped at 4°C but should be transferred to lower temperatures upon receipt .

• Avoid repeated freeze-thaw cycles: Aliquot the antibody upon receipt to minimize freeze-thaw cycles, which can degrade both the antibody and the biotin conjugate .

• Buffer composition: The standard storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 helps maintain stability . Avoid buffers containing sodium azide as it can inhibit HRP activity if using streptavidin-HRP for detection.

• Working dilution stability: Diluted working solutions can be stored at 4°C for up to one week, but preparing fresh dilutions for each experiment is recommended for optimal results.

• Light sensitivity: Protect biotin-conjugated antibodies from prolonged exposure to light, particularly if used in conjunction with fluorescent detection systems.

How can TMEM240 expression analysis be integrated into comprehensive cancer biomarker panels?

Integration of TMEM240 expression analysis into cancer biomarker panels represents an advanced application of this research:

• Multiplex IHC approaches: Biotin-conjugated TMEM240 antibodies can be combined with other differently labeled antibodies targeting additional cancer biomarkers. This requires careful optimization of antibody combinations and detection systems to avoid cross-reactivity.

• Circulating biomarker detection: Research indicates that circulating cell-free methylated TMEM240 was detected in 52.0% (13/25) of Taiwanese colorectal cancer patients compared to 28.6% in healthy controls, suggesting potential for liquid biopsy applications .

• Predictive and prognostic modeling: TMEM240 hypermethylation and expression data can be incorporated into multi-parameter models for cancer prognosis and treatment response prediction.

• Correlation with genomic data: Integrating TMEM240 expression with genomic alterations (mutations, copy number variations) can provide deeper insights into cancer biology and patient stratification.

• Therapeutic target assessment: Evaluating TMEM240 restoration as a potential therapeutic approach requires sophisticated in vitro and in vivo models, potentially using biotin-conjugated antibodies to track expression changes.

What are the methodological considerations for studying TMEM240 in relation to cell cycle regulation?

Advanced methodological approaches for studying TMEM240's role in cell cycle regulation include:

• Synchronization protocols: To study phase-specific effects, researchers should synchronize cells using methods appropriate for the specific phase under investigation (e.g., double thymidine block for G1/S transition, nocodazole for G2/M).

• Real-time cell cycle analysis: Live-cell imaging with fluorescent reporters for cell cycle phases (FUCCI system) combined with TMEM240 expression manipulation can reveal dynamic relationships.

• Cell cycle protein interactions: Co-immunoprecipitation using biotin-conjugated TMEM240 antibodies can identify protein interaction partners involved in cell cycle regulation.

• Phosphorylation analysis: Since cell cycle regulation often involves phosphorylation cascades, researchers should investigate whether TMEM240 undergoes cell cycle-dependent phosphorylation or affects the phosphorylation of key cell cycle regulators.

• Cell cycle checkpoint analysis: Given that TMEM240 induces G1 arrest, detailed analysis of G1 checkpoint proteins (p21, p27, cyclins, CDKs) should be performed following TMEM240 overexpression or knockdown .

• Mathematical modeling: For advanced understanding, experimental data can be incorporated into mathematical models of cell cycle progression to predict the impact of TMEM240 alterations in different cellular contexts.

How does the epigenetic regulation of TMEM240 compare across different cancer types?

Research indicates varied patterns of TMEM240 epigenetic regulation across cancer types that warrant deeper investigation:

Advanced methodological considerations for comparative epigenetic studies include:

• Genome-wide methylation profiling: Beyond targeted analysis, whole-genome bisulfite sequencing or methylation arrays can reveal comprehensive methylation landscapes.

• Integration with chromatin structure data: Combining DNA methylation data with histone modification patterns and chromatin accessibility information provides deeper insights into epigenetic regulation mechanisms.

• Single-cell approaches: Single-cell methylation and expression analysis can reveal heterogeneity within tumors that might be missed in bulk analysis.

• Longitudinal sampling: Analyzing TMEM240 methylation changes during cancer progression, treatment, and potential recurrence can provide insights into dynamic epigenetic regulation.

• Epigenetic editing: Using CRISPR-based epigenetic editing tools to specifically alter TMEM240 methylation can establish causality between methylation status and expression/function .