TYMS Antibody

What is TYMS and what is its biological significance?

TYMS (Thymidylate Synthase) is a critical enzyme that catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to thymidine 5'-monophosphate (dTMP), using 5,10-methylenetetrahydrofolate (CH2H4folate) as a 1-carbon donor and reductant . This conversion is essential for DNA synthesis as it represents the only de novo pathway for thymidine production . TYMS plays a crucial role in regulating the supply of all four DNA precursors necessary for DNA replication and repair . It is also the only enzyme in folate metabolism that can oxidize 5,10-methylenetetrahydrofolate during one-carbon transfer, making it essential for folate metabolism regulation . In-vitro studies have demonstrated that upregulation of TYMS is sufficient to transform immortalized mammalian cells to a malignant phenotype, highlighting its potential role in carcinogenesis .

What applications are TYMS antibodies typically used for in research?

TYMS antibodies are versatile research tools applicable across multiple experimental methods including:

• Western blot analysis (recommended dilution range 1:500-1:1000)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Immunofluorescence analysis (recommended starting dilution 1:500)

• Flow cytometry for analyzing cellular expression patterns

• Protein array analysis for high-throughput screening

TYMS antibodies are particularly valuable in cancer research for evaluating expression levels in tumor tissues, correlating expression with clinical outcomes, and investigating TYMS-targeting drug efficacy . The antibodies enable researchers to study both mRNA and protein-level expression changes in response to experimental treatments .

What are the recommended positive and negative controls for TYMS antibody validation?

For proper validation of TYMS antibodies in immunohistochemical applications:

Positive control: Lymph node tissue should show strong TYMS staining in a fraction of lymphocytic cells, particularly in germinal centers . Additional suitable positive controls include thymus, bone marrow, and tonsil tissues, which naturally express higher levels of TYMS .

Negative control: Prostate tissue serves as an effective negative control, as TYMS staining should be absent in normal epithelial cells . Most normal tissues exhibit TYMS expression levels too low for detection by standard immunohistochemistry, making them suitable negative controls as well .

When validating by Western blot, recombinant human TYMS protein can serve as a positive control, with expected molecular weight of approximately 32-35kD .

How should TYMS antibody staining be quantified in tissue samples?

Semi-quantitative classification of TYMS immunohistochemical staining should follow a standardized scoring system based on:

• Percentage of positive cells (PP) scored as:

  • 0: negative

  • 1: <25% positive cells

  • 2: 25-75% positive cells

  • 3: >75% positive cells

• Staining intensity (SI) scored as:

  • 0: negative

  • 1: weak

  • 2: moderate

  • 3: strong

An immunoreactivity score (IRS) is calculated by multiplying PP by SI. Samples with IRS=0 are considered "negative," while those with IRS>0 are considered "positive" . This standardized approach enables consistent evaluation across different studies and laboratories. Assessment should ideally be performed by two independent pathologists blinded to clinical information to minimize bias .

How does TYMS expression correlate with cancer prognosis and treatment response?

TYMS expression has significant implications for both cancer prognosis and treatment efficacy:

What mechanisms regulate TYMS expression in cancer cells and how can they be experimentally manipulated?

TYMS expression in cancer cells is regulated through multiple mechanisms that can be experimentally modified:

• Small molecule inhibitors: Sodium butyrate (NaB) has been shown to inhibit both mRNA and protein expression of TYMS in colorectal cancer cells (HCT116 and LoVo) . In experimental settings, NaB (2 mM) clearly reduced TYMS mRNA and protein levels, providing a potential approach for modulating TYMS expression .

• Genetic knockdown approaches: Lentivirus-mediated short hairpin RNA (shRNA) targeting TYMS has been successfully used to downregulate TYMS expression . This approach allows researchers to study the functional consequences of TYMS reduction, including effects on cell proliferation, apoptosis, cell cycle progression, and cell migration/invasion .

• 5-FU treatment effects: Treatment with 5-FU dramatically increases TYMS expression in colorectal cancer cells, particularly the "bound" form of TYMS (visible as an upper band in Western blots) . Combined treatment with NaB significantly alleviates this 5-FU-induced increase in TYMS, suggesting potential therapeutic combinations for overcoming treatment resistance .

Protein microarray analysis followed by Western blot validation provides an effective approach for examining the downstream mechanisms affected by TYMS modulation .

How do different TYMS antibody clones compare in specificity and sensitivity across applications?

When selecting a TYMS antibody for research applications, researchers should consider these clone-specific characteristics:

Monoclonal antibody clone PAT1S5AT:

• Derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human TYMS amino acids 1-313

• Isotype: Mouse IgG2b heavy chain and κ light chain

• Purification method: Protein-G affinity chromatography from mouse ascitic fluids

• Recommended dilution: 1:500-1:1000 for Western blot and immunofluorescence

Recombinant Rabbit monoclonal clone HMV305:

• Optimized for immunohistochemistry applications

• Recommended dilution: 1:100-1:200 for IHC applications

• Cellular localization detection: Intracellular

Mouse Monoclonal clone TYMS/1884:

• Immunogen corresponding to recombinant fragment protein within Human TYMS aa 50-200

• Successfully validated for multiple applications including ICC, Flow Cytometry, Western blot, Protein Array, and IHC-P

• Demonstrates specific staining in flow cytometry analysis of PFA-fixed leukemia and lymphoma cell lines

Antibody validation should include positive and negative controls as discussed previously, and selection should be based on the intended application and required sensitivity.

What experimental considerations are important when studying TYMS in 5-FU resistant cancer models?

When investigating TYMS in 5-FU resistant cancer models, researchers should consider several critical experimental factors:

• Baseline TYMS expression patterns: 5-FU resistant cells (e.g., HCT116-5-FUR) exhibit different baseline TYMS expression patterns compared to their parental cell lines, with higher levels of bound TYMS (visualized as the upper band in Western blots) even without 5-FU treatment . These baseline differences should be carefully documented.

• Differential response monitoring: Unlike parental cells, 5-FU resistant cells may show distinct responses to TYMS-modulating compounds. For instance, in HCT116-5-FUR cells, NaB treatment alone may not significantly inhibit baseline TYMS expression compared to control, but can still inhibit 5-FU-induced TYMS upregulation .

• Apoptosis markers: Include analysis of apoptosis markers such as cleaved PARP, which may show different patterns in 5-FU resistant cells compared to parental lines following TYMS-modulating treatments .

• Storage and treatment conditions: When using compounds like NaB, observe proper storage procedures (store at 4°C for periods up to 1 month, or at -20°C for longer periods) and prevent freeze-thaw cycles to maintain reagent efficacy .

• Combination treatments: Experimental designs should include both single-agent and combination treatments (e.g., 5-FU alone, TYMS modulator alone, and combination) to fully characterize the interactions between 5-FU and TYMS-targeting approaches .

How does TYMS relate to other enzymes in the folate metabolism and pyrimidine synthesis pathways?

TYMS functions within interconnected metabolic networks that can be experimentally investigated:

TYMS interacts with multiple enzymes involved in 5-FU metabolism and folate-mediated one-carbon transfer:

• Methylenetetrahydrofolate reductase (MTHFR): This enzyme's activity affects the availability of 5,10-methylenetetrahydrofolate, which TYMS uses as a cofactor. Research shows that NaB treatment increases MTHFR mRNA levels in colorectal cancer cells, potentially affecting the metabolite pool available for TYMS activity .

• Dihydropyrimidine dehydrogenase (DPYD): DPYD is involved in the intracellular metabolism of 5-FU. Studies indicate that NaB treatment increases DPYD mRNA levels in HCT116 and LoVo cells, suggesting coordinated regulation with TYMS .

• Folate metabolic pathway: TYMS holds a unique position as the only enzyme in folate metabolism that can oxidize 5,10-methylenetetrahydrofolate during one-carbon transfer, making it a critical regulator of folate metabolism .

Experimental designs examining TYMS should consider measuring these related enzymes to provide a comprehensive understanding of pathway interactions. RT-qPCR can effectively quantify mRNA expression levels of these multiple metabolic enzymes simultaneously, while protein interaction studies may require co-immunoprecipitation or proximity ligation assays to detect physical associations between pathway components .

What are the optimal storage and handling conditions for TYMS antibodies?

To maintain TYMS antibody integrity and performance, researchers should adhere to these storage and handling guidelines:

• Short-term storage (up to 1 month): Store at 4°C

• Long-term storage: Store at -20°C

• Critical precaution: Prevent freeze-thaw cycles that can degrade antibody quality

• Shelf life: Typically 12 months at -20°C and 1 month at 4°C when properly stored

• Formulation considerations: Many commercial TYMS antibodies are formulated at 1mg/ml in PBS (pH 7.4) with 0.1% Sodium Azide as preservative

For optimal experimental results, always bring antibodies to room temperature before use and centrifuge briefly to collect contents at the bottom of the tube. Working aliquots can be prepared to minimize freeze-thaw cycles of the main stock .

How should researchers troubleshoot non-specific binding or weak signals when using TYMS antibodies?

When encountering issues with TYMS antibody performance, implement these troubleshooting approaches:

For non-specific binding:

• Optimize antibody dilution: Test a range around the recommended dilution (e.g., 1:250, 1:500, 1:1000, 1:2000) to identify the optimal concentration that maximizes specific signal while minimizing background .

• Blocking optimization: Increase blocking time or try alternative blocking agents (BSA, normal serum, commercial blockers) to reduce non-specific binding.

• Wash protocol adjustment: Increase washing duration or add additional wash steps using appropriate buffers.

• Validate with additional controls: Include isotype controls and examine staining in tissues known to be negative for TYMS .

For weak signals:

• Sample preparation assessment: Ensure proper fixation and antigen retrieval protocols for IHC applications.

• Signal amplification: Consider using polymer-based detection systems or tyramide signal amplification.

• Antibody quality check: Confirm antibody viability by testing on known positive controls like lymph node tissue for IHC or appropriate cell lysates for Western blot .

• Protocol modification: For Western blot, adjust transfer conditions, primary antibody incubation time (overnight at 4°C may improve signal), or detection method sensitivity.

Document all optimization steps methodically to establish reproducible protocols for your specific experimental systems.

What are the recommended protocols for TYMS knockdown or inhibition experiments?

For researchers conducting TYMS inhibition studies, these validated approaches provide effective experimental frameworks:

Genetic knockdown approaches:

• shRNA-mediated knockdown: Lentivirus-mediated short hairpin RNA targeting TYMS has been successfully implemented to investigate TYMS function . This approach allows for stable knockdown and evaluation of long-term effects on cell behaviors including proliferation, apoptosis, cell cycle progression, and migration/invasion .

• siRNA transient knockdown: For short-term experiments, synthetic siRNA transfection provides a more rapid approach to assess acute effects of TYMS reduction.

Chemical inhibition approaches:

• Sodium Butyrate (NaB) treatment: NaB at 2mM concentration effectively reduces TYMS expression in colorectal cancer cells . The recommended treatment concentration range for in vitro models is 0-10mM .

• 5-FU and combination approaches: Treatment with 5-FU followed by evaluation of TYMS binding (visible as upper band increases in Western blots) provides a model for studying TYMS inhibition mechanisms . Combination of NaB with 5-FU shows enhanced effectiveness in reducing cell viability compared to either treatment alone .

Experimental validation:
Following knockdown or inhibition, comprehensive validation should include:

• RT-qPCR to confirm mRNA reduction

• Western blot to verify protein level changes (both free and bound forms of TYMS)

• Functional assays to assess biological consequences (proliferation, apoptosis, cell cycle)

How can TYMS expression be accurately quantified at both mRNA and protein levels?

For comprehensive TYMS expression analysis, researchers should employ these complementary quantification methods:

mRNA level quantification:

• RT-qPCR: The gold standard for mRNA quantification, providing sensitive detection of TYMS transcript levels . Normalization to appropriate housekeeping genes is essential for reliable comparison between samples.

• RNA-Seq: For broader analysis of TYMS in the context of global gene expression changes, RNA-sequencing provides comprehensive insights into transcriptional networks.

Protein level quantification:

• Western blot analysis: Enables distinction between different forms of TYMS protein, particularly the "free" (lower band) and "bound" (upper band) forms that have functional significance in drug response studies . Recommended antibody dilution typically ranges from 1:500 to 1:1000 .

• Immunohistochemistry scoring: Semi-quantitative assessment using the percentage of positive cells (PP) and staining intensity (SI) to calculate an immunoreactivity score (IRS) . This approach is particularly valuable for tissue-based studies and clinical correlations.

• Flow cytometry: Enables quantification of TYMS protein at the single-cell level, allowing for analysis of expression heterogeneity within populations .

For comprehensive studies, combining both mRNA and protein analyses provides the most complete picture of TYMS expression regulation, as post-transcriptional mechanisms may result in discrepancies between transcript and protein levels.