AITRL Human, His

What is AITRL Human, His and how is it structurally characterized?

AITRL Human, His is a recombinant form of human AITRL (Activation-Inducible TNF-Related Ligand) protein engineered with a histidine tag to facilitate purification and detection. The protein has a molecular weight of approximately 15.7 kDa and contains 125 amino acid residues when produced in E. coli expression systems .

As a member of the TNF superfamily, AITRL adopts a characteristic trimeric structure essential for receptor binding and downstream signaling. The His-tag modification, while not naturally occurring, provides minimal interference with the protein's native conformation when properly positioned at either the N- or C-terminus. Researchers should note that proper disulfide bond formation is critical for maintaining the protein's tertiary structure and biological activity.

For structural analysis, techniques such as circular dichroism, size-exclusion chromatography, and dynamic light scattering can verify proper folding and oligomerization states. When comparing with native AITRL, researchers should consider potential differences in glycosylation patterns, as E. coli-produced proteins lack post-translational modifications found in mammalian expression systems.

How does the His-tag affect AITRL protein functionality in experimental systems?

The histidine tag on AITRL Human, His serves primarily as a purification tool but may influence experimental outcomes in several ways that researchers must account for:

• Protein solubility and stability: The His-tag can enhance solubility due to the charged nature of histidine residues, potentially altering the protein's stability profile compared to untagged variants.

• Biological activity: While generally minimal, His-tags may occasionally interfere with receptor binding, particularly if located near functional domains. Empirical validation of activity compared to untagged proteins is therefore recommended.

• Detection specificity: The tag enables specific detection using anti-His antibodies, providing an alternative to direct AITRL antibodies that may have variable epitope recognition.

• Experimental artifacts: Potential tag-mediated protein aggregation or non-specific interactions with other biomolecules should be controlled for through appropriate experimental design.

To minimize tag-related artifacts, researchers should consider enzymatic tag removal when absolute native conformation is required, particularly for structural studies or when inconsistent results are observed between tagged and untagged variants.

What are the optimal conditions for reconstitution and storage of AITRL Human, His?

Optimal handling of AITRL Human, His follows protocols similar to those established for other histidine-tagged recombinant proteins in the TNF family. Based on established methodologies for structurally similar proteins:

Reconstitution Protocol:

• Allow the lyophilized protein to equilibrate to room temperature (20-25°C) before opening.

• Reconstitute in sterile, ultrapure water or buffer (typically PBS with pH 7.2-7.4) to a concentration of 0.1-1.0 mg/mL.

• Gently swirl or rotate the vial until completely dissolved; avoid vigorous shaking or vortexing that may cause protein denaturation.

• Allow the solution to stand for 10-15 minutes at room temperature for complete rehydration.

Storage Recommendations:

• Short-term (≤1 week): Store at 2-8°C.

• Long-term: Aliquot and store at -20°C to -80°C, avoiding repeated freeze-thaw cycles (limit to ≤5 cycles).

• Consider adding carrier proteins (e.g., 0.1% BSA) for dilute solutions to prevent adsorption to container surfaces.

• For working solutions, maintain at 4°C and use within 24 hours to ensure optimal activity.

Researchers should validate protein stability using functional assays after storage periods to ensure experimental reproducibility.

How can AITRL Human, His be effectively incorporated into immunological assays?

For optimal incorporation of AITRL Human, His into immunological assays, researchers should consider the following methodological approaches:

Cell-Based Functional Assays:

• Receptor binding studies: Use 0.1-10 μg/mL of AITRL Human, His in binding buffer (PBS with 1-2% BSA) when assessing interactions with its receptor (TNFRSF18/GITR).

• Dose titration: Always perform preliminary dose-response experiments (0.1-1000 ng/mL) to determine optimal concentrations for specific cell lines and readouts.

• Incubation conditions: Standard conditions include 37°C, 5% CO₂, for 24-72 hours depending on the cellular response being measured.

Flow Cytometry Applications:

• For direct binding detection, conjugate AITRL Human, His with fluorophores using commercial kits designed for His-tagged proteins.

• As a positive control, include parallel samples with commercially validated anti-GITR antibodies.

• For competitive binding assays, pre-incubate cells with unlabeled AITRL Human, His before adding labeled detection antibodies.

ELISA-Based Detection:

• Capture phase: Immobilize AITRL Human, His (1-5 μg/mL) on Ni-NTA or anti-His coated plates.

• Detection phase: Use biotinylated receptor proteins or specific antibodies against AITRL.

• Standard curve generation: Create a dilution series (0.1-1000 ng/mL) of purified AITRL Human, His for quantitative analysis.

These methodologies should be optimized for each experimental system, with careful attention to appropriate controls for His-tag interference.

What experimental controls are essential when using AITRL Human, His in signaling pathway studies?

When investigating signaling pathways mediated by AITRL Human, His, the following controls are essential to ensure scientific rigor and validity:

Negative Controls:

• His-tagged irrelevant protein: Include a similarly sized, His-tagged protein with no known binding to GITR to control for non-specific effects of the His tag.

• Heat-inactivated AITRL Human, His: Prepare by heating at 95°C for 10 minutes to serve as a denatured protein control.

• Receptor-negative cell lines: Use cell lines lacking GITR expression to confirm signaling specificity.

Positive Controls:

• Commercial AITRL without His-tag, if available, to benchmark activity.

• Validated GITR agonist antibodies with established signaling outcomes.

• Known downstream signaling activators (e.g., TNFα for NF-κB pathway) as pathway-specific positive controls.

Validation Controls:

• Receptor blocking: Pre-incubate cells with anti-GITR neutralizing antibodies before AITRL Human, His treatment.

• Dose-response assessment: Use at least 5-6 concentration points (typically 0.1-100 ng/mL) to establish response curves.

• Time-course experiments: Sample at multiple time points (15 min, 30 min, 1h, 2h, 6h, 24h) to capture both early and late signaling events.

• Pathway inhibitors: Include specific inhibitors of predicted downstream pathways (e.g., NF-κB, MAPK, JAK/STAT inhibitors) to confirm mechanism.

These controls collectively address potential confounding factors including His-tag artifacts, non-specific stimulation, and technical variability.

How do you troubleshoot inconsistent results in AITRL-mediated cellular responses?

Inconsistent results when working with AITRL Human, His often stem from several common challenges that can be systematically addressed:

Protein Quality Issues:

• Degradation assessment: Run SDS-PAGE to check for protein fragmentation; freshly thawed aliquots should show a single dominant band at 15.7 kDa .

• Aggregation testing: Perform dynamic light scattering or size-exclusion chromatography to detect aggregates that may alter activity.

• Activity validation: Include a functional assay control with each new lot or after extended storage periods.

Cell-Related Variables:

• Receptor expression: Quantify GITR levels via flow cytometry or Western blot, as expression can vary with cell passage or culture conditions.

• Cell cycle synchronization: For cell cycle-dependent responses, synchronize cells using serum starvation (0.5% FBS for 24h) before treatment.

• Confluence effects: Maintain consistent cell density (typically 60-80% confluence) as receptor expression and signaling sensitivity often vary with cell density.

Experimental Design Refinements:

• Standardize protein handling: Minimize freeze-thaw cycles and prepare fresh working dilutions for each experiment.

• Control environmental variables: Monitor incubator CO₂ levels, humidity, and temperature stability.

• Refine detection timing: For temporally dynamic responses, perform detailed time-course experiments to identify optimal measurement windows.

Reagent Interactions:

• Media components: Test for interference from serum components by comparing responses in serum-free versus serum-containing conditions.

• Buffer compatibility: Verify that reconstitution buffers do not contain components (e.g., certain detergents or metal chelators) that might affect protein activity.

Implementing these troubleshooting approaches systematically can help identify and eliminate sources of variability in AITRL Human, His experimental outcomes.

How does AITRL Human, His compare functionally to other similar recombinant proteins?

AITRL Human, His shares functional characteristics with other TNF superfamily members while maintaining distinct activity profiles. Understanding these comparative aspects helps researchers contextualize experimental findings:

Comparative Analysis with Related Recombinant Proteins:

When designing experiments with AITRL Human, His, researchers should consider:

• Target cell selection: While HGF primarily targets epithelial and endothelial cells , AITRL primarily affects T cells and antigen-presenting cells, requiring appropriate immune cell models.

• Functional readouts: Unlike HGF which promotes cell migration and proliferation through c-Met signaling , AITRL modulates immune activation through NF-κB and MAPK pathways, necessitating distinct functional assays.

• Combination studies: When investigating potential synergies between multiple recombinant proteins (e.g., AITRL with cytokines), careful titration experiments should account for potential cooperative or antagonistic signaling effects.

This contextual understanding ensures appropriate experimental design and accurate interpretation of AITRL Human, His-mediated effects relative to other recombinant proteins in research applications.

What are the emerging applications of AITRL Human, His in immunotherapy and cancer research?

AITRL Human, His is increasingly employed in cutting-edge immunotherapy research, with several promising applications that extend beyond traditional immunological studies:

Emerging Research Applications:

• Tumor microenvironment modulation:

  • AITRL Human, His serves as a valuable tool for investigating GITR-mediated reversal of immunosuppression within tumor microenvironments.

  • Researchers use the protein to stimulate effector T cells while simultaneously inhibiting regulatory T cell function in ex vivo tumor models.

  • Functional studies employing AITRL Human, His help delineate mechanisms by which GITR targeting enhances anti-tumor immunity.

• Chimeric antigen receptor (CAR) T cell enhancement:

  • AITRL incorporation into CAR constructs represents an emerging application where the recombinant protein serves as both research tool and potential therapeutic component.

  • For such applications, researchers must carefully evaluate His-tag effects on CAR assembly and function.

• Bi-specific antibody development:

  • AITRL Human, His functions as a critical positive control when evaluating novel bi-specific antibodies targeting GITR and other co-stimulatory receptors.

  • Comparative activity assays between soluble AITRL Human, His and antibody candidates help establish relative potency and mechanism of action.

• Imaging and biodistribution studies:

  • The His-tag facilitates direct labeling for tracking AITRL distribution in complex systems.

  • Researchers can leverage this property for in vivo imaging when studying GITR expression dynamics during immune responses.

These applications highlight the versatility of AITRL Human, His in both fundamental immunology research and translational cancer immunotherapy development, positioning it as a valuable reagent for researchers across multiple disciplines.

What approaches can distinguish direct versus indirect effects of AITRL in complex cellular systems?

Distinguishing direct from indirect effects of AITRL Human, His in complex cellular environments requires rigorous experimental approaches:

Cell-Type Specific Isolation:

• Magnetic or flow cytometry-based cell sorting to isolate distinct populations before and after AITRL Human, His treatment.

• Analysis of changes in each population independently to identify primary responders versus secondary effect recipients.

Temporal Resolution Approaches:

• Early timepoint sampling (5-30 minutes) to capture immediate receptor-proximal events likely representing direct effects.

• Time-course experiments extending to later points (24-72 hours) to monitor secondary cytokine production and paracrine signaling.

Receptor Knockdown/Knockout Validation:

• CRISPR-Cas9 mediated GITR knockout in specific cell populations.

• siRNA-mediated transient receptor knockdown with subsequent functional assessment.

• Comparison of responses between receptor-positive and receptor-negative cells within the same culture environment.

Signaling Pathway Dissection:

• Phospho-flow cytometry to simultaneously assess signaling in multiple cell types following AITRL exposure.

• Single-cell RNA sequencing to identify cell-specific transcriptional responses distinguishing primary from secondary responders.

• Application of pathway-specific inhibitors at concentrations that block specific signaling cascades without affecting cell viability.

Co-culture Systems with Physical Separation:

• Transwell assays to separate GITR+ and GITR- populations while allowing soluble factor exchange.

• Comparison of responses in direct co-culture versus transwell systems to delineate contact-dependent versus soluble mediator-dependent effects.

These methodological approaches collectively provide robust strategies for distinguishing the direct effects of AITRL Human, His from secondary phenomena in complex cellular systems.

How should dose-response experiments with AITRL Human, His be optimized for reproducibility?

Optimizing dose-response experiments with AITRL Human, His requires careful attention to several key methodological elements:

Concentration Range Selection:

• Employ a logarithmic concentration series spanning at least 5 orders of magnitude (e.g., 0.01 ng/mL to 1000 ng/mL).

• Include a minimum of 8 concentration points to enable accurate curve fitting.

• Conduct preliminary range-finding experiments to identify the potential EC₅₀ value, then design the main experiment with concentrations centered around this value.

Technical Replication Requirements:

• Perform technical triplicates at minimum for each concentration.

• Include biological replicates (different cell passages, donors, or experimental days) to establish reproducibility across biological variation.

• Calculate statistical power before experiments to determine appropriate sample sizes for detecting biologically significant differences.

Protocol Standardization:

• Standardize protein reconstitution procedures, including consistent buffer composition and protein handling.

• Prepare master stocks of AITRL Human, His at high concentration (≥100 μg/mL), then create working dilutions fresh for each experiment.

• Document equilibration times and temperature conditions precisely in protocols.

Response Normalization Strategies:

• Include both positive controls (maximal stimulation) and negative controls (vehicle only) to enable percent response normalization.

• Consider using internal normalization controls (housekeeping proteins for Western blots, reference genes for qPCR) to account for technical variation.

Data Analysis Considerations:

• Apply appropriate curve-fitting models (typically four-parameter logistic regression for receptor-ligand interactions).

• Report both EC₅₀ values and Hill slopes to characterize response dynamics fully.

• Present complete dose-response data rather than selected concentrations to enable critical evaluation.

Implementation of these methodological considerations will significantly enhance reproducibility of AITRL Human, His dose-response experiments across different research settings.

What are the most promising future applications for AITRL Human, His in immunology research?

The continuing evolution of immunology research presents several promising future applications for AITRL Human, His:

• Single-cell analysis of GITR-mediated signaling heterogeneity will likely expand our understanding of variable responses within seemingly homogeneous immune cell populations.

• Integration with advanced organoid and 3D culture systems will enable more physiologically relevant modeling of AITRL-mediated immune modulation.

• Development of controlled-release formulations incorporating AITRL Human, His may facilitate extended stimulation studies that better recapitulate chronic immune activation scenarios.

• CRISPR-based screens using AITRL Human, His as a selective pressure could identify novel components of GITR signaling pathways and regulatory mechanisms.

• Combination approaches pairing AITRL Human, His with checkpoint inhibitors or other immunomodulatory agents will continue to provide insights into potential therapeutic synergies.