CTPS1 Antibody

What is CTPS1 and why is it significant in cellular metabolism?

CTPS1 is a 591 amino acid enzyme that catalyzes the ATP-dependent conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP). This reaction represents a rate-limiting step in nucleic acid synthesis, making CTPS1 essential for proper cell growth and development. Located primarily in the cytoplasm, CTPS1 facilitates the availability of CTP, which serves as a critical building block for RNA synthesis and cellular energy metabolism . Its regulation involves complex post-translational modifications, including phosphorylation by protein kinase C, which modulates CTPS1 activity and influences cellular responses to growth signals . The gene encoding CTPS1 is situated on chromosome 1, a region frequently implicated in cancer progression .

How do CTPS1 antibodies differ from other nucleotide metabolism research tools?

CTPS1 antibodies offer unique advantages over other nucleotide metabolism research tools due to their high specificity for a rate-limiting enzyme in the pyrimidine synthesis pathway. Unlike inhibitors that may affect multiple nucleotide synthesis enzymes, CTPS1-specific antibodies enable precise detection and analysis of this particular enzyme. Current commercially available CTPS1 antibodies, such as the mouse monoclonal IgG2b kappa light chain antibody (2G7-1D10), can detect CTPS1 protein across multiple species (mouse, rat, human) and are validated for western blotting, immunoprecipitation, and ELISA applications . This multi-species reactivity facilitates translational research across model organisms, while other nucleotide metabolism tools may be more species-restricted.

What experimental models are most appropriate for CTPS1 antibody research?

Based on current literature, the most appropriate experimental models for CTPS1 antibody research include:

• Cancer cell lines - Particularly those derived from acute myeloid leukemia (AML), where CTPS1 shows preferential essentiality compared to other cancers

• Primary T and B lymphocytes - CTPS1 is strongly upregulated in activated T cells upon T-cell receptor (TCR) stimulation and is critical for lymphocyte proliferation

• Mouse models with tissue-selective CTPS1 inactivation - These provide valuable insights into the in vivo role of CTPS1, particularly in immune responses and germinal center formation

• Human patient samples - Especially from individuals with CTPS1 deficiency, which causes a combined immunodeficiency characterized by susceptibility to viral infections

The selection of an appropriate model should be guided by the specific research question, with consideration of CTPS1's differential expression and functionality across tissues and cell types.

How can CTPS1 antibodies be utilized to investigate immune cell proliferation?

CTPS1 antibodies provide crucial insights into immune cell proliferation mechanisms through several methodological approaches:

• Flow cytometry analysis: Researchers can combine CTPS1 antibody staining with proliferation markers (Ki-67, CFSE dilution) to correlate CTPS1 expression with cell division phases in activated T and B lymphocytes.

• Western blotting time-course studies: By analyzing CTPS1 protein levels at various time points after TCR or BCR stimulation, researchers can establish the temporal relationship between CTPS1 upregulation and proliferation initiation .

• Immunofluorescence microscopy: This technique allows visualization of CTPS1 subcellular localization changes during lymphocyte activation and proliferation, revealing potential interactions with other proteins or organelles .

• Chromatin immunoprecipitation (ChIP) assays: When combined with transcription factor antibodies, these assays can identify regulators controlling CTPS1 expression during immune cell activation.

Studies using these approaches have demonstrated that CTPS1 expression is strongly induced following T cell activation and is essential for proliferation while leaving effector functions like cytotoxicity largely intact . This selective requirement for proliferation makes CTPS1 a potentially valuable target for modulating immune responses.

What is the role of CTPS1 antibodies in cancer research, particularly for hematological malignancies?

CTPS1 antibodies serve multiple critical functions in cancer research, with particular relevance to hematological malignancies:

The particular importance of CTPS1 in hematological malignancies is supported by Depmap dataset analysis showing that CTPS1 activity is preferentially required for AML cell viability compared to other cancer types .

How can CTPS1 antibodies be employed to study nucleotide metabolism dysregulation?

CTPS1 antibodies offer several methodological approaches for studying nucleotide metabolism dysregulation:

• Co-immunoprecipitation studies: These can identify protein complexes containing CTPS1, revealing regulatory interactions that control nucleotide synthesis. Researchers can precipitate CTPS1 using specific antibodies and identify binding partners through mass spectrometry .

• Metabolic flux analysis: By combining CTPS1 antibody-based protein quantification with metabolomic measurements of nucleotide intermediates, researchers can correlate enzyme levels with pathway flux.

• Immunohistochemistry of tissue sections: This reveals the spatial distribution of CTPS1 expression across different cell types in healthy and diseased tissues, indicating where nucleotide metabolism may be particularly active .

• Phospho-specific CTPS1 antibodies: These specialized antibodies can detect post-translational modifications of CTPS1, revealing regulatory mechanisms such as phosphorylation by protein kinase C that modulate enzyme activity in response to cellular signals .

These approaches have revealed that CTPS1's role extends beyond simply catalyzing a biochemical reaction—it serves as a regulatory node integrating cellular proliferation signals with nucleotide availability, explaining why its dysregulation contributes to pathological states ranging from immunodeficiency to cancer.

What protocols optimize CTPS1 antibody performance in immunoblotting applications?

Optimizing CTPS1 antibody performance in immunoblotting requires attention to several critical parameters:

Sample Preparation Protocol:

• Extract total protein using RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if phosphorylated forms of CTPS1 are being studied

• Sonicate lysates briefly (3 × 10s pulses) to reduce sample viscosity

• Heat samples at 95°C for 5 minutes in Laemmli buffer with DTT

Electrophoresis and Transfer Conditions:

• Use 10% SDS-PAGE gels for optimal resolution of the 66.69 kDa CTPS1 protein

• Transfer to PVDF membranes (rather than nitrocellulose) at 100V for 90 minutes in cold transfer buffer containing 10% methanol

• Verify transfer efficiency with reversible Ponceau S staining

Antibody Incubation Parameters:

• Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Dilute primary CTPS1 antibody (e.g., 2G7-1D10 clone) at 1:1000 in 5% BSA/TBST

• Incubate overnight at 4°C with gentle rocking

• Wash 4 × 5 minutes with TBST

• Incubate with appropriate HRP-conjugated secondary antibody at 1:5000 for 1 hour

• Perform enhanced chemiluminescence detection with appropriate exposure times

For detecting low levels of CTPS1, researchers may benefit from signal amplification using biotinylated secondary antibodies and streptavidin-HRP conjugates. When analyzing activated lymphocytes, collecting samples at 24, 48, and 72 hours post-stimulation provides optimal detection of induced CTPS1 expression .

What controls are essential for validating CTPS1 antibody specificity?

Validating CTPS1 antibody specificity requires implementation of multiple complementary controls:

Positive Controls:

• Cell lines with confirmed high CTPS1 expression (e.g., activated T lymphocytes, AML cell lines)

• Recombinant CTPS1 protein as a reference standard

• Tissues known to express CTPS1 (e.g., thymus, germinal centers of lymphoid organs)

Negative Controls:

• CTPS1 knockout cell lines generated using CRISPR-Cas9

• Tissues from CTPS1-deficient mice (Ctps1 ko/ko)

• Cell lines where CTPS1 has been silenced using siRNA or shRNA

Specificity Controls:

• Pre-absorption control: Pre-incubate the antibody with purified CTPS1 protein before application to samples

• Isotype control: Use non-specific antibodies of the same isotype (e.g., mouse IgG2b for 2G7-1D10 clone)

• Cross-reactivity assessment: Test reactivity against recombinant CTPS2 to confirm isoform specificity

Validation Across Applications:

• Compare results across different applications (WB, IP, ELISA) to confirm consistent target recognition

• Verify antibody performance in samples from multiple species if cross-reactivity is claimed (human, mouse, rat)

Implementing these controls systematically ensures reliable interpretation of CTPS1 antibody-based experimental results and prevents publication of artifacts that could mislead the field.

How should researchers interpret contradictory results when using different CTPS1 antibody clones?

When faced with contradictory results using different CTPS1 antibody clones, researchers should implement a systematic troubleshooting approach:

• Epitope mapping analysis:

  • Determine the specific epitopes recognized by each antibody clone

  • Assess whether epitopes might be masked by protein-protein interactions

  • Consider whether post-translational modifications might affect epitope accessibility

  • Examine if different antibodies detect distinct isoforms or splice variants

• Validation using orthogonal techniques:

  • Confirm CTPS1 expression using mRNA quantification (qRT-PCR)

  • Employ mass spectrometry to verify protein identity and abundance

  • Use CRISPR-Cas9 gene editing to generate true negative controls

• Comprehensive comparison table:

  Antibody Parameter	Clone A (e.g., 2G7-1D10)	Clone B	Clone C
  Epitope location	N-terminal/C-terminal	?	?
  Antibody format	Non-conjugated	?	?
  Host species	Mouse	?	?
  Validated applications	WB, IP, ELISA	?	?
  Detects phosphorylated CTPS1	Yes/No	?	?
  Cross-reactivity with CTPS2	Minimal/Significant	?	?

• Biological context consideration:

  • Evaluate whether contradictions arise from different cell types or treatments

  • Consider whether CTPS1 forms different protein complexes across contexts

  • Assess whether contradictions reflect real biological variability rather than technical artifacts

When publishing, researchers should transparently report all antibody validation steps and clearly specify which clone was used for each experiment, facilitating reproducibility across the scientific community.

How does the dual targeting of CTPS1 and immune checkpoint inhibitors show promise in cancer immunotherapy?

The combination of CTPS1 targeting and immune checkpoint inhibitors (ICIs) represents a promising therapeutic strategy based on complementary mechanisms:

Mechanistic Basis for Synergy:

• CTPS1 inhibition selectively impacts rapidly proliferating cells, including cancer cells and certain immune populations

• Analysis of BEAT AML dataset revealed negative correlation between CTPS1 levels and cytotoxic T lymphocyte (CTL) scores, suggesting CTPS1's immunosuppressive role

• ICIs (e.g., anti-PD-1, anti-CTLA-4) remove inhibitory signals that prevent T cell activation

• CTPS1 targeting may enhance the antigen presentation environment while ICIs simultaneously boost T cell effector functions

Experimental Evidence Supporting This Approach:
Recent studies indicate that targeting CTPS1 promotes anti-AML immunity, and when combined with ICIs, may provide a novel therapeutic strategy against AML . The selective effect of CTPS1 inhibition on proliferation while preserving cytotoxic effector functions creates a unique therapeutic window that complements ICI mechanisms.

Methodological Considerations for Investigation:

• Flow cytometric analysis should evaluate both tumor burden reduction and changes in immune cell populations

• Single-cell RNA sequencing can identify specific cell populations affected by combination therapy

• Spatial transcriptomics provides insights into tumor-immune interactions in the microenvironment

• In vivo models should assess tumor regression, survival benefits, and potential development of immunological memory

This dual-targeting approach represents a paradigm shift from either pure cytotoxic therapy or immunotherapy alone, potentially addressing the limitations of ICIs in AML and other hematological malignancies.

What insights have emerged from studying patients with CTPS1 deficiency for therapeutic applications?

Studies of patients with CTPS1 deficiency have yielded several critical insights with therapeutic implications:

Clinical Phenotype and Immunological Features:
Patients with CTPS1 deficiency develop a combined immunodeficiency characterized by:

• High susceptibility to viral infections, particularly Epstein-Barr virus

• Impaired capacity of T lymphocytes to proliferate while maintaining effector functions (cytotoxicity, cytokine production except IL-2)

• Defective B cell proliferation when stimulated through the B-cell receptor

• Absence of other major clinical symptoms, suggesting CTPS1's selective importance in immune cells

Molecular Mechanism:
The identified mutation leads to exon 18 skipping, resulting in an unstable protein with >80% reduction in total CTPS1 expression but preserving 10-20% residual CTP synthetase activity . This selective defect in proliferation upon activation provides a blueprint for therapeutic targeting.

Therapeutic Applications:
These findings have positioned CTPS1 as an attractive therapeutic target for:

• Autoimmune diseases

• T cell lymphoma/leukemia

• Graft versus host disease (GvHD)

• Organ transplantation

A selective CTPS1 inhibitor is currently being evaluated in clinical trials for relapsed and refractory T and B cell lymphoma patients (NCT05463263) , directly translating observations from these rare patients to broader therapeutic applications.

This represents a compelling example of how studying rare genetic disorders can reveal new therapeutic targets with broader applications in common diseases.

What technical considerations are important when using CTPS1 antibodies to study its role in germinal center reactions?

Studying CTPS1's role in germinal center (GC) reactions with antibodies requires specialized methodological approaches:

Sample Preparation for Optimal GC Visualization:

• Use fresh-frozen rather than formalin-fixed tissues to preserve CTPS1 epitopes

• Consider dual immunofluorescence approaches with GC markers (GL7, CD95) for precise localization

• Implement thin sections (4-5μm) to achieve optimal resolution of GC architecture

Experimental Design for GC Studies:
Mouse models have revealed that CTPS1 plays a key role in expansion of activated B cells and T follicular helper (TFH) cells in GCs. Experiments demonstrated significant reductions in CD19+CD95+GL-7+ GC B cells and CXCR5+PD-1+ TFH cells in Ctps1 ko/ko mice . When designing similar studies, researchers should:

• Incorporate appropriate immunization protocols (e.g., NP-CGG adsorbed on alum)

• Plan time-course analyses (e.g., day 7 and day 14 post-immunization)

• Utilize multi-parameter flow cytometry to simultaneously assess proliferation markers

• Measure antigen-specific antibody responses (e.g., NP-specific IgM and IgG1 by ELISA)

Analytical Approaches:

• Distinguish between effects on GC initiation versus maintenance

• Quantify both percentage and absolute numbers of GC B cells and TFH cells

• Assess proliferation (Ki-67) versus survival (Bcl-2, active caspase-3) effects

• Evaluate spatial organization of GC light and dark zones using confocal microscopy

Data Interpretation Challenges:
When using CTPS1 antibodies in conditional knockout models like tamoxifen-inducible systems, researchers must account for potential incomplete deletion. As observed in some studies, residual CTPS1 expression in Ctps1 ko/ko T cells can complicate interpretation of results. Quantitative western blot analysis should accompany functional studies to determine the extent of CTPS1 deletion.

How can researchers address non-specific binding issues with CTPS1 antibodies?

Non-specific binding represents a common challenge when working with CTPS1 antibodies. Researchers can implement several strategies to minimize this issue:

Optimization of Blocking Conditions:

• Test multiple blocking agents (BSA, non-fat milk, normal serum, commercial blockers)

• Extend blocking time to 2 hours at room temperature or overnight at 4°C

• Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions

• Consider dual blocking with combination of protein and detergent-based agents

Antibody Dilution Optimization:

• Perform titration experiments to determine optimal antibody concentration

• For the 2G7-1D10 clone, start with manufacturer-recommended dilutions (typically 1:100 to 1:1000) and adjust based on signal-to-noise ratio

• Dilute antibodies in blocking buffer rather than plain buffer

Sample Processing Improvements:

• Increase washing frequency and duration (minimum 5 × 5 minutes with agitation)

• Pre-absorb antibodies with proteins from the species being studied

• For tissue sections, treat with hydrogen peroxide to block endogenous peroxidases

Validated Alternatives:
If non-specific binding persists despite optimization, consider:

• Testing alternative CTPS1 antibody clones that recognize different epitopes

• Using highly purified biotechnology-grade antibodies that undergo additional purification steps

• Implementing more specific detection methods like proximity ligation assay (PLA)

Systematic documentation of optimization steps in laboratory notebooks facilitates reproducibility and prevents repeated troubleshooting of the same issues.

What are the critical factors affecting CTPS1 antibody performance in immunohistochemistry applications?

Several critical factors significantly impact CTPS1 antibody performance in immunohistochemistry (IHC):

Tissue Fixation and Processing:

• Overfixation with formalin can mask CTPS1 epitopes; limit fixation to 24 hours

• Consider alternative fixatives (zinc-based) that better preserve protein epitopes

• Implement antigen retrieval methods optimized for CTPS1:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Enzymatic retrieval with proteinase K for certain tissue types

Antibody Selection and Validation:

• Verify IHC validation of the specific CTPS1 antibody clone being used

• Consider antibodies specifically validated for IHC-P applications

• Test multiple antibody dilutions on control tissues with known CTPS1 expression

Detection System Considerations:

• For low CTPS1 expression, employ signal amplification methods:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems rather than traditional ABC method

• Balance sensitivity with potential background increases

Tissue-Specific Factors:
CTPS1 expression varies significantly across tissues, with particularly high expression in:

• Lymphoid tissues (especially germinal centers)

• Rapidly proliferating epithelial cells

• Certain tumor types, especially hematological malignancies

When examining these tissues, adjust antibody dilutions accordingly and include appropriate positive controls. For tissues with endogenous biotin (liver, kidney), use biotin-free detection systems to avoid false positives.

Quantification Approaches:
For accurate assessment of CTPS1 expression in IHC:

• Implement digital image analysis rather than subjective scoring

• Establish clear scoring criteria based on staining intensity and percentage of positive cells

• Consider multiplexed IHC to correlate CTPS1 with proliferation markers

How should researchers design experiments to differentiate between CTPS1 and CTPS2 functions using antibodies?

Differentiating between CTPS1 and CTPS2 functions requires careful experimental design with highly specific antibodies:

Antibody Selection and Validation:

• Obtain antibodies with validated specificity for either CTPS1 or CTPS2

• Confirm isoform specificity by Western blotting in:

  • Cells overexpressing each isoform individually

  • CTPS1 or CTPS2 knockout cells as negative controls

• Sequence-based epitope analysis to ensure targeting of divergent regions

Comparative Expression Analysis:

• Perform parallel immunoblotting for both isoforms across tissues/cell types

• Use RT-qPCR to correlate protein with mRNA expression patterns

• Consider single-cell approaches to assess cell-type specific expression patterns

Functional Studies Design:

• Knockdown/Knockout Approaches:

  • Use isoform-specific siRNAs with validation of selective targeting

  • Implement CRISPR-Cas9 knockout of each isoform individually

  • Create dual knockout systems with rescue experiments using each isoform

• Differential Activity Assessment:

  Parameter	CTPS1	CTPS2	Experimental Approach
  Cell type specificity	Immune cells	Broader expression	Multi-tissue Western blot
  Inducibility	Highly inducible upon activation	Constitutive/less inducible	Time-course after stimulation
  Subcellular localization	Cytoplasmic	Cytoplasmic/Other	Subcellular fractionation
  Impact on proliferation	Critical for lymphocyte proliferation	Variable by cell type	BrdU incorporation assay

• Rescue Experiments:

  • Express CTPS1 in CTPS1-deficient cells to confirm specificity of phenotype

  • Test whether CTPS2 overexpression can compensate for CTPS1 deficiency

  • Create chimeric proteins to map functional domains

Mouse models have revealed distinct but overlapping functions, with CTPS1 being particularly critical for lymphocyte proliferation. Studies of CTPS1-deficient patients also highlight the non-redundant role of CTPS1 in immune function, despite the presence of CTPS2 . These observations underscore the importance of isoform-specific antibodies in delineating their respective functions.