RASA2 Antibody, FITC conjugated

Basic Research Questions

• What is RASA2 and why is it important in T cell research?

RASA2 (RAS p21 protein activator 2) is a RAS GTPase-activating protein (RasGAP) that functions as an inhibitory regulator of the RAS-cyclic AMP pathway. It has emerged as a significant signaling checkpoint in human T cells that modulates T cell activation and function .

RASA2 works by accelerating the hydrolysis of active RAS-GTP to RAS-GDP, effectively suppressing RAS signal output. Research has shown that RASA2 is selectively expressed in CD8+ and CD4+ human T cells compared to other RasGAP family members . Studies have demonstrated that RASA2 is downregulated upon acute T cell receptor stimulation but can increase gradually with chronic antigen exposure, suggesting its role as a checkpoint to restrain T cell responses in settings of chronic stimulation .

The importance of RASA2 in T cell research has increased since studies revealed that RASA2 ablation enhances sensitivity to antigen and improves both effector function and persistence of CAR T and TCR T cells, presenting a potential target for improving immunotherapy efficacy .

• What experimental applications is RASA2 Antibody, FITC conjugated suitable for?

RASA2 Antibody, FITC conjugated is suitable for several experimental applications in immunology and cancer research:

The FITC conjugation (fluorescein isothiocyanate) enables direct visualization with excitation at approximately 490nm and emission at 525nm, making it particularly valuable for flow cytometry and fluorescence microscopy applications without requiring secondary antibody detection steps .

• How should researchers validate the specificity of RASA2 Antibody, FITC conjugated?

Validating antibody specificity is crucial for reliable research results. For RASA2 Antibody, FITC conjugated, researchers should employ multiple validation approaches:

• Knockout/Knockdown Controls: Compare staining in wild-type cells versus CRISPR-Cas9 RASA2 knockout cells . This provides the strongest validation of specificity.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before staining to confirm binding specificity.

• Western Blot Correlation: When possible, correlate flow cytometry or IHC results with western blot analysis using the same antibody (unconjugated version) to confirm the correct molecular weight (approximately 97 kDa for RASA2) .

• Positive and Negative Control Tissues: Use tissues known to express high levels of RASA2 (T cells, endometrium) versus tissues with minimal expression .

• Comparative Analysis: Compare staining patterns with alternative RASA2 antibodies from different sources or clones.

Researchers should document that the staining pattern correlates with the known subcellular localization of RASA2 (cytoplasmic and perinuclear regions) .

• What are the optimal storage and handling conditions for RASA2 Antibody, FITC conjugated?

Proper storage and handling of FITC-conjugated antibodies is essential to maintain fluorescence activity and binding specificity:

When working with the antibody, minimize exposure to bright light, especially UV sources, and handle on ice when possible to preserve both protein integrity and FITC fluorescence.

• What controls should be included when using RASA2 Antibody, FITC conjugated in flow cytometry?

Robust controls are essential for accurate interpretation of flow cytometry data using RASA2 Antibody, FITC conjugated:

Essential Controls:

• Unstained Cells: To establish baseline autofluorescence

• Isotype Control, FITC-Conjugated: Rabbit IgG-FITC control antibody to assess non-specific binding

• Single-Color Controls: If performing multicolor flow cytometry, for compensation setup

• Fluorescence Minus One (FMO) Control: All antibodies except RASA2-FITC to establish gating boundaries

Biological Controls:

• Positive Control: Cell type known to express RASA2 (e.g., primary T cells)

• Negative Control: Cell line with minimal RASA2 expression or RASA2 knockout cells

• Stimulation Control: Compare unstimulated vs. TCR-stimulated T cells, as RASA2 expression changes with stimulation

Technical Validation:

• Titration Series: Test different antibody concentrations to determine optimal signal-to-noise ratio

• Blocking Control: Pre-incubation with the immunizing peptide to confirm specificity

For quantitative analysis, consider including calibration beads with known quantities of FITC molecules to standardize fluorescence intensity measurements across experiments.

Advanced Research Questions

• How can researchers use RASA2 Antibody, FITC conjugated to study the relationship between RASA2 expression and T cell dysfunction?

To investigate the relationship between RASA2 expression and T cell dysfunction, researchers can employ several advanced methodological approaches using RASA2 Antibody, FITC conjugated:

Experimental Design for Chronic Stimulation Studies:

• Sequential Time-Point Analysis: Culture primary T cells with repeated antigen stimulation over multiple time points (e.g., 1, 3, 5, 7 days)

• Flow Cytometry Panel: Combine RASA2-FITC antibody with markers of:

  • T cell exhaustion (PD-1, LAG-3, TIM-3, TIGIT)

  • Activation status (CD69, CD25, CD154)

  • Effector function (intracellular IFN-γ, TNF-α, perforin)

Quantitative Analysis Methodology:

• Use mean fluorescence intensity (MFI) to quantify RASA2 expression levels

• Perform correlation analysis between RASA2 expression and exhaustion marker levels

• Compare RASA2 expression between responding vs. non-responding T cells

Research indicates that RASA2 expression increases with chronic antigen exposure and correlates with diminished T cell function . This approach allows researchers to track temporal changes in RASA2 expression during the development of T cell dysfunction and establish potential causal relationships.

For more advanced studies, researchers can compare RASA2 expression in T cells from cancer patients versus healthy donors, correlating expression with clinical outcomes and treatment response.

• What methodological approaches can be used to study how RASA2 affects RAS-MAPK signaling in T cells?

To investigate how RASA2 influences RAS-MAPK signaling in T cells, researchers can employ several sophisticated methodological approaches using RASA2 Antibody, FITC conjugated:

Multi-parameter Phospho-Flow Cytometry Protocol:

• Stimulation Time Course: Stimulate T cells with anti-CD3/CD28 for various time points (0, 2, 5, 10, 30 min)

• Fixation and Permeabilization: Use paraformaldehyde fixation followed by methanol permeabilization to preserve phospho-epitopes

• Antibody Panel: Combine RASA2-FITC with antibodies against:

  • Phospho-ERK (pT202/pY204)

  • Phospho-MEK (pS217/pS221)

  • Phospho-S6 (pS235/pS236)

  • RAS-GTP (using GST-RBD fusion proteins)

• Comparative Analysis: Analyze cells with high versus low RASA2 expression or compare wild-type versus RASA2-knockout cells

This approach enables direct correlation between RASA2 levels and downstream MAPK pathway activation at the single-cell level .

Advanced Signaling Analysis:

• Dose-Response Studies: Analyze signaling across a range of TCR stimulation strengths to assess how RASA2 affects antigen sensitivity thresholds

• Temporal Resolution: Compare early (1-10 min) versus sustained (30-120 min) signaling phases

• Cross-Pathway Integration: Include markers for PI3K-AKT pathway to understand pathway crosstalk

Research has shown that RASA2-deficient T cells exhibit higher levels of RAS-GTP, phospho-ERK, and phospho-S6 following TCR stimulation, with enhanced sensitivity to low levels of antigen stimulation .

• How can researchers use RASA2 Antibody, FITC conjugated to evaluate the effect of RASA2 ablation on CAR T cell persistence and function?

To evaluate how RASA2 ablation affects CAR T cell persistence and function, researchers can implement a comprehensive experimental approach using RASA2 Antibody, FITC conjugated:

Ex Vivo Analysis Protocol:

• CAR T Cell Engineering: Generate CD19-specific CAR T cells with CRISPR/Cas9-mediated RASA2 knockout versus control

• Verification: Use RASA2-FITC antibody to confirm knockout efficiency by flow cytometry

• In Vitro Functional Assessment:

  • Cytotoxicity Assay: Co-culture with tumor cells expressing varied levels of target antigen (e.g., CD19-high and CD19-low)

  • Serial Killing Assay: Assess multiple rounds of tumor cell killing capacity

  • Metabolic Function: Measure mitochondrial fitness using Seahorse analyzer

In Vivo Experimental Design:

• Adoptive Transfer: Inject RASA2-knockout versus control CAR T cells into tumor-bearing mice

• Sequential Monitoring:

  • Tumor burden measurement

  • Peripheral blood sampling at multiple timepoints

• Ex Vivo Analysis of Harvested Cells:

  • RASA2-FITC staining to verify maintained knockout

  • Phenotypic analysis (exhaustion markers, memory markers)

  • Re-challenge with tumor cells to assess preserved function

Research has demonstrated that RASA2-knockout CAR T cells maintain their cancer cell-killing capacity following repeated antigen exposure, whereas control CAR T cells gradually lose this ability . This approach allows researchers to comprehensively evaluate how RASA2 ablation impacts CAR T cell longevity and functional persistence in both in vitro and in vivo settings.

• What considerations are important when using RASA2 Antibody, FITC conjugated in multicolor flow cytometry panels?

When incorporating RASA2 Antibody, FITC conjugated into multicolor flow cytometry panels, researchers should address several critical technical considerations:

Spectral Overlap Considerations:

• FITC emission spectrum (peak ~525nm) overlaps significantly with PE (phycoerythrin) and potentially with other green fluorophores

• When designing panels, place markers of lower abundance on brighter fluorophores than FITC

• Perform proper compensation using single-stained controls

Panel Design Strategy:

Fixation and Permeabilization Protocol:

• Fix cells using 2-4% paraformaldehyde (10 min, room temperature)

• Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer

• Perform surface staining before fixation

• Add RASA2-FITC antibody during the intracellular staining step

RASA2 is primarily cytoplasmic and perinuclear , requiring effective permeabilization for optimal staining. When analyzing T cell activation, researchers should be aware that RASA2 expression decreases after acute TCR stimulation but increases with chronic stimulation , which may affect staining intensity depending on the experimental timepoint.

• What methodological approaches can be used to study RASA2 in patient-derived T cells for clinical research applications?

For clinical research applications using patient-derived T cells, several methodological approaches can be implemented with RASA2 Antibody, FITC conjugated:

Clinical Sample Processing Protocol:

• Isolation: Purify peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation

• T Cell Enrichment: Use negative selection for untouched T cells or specific subsets (CD8+, CD4+)

• Cryopreservation Considerations: Use controlled-rate freezing to maintain viability and protein expression

• Thawing Protocol: Rapid thawing with dropwise addition of warm media containing DNase to prevent clumping

Analytical Framework for Patient Samples:

Advanced Applications:

• Ex Vivo Drug Screening: Test how pharmacological modulators affect RASA2 expression and T cell function

• Genetic Modification Assessment: Evaluate the feasibility of RASA2 targeting in patient T cells for adoptive cell therapy

• Biomarker Development: Assess whether RASA2 expression correlates with clinical response to immunotherapy

Research has shown that RASA2 expression patterns may be relevant for predicting T cell dysfunction and potential response to immunotherapy, making this a valuable target for clinical research applications .

• How can researchers quantitatively measure the effect of RASA2 expression on antigen sensitivity thresholds in T cells?

To quantitatively measure how RASA2 expression influences antigen sensitivity thresholds in T cells, researchers can implement the following methodological approach using RASA2 Antibody, FITC conjugated:

Dose-Response Experimental Protocol:

• T Cell Preparation: Isolate primary T cells or use TCR-transduced T cells with known antigen specificity

• RASA2 Expression Analysis: Stain with RASA2-FITC antibody and sort into RASA2-high and RASA2-low populations (or compare wild-type vs. RASA2-knockout cells)

• Antigen Titration Series: Expose T cells to target cells loaded with decreasing concentrations of cognate peptide (e.g., 10-fold dilutions from 10μM to 0.1nM)

• Activation Readouts:

  • Early: Phospho-ERK by flow cytometry (5-15 minutes)

  • Intermediate: CD69 expression (6-16 hours)

  • Late: Proliferation by CFSE dilution (72-96 hours)

Data Analysis Framework:

Research has demonstrated that RASA2-knockout T cells show increased phospho-ERK signaling and proliferation across a range of anti-CD3/CD28 concentrations compared to control T cells . Similarly, NY-ESO-1 antigen-specific T cells lacking RASA2 exhibited higher levels of phospho-ERK across a range of peptide concentrations, effectively increasing sensitivity to antigen .

This methodological approach allows precise quantification of how RASA2 modulates the minimum antigen density required for T cell activation, which has implications for targeting low-antigen-expressing tumor cells.

• What methods can be used to investigate the relationship between RASA2 expression and metabolic reprogramming in T cells?

To investigate the relationship between RASA2 expression and metabolic reprogramming in T cells, researchers can employ a comprehensive suite of methods incorporating RASA2 Antibody, FITC conjugated:

Integrated Metabolic Analysis Protocol:

• T Cell Classification: Use flow cytometry with RASA2-FITC antibody to sort cells based on RASA2 expression levels

• Seahorse XF Analysis: Measure:

  • Oxygen consumption rate (OCR) - indicator of mitochondrial respiration

  • Extracellular acidification rate (ECAR) - indicator of glycolysis

  • Spare respiratory capacity - indicator of metabolic fitness

• Metabolite Profiling: Conduct targeted metabolomics on sorted populations for:

  • Glycolytic intermediates

  • TCA cycle metabolites

  • Amino acids and fatty acids

Flow Cytometry-Based Metabolic Assays:

Research has shown that RASA2-knockout T cells demonstrate transcriptional reprogramming toward metabolic states favoring oxidative phosphorylation after chronic antigen exposure, suggesting that RASA2 ablation may prevent T cell dysfunction partly through altering metabolic profiles . This is particularly relevant for maintaining T cell function during repeated tumor antigen exposures, where metabolic exhaustion often limits efficacy.

These methodological approaches enable researchers to establish mechanistic links between RASA2's role in RAS signaling and downstream metabolic pathways that influence T cell persistence and function.

• What techniques can researchers use to study the effect of RASA2 on memory T cell formation and long-term persistence?

To investigate how RASA2 influences memory T cell formation and long-term persistence, researchers can employ several sophisticated techniques using RASA2 Antibody, FITC conjugated:

Longitudinal Memory Development Protocol:

• Initial Activation: Stimulate T cells with antigen or anti-CD3/CD28, tracking RASA2 expression via RASA2-FITC antibody

• Rest Phase: Culture cells in low-dose IL-7/IL-15 to promote memory formation

• Re-challenge: Perform secondary stimulation to assess recall response

• Phenotypic Analysis: Design a comprehensive flow cytometry panel including:

In Vivo Memory Study Design:

• Adoptive Transfer: Introduce RASA2-knockout versus control antigen-specific T cells into mice

• Primary Response: Measure expansion, contraction, and phenotype

• Memory Phase: Analyze persistence and RASA2 expression in surviving cells at 30, 60, and 90 days

• Re-challenge: Assess secondary expansion capacity and functional response

Research indicates that RASA2-deficient T cells exhibit features of enhanced effector-memory differentiation compared to control cells, and maintain functional capacity after multiple stimulations . This suggests RASA2 may regulate the balance between terminal effector differentiation and memory formation.

These approaches allow researchers to determine whether RASA2 expression correlates with or causally influences memory T cell development, which has significant implications for developing more persistent and effective T cell therapies.

• How can RASA2 Antibody, FITC conjugated be used in imaging studies to investigate RASA2 localization during T cell activation?

For investigating RASA2 subcellular localization during T cell activation, researchers can employ several advanced imaging techniques using RASA2 Antibody, FITC conjugated:

Confocal Microscopy Experimental Protocol:

• T Cell Activation System:

  • Option A: T cells on anti-CD3/CD28-coated coverslips

  • Option B: T cell:APC conjugates with cognate antigen

• Time Course Analysis: Fix cells at multiple timepoints (0, 5, 15, 30, 60 min) post-stimulation

• Immunofluorescence Staining:

  • RASA2-FITC antibody (green channel)

  • Membrane marker (CD45-APC or WGA-Alexa647)

  • Nuclear stain (DAPI)

  • RAS or downstream effector proteins (e.g., phospho-ERK)

Advanced Imaging Methods:

Image Analysis Approach:

• Quantify RASA2 redistribution using intensity profiles across cellular compartments

• Measure colocalization with signaling components using Pearson's correlation coefficient

• Analyze temporal dynamics of RASA2 localization relative to TCR signaling events

RASA2 has been characterized as primarily cytoplasmic and perinuclear . Research suggests that RASA2's RasGAP activity may depend on recruitment to the plasma membrane via its PH domain, which binds phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated during T cell activation . This membrane recruitment mechanism could be visually confirmed using these imaging approaches.

• What experimental approaches can be used to study RASA2's role in cancer therapy resistance mechanisms?

To investigate RASA2's potential role in cancer therapy resistance mechanisms, researchers can implement several sophisticated experimental approaches using RASA2 Antibody, FITC conjugated:

Clinical Sample Analysis Protocol:

• Patient Cohort Selection: Obtain samples from responders vs. non-responders to immunotherapy

• Flow Cytometry Panel Design:

  • RASA2-FITC antibody for expression level quantification

  • T cell exhaustion markers (PD-1, TIM-3, LAG-3)

  • Activation markers (CD25, CD69, HLA-DR)

  • Cytotoxic molecules (Granzyme B, Perforin)

• Comparative Analysis: Assess correlation between RASA2 expression levels in tumor-infiltrating lymphocytes and therapy response

Mechanistic Investigation Models:

Therapeutic Modulation Strategy Testing:

• Pharmacological Approach: Test compounds that may indirectly modulate RASA2 function

• Genetic Engineering: Compare different RASA2 targeting strategies (knockout, knockdown, domain mutations)

• Combinatorial Therapy: Test RASA2 modification combined with checkpoint inhibitors

Research suggests that RASA2 deficiency renders T cells more resistant to dysfunction during chronic antigen exposure, which is relevant to the tumor microenvironment context . Additionally, RASA2-knockout CAR T cells showed enhanced killing of cancer cells with low target antigen expression, addressing a major mechanism of therapy resistance .

These approaches enable researchers to comprehensively evaluate whether RASA2 targeting could overcome resistance mechanisms in current cancer immunotherapies.