Phospho-IL2RB (Tyr364) Antibody

What is IL-2Rβ (CD122) and its role in T cell signaling?

IL-2Rβ (also known as CD122) is a critical subunit of the interleukin-2 receptor, which mediates T cell-mediated immune responses. The IL-2 receptor exists in three distinct forms with varying binding affinities to IL-2:

• Low affinity: Alpha subunit monomer (not involved in signal transduction)

• Intermediate affinity: Alpha/beta subunit heterodimer

• High affinity: Alpha/beta/gamma subunit heterotrimer

The beta subunit (IL-2Rβ) plays an essential role in both receptor-mediated endocytosis and transduction of mitogenic signals from IL-2. It is primarily expressed in the hematopoietic system and functions as a type I membrane protein . When IL-2 binds to its receptor complex, the IL-2Rβ chain becomes phosphorylated at specific tyrosine residues, including Tyr364, which initiates downstream signaling cascades essential for T cell proliferation, differentiation, and effector function .

What is the specificity of Phospho-IL2RB (Tyr364) Antibody?

Phospho-IL2RB (Tyr364) Antibody specifically detects endogenous levels of IL-2Rβ protein only when phosphorylated at tyrosine 364 . This high specificity makes it an invaluable tool for studying IL-2 signaling pathways. The antibody is typically generated by immunizing rabbits with synthetic phosphopeptides derived from human IL-2Rβ around the phosphorylation site of Tyr364 (sequence: Q-G-YP-F-F) .

To ensure specificity, these antibodies undergo rigorous purification processes:

• Affinity-purification from rabbit antiserum using epitope-specific phosphopeptide

• Removal of non-phospho specific antibodies by chromatography using non-phosphopeptide corresponding to the phosphorylation site

This dual purification approach ensures that the antibody exclusively recognizes the phosphorylated form of the protein at this specific site.

What are the standard applications for Phospho-IL2RB (Tyr364) Antibody?

Phospho-IL2RB (Tyr364) Antibody has several validated research applications:

Most commercial preparations have been validated for reactivity with human, mouse, and rat samples . For optimal results in Western blotting, researchers should optimize antibody concentration based on their specific sample types and detection methods.

What are the proper storage and handling conditions for Phospho-IL2RB (Tyr364) Antibody?

For maximum stability and activity retention:

• Store at -20°C for long-term storage (up to 1 year)

• Avoid repeated freeze-thaw cycles that can compromise antibody activity

• Upon receipt, consider aliquoting the antibody into smaller volumes to minimize freeze-thaw cycles

• Most commercial preparations are supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% sodium azide, and 50% glycerol

The presence of glycerol and stabilizers in the formulation helps maintain antibody integrity during freeze-thaw cycles when they cannot be avoided.

How can Phospho-IL2RB (Tyr364) Antibody be used to study differential IL-2 signaling dynamics between CD4⁺ and CD8⁺ T cells?

Recent research has revealed distinct IL-2 signaling dynamics between CD4⁺ and CD8⁺ T cells, with CD8⁺ T cells demonstrating more sustained signaling responses. Phospho-IL2RB (Tyr364) Antibody can be instrumental in investigating these differences through:

• Time-course analysis: Monitor phosphorylation at different time points after IL-2 stimulation in both cell types. CD8⁺ T cells maintain phospho-STAT5 signaling longer than CD4⁺ T cells despite similar initial activation (EC₅₀ values of 0.28 u/mL vs. 0.35 u/mL) .

• Co-treatment with inhibitors: Combine IL-2 stimulation with protein synthesis inhibitors (cycloheximide) or vesicular transport inhibitors (brefeldin A) to distinguish between receptor recycling versus new synthesis mechanisms .

• Receptor abundance correlation: Compare IL-2Rβ phosphorylation levels with receptor abundance using flow cytometry or Western blotting to determine if signaling differences stem from receptor density variations.

A comprehensive experimental design would include:

• Isolation of CD4⁺ and CD8⁺ T cells from the same donor

• IL-2 stimulation time course (0, 15, 30, 60, 120, 240 minutes)

• Western blotting with Phospho-IL2RB (Tyr364) Antibody

• Parallel analysis of downstream signaling markers (pSTAT5)

• Flow cytometry to quantify total receptor surface expression

How does phosphorylation at Tyr364 integrate with other phosphorylation sites on IL-2Rβ?

IL-2Rβ contains multiple phosphorylation sites that coordinate complex signaling responses. Tyr364 phosphorylation functions within a network of other phosphorylation events:

To study the interrelationship between these phosphorylation sites:

• Sequential immunoprecipitation: Use Phospho-IL2RB (Tyr364) Antibody for initial pulldown, followed by Western blotting with antibodies against other phosphorylation sites.

• Mutational analysis: Compare signaling in cells expressing wild-type IL-2Rβ versus those with Y364F mutations to determine the specific contribution of this phosphorylation site.

• Phosphatase inhibition studies: Treat cells with phosphatase inhibitors followed by IL-2 stimulation to determine if Tyr364 phosphorylation has different kinetics or sensitivity to phosphatase action compared to other sites.

The timing and duration of Tyr364 phosphorylation may be critical for sustaining IL-2 signaling required for cell fate decisions, which can require hours to days of continuous stimulation .

What protocols are recommended for optimizing Phospho-IL2RB (Tyr364) Antibody use in Western blot applications?

For optimal Western blot results with Phospho-IL2RB (Tyr364) Antibody:

Sample Preparation:

• Stimulate cells with IL-2 (concentrations ranging from 1-100 U/mL) for 5-15 minutes to achieve maximum phosphorylation

• Lyse cells in buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

• Include protease inhibitors to prevent protein degradation

• Use quantitative protein determination methods to ensure equal loading

Western Blotting Protocol:

• Load 20-50 μg total protein per lane

• Use 8-10% SDS-PAGE gels (IL-2Rβ has a molecular weight of ~61 kDa)

• Transfer to PVDF membrane (preferred over nitrocellulose for phospho-proteins)

• Block with 5% BSA in TBST (not milk, which contains phospho-proteins)

• Incubate with Phospho-IL2RB (Tyr364) Antibody at 1:500-1:1000 dilution overnight at 4°C

• Wash 4-5 times with TBST

• Incubate with HRP-conjugated anti-rabbit secondary antibody

• Develop using enhanced chemiluminescence

Controls:

• Positive control: Lysate from IL-2 stimulated T cells

• Negative control: Lysate from unstimulated cells

• Phosphatase-treated control: Sample treated with lambda phosphatase

• Loading control: Probe for total IL-2Rβ or housekeeping protein

How can Phospho-IL2RB (Tyr364) Antibody be used to investigate receptor internalization dynamics?

IL-2 receptor internalization is a critical regulatory mechanism in T cell signaling. Upon IL-2 binding, the receptor complex is rapidly internalized, and IL-2Rβ and IL-2Rγ are degraded, potentially limiting sustained signaling . Phospho-IL2RB (Tyr364) Antibody can be used to track this process:

Subcellular Fractionation Approach:

• Stimulate cells with IL-2 for various time points (0-60 minutes)

• Perform subcellular fractionation to separate membrane, cytosolic, and endosomal fractions

• Analyze each fraction by Western blotting with both phospho-specific and total IL-2Rβ antibodies

• Quantify the ratio of phosphorylated to total IL-2Rβ in each fraction over time

Flow Cytometry Method:

• Stimulate cells with IL-2

• At various time points, fix and permeabilize cells

• Double-stain with fluorescently labeled antibodies against:

  • Surface IL-2Rβ (to track remaining surface receptors)

  • Phospho-IL2RB (Tyr364) (to track activated receptors)

• Analyze by flow cytometry to determine the kinetics of receptor activation versus internalization

Inhibitor Studies:

• Compare normal conditions with cells treated with:

  • Brefeldin A (blocks vesicular transport)

  • Dynamin inhibitors (block endocytosis)

  • Cycloheximide (blocks protein synthesis)

This approach can reveal whether sustained signaling requires new receptor synthesis or recycling of internalized receptors, and how phosphorylation at Tyr364 correlates with these processes.

What methods can be used to validate the specificity of Phospho-IL2RB (Tyr364) Antibody in experimental systems?

Ensuring antibody specificity is crucial for reliable research results. Several validation approaches are recommended:

Peptide Competition Assay:

• Pre-incubate Phospho-IL2RB (Tyr364) Antibody with:

  • Phosphorylated immunizing peptide

  • Non-phosphorylated version of the same peptide

• Use these pre-absorbed antibodies in parallel Western blots

• Specific binding should be blocked only by the phosphorylated peptide

Genetic Approaches:

• Use CRISPR/Cas9 to generate IL-2Rβ knockout cells as negative controls

• Create Y364F mutant (tyrosine to phenylalanine) cell lines that cannot be phosphorylated at this site

• Test antibody reactivity in both systems - signal should be absent

Phosphatase Treatment:

• Divide cell lysates from IL-2 stimulated cells into two portions

• Treat one portion with lambda phosphatase

• Run both samples on Western blot

• Signal should be present in untreated sample but absent in phosphatase-treated sample

Correlation with Functional Assays:

• Monitor IL-2Rβ phosphorylation using the antibody

• In parallel, measure downstream functional readouts (pSTAT5, cell proliferation)

• Establish temporal correlation between phosphorylation and functional outcomes

• Use JAK inhibitors to block phosphorylation and confirm concurrent loss of function

These validation techniques ensure that experimental observations truly reflect IL-2Rβ phosphorylation at Tyr364 rather than non-specific antibody binding.

What are recommended protocols for sample preparation to preserve phosphorylation status?

Phosphorylation states are highly labile and can be rapidly lost during sample preparation. To preserve phosphorylation at Tyr364:

Cell Lysis Protocol:

• Stimulate cells with IL-2 (10-100 U/mL) for optimal time (typically 5-15 minutes)

• Immediately wash cells with ice-cold PBS containing phosphatase inhibitors (2 mM sodium orthovanadate, 10 mM sodium fluoride)

• Lyse cells in buffer containing:

  • 1% NP-40 or Triton X-100

  • 150 mM NaCl

  • 50 mM Tris-HCl (pH 7.4)

  • 1 mM EDTA

  • 1 mM EGTA

  • 5 mM sodium pyrophosphate

  • 10 mM β-glycerophosphate

  • 1 mM sodium orthovanadate

  • 1 mM sodium fluoride

  • Protease inhibitor cocktail

• Keep samples on ice throughout processing

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and add 4× Laemmli sample buffer

• Heat at 95°C for 5 minutes

Tissue Sample Processing:

• Snap-freeze tissues in liquid nitrogen immediately after collection

• Pulverize frozen tissue under liquid nitrogen using a mortar and pestle

• Transfer powdered tissue directly into lysis buffer (as above)

• Homogenize briefly while maintaining cold temperature

• Proceed as with cell lysates

These methods maximize phosphorylation preservation by rapidly inactivating phosphatases that would otherwise dephosphorylate IL-2Rβ at Tyr364.

How can Phospho-IL2RB (Tyr364) Antibody be used to study IL-2 signaling in primary human samples?

Working with primary human samples presents unique challenges but offers physiologically relevant insights:

Isolation and Stimulation Protocol:

• Isolate peripheral blood mononuclear cells (PBMCs) from whole blood using Ficoll density gradient centrifugation

• Further isolate T cell subsets using negative selection magnetic bead methods

• Rest cells for 1-2 hours in serum-free media

• Stimulate with recombinant human IL-2 (1-100 U/mL) for time course analysis

• Process for Western blot or flow cytometry as described earlier

Flow Cytometry for Rare Cell Populations:

• Stimulate whole PBMCs with IL-2

• Fix with 1.5% paraformaldehyde for 10 minutes at room temperature

• Permeabilize with ice-cold 90% methanol for 30 minutes

• Stain with cell surface markers (CD3, CD4, CD8, CD25) and Phospho-IL2RB (Tyr364) Antibody

• Analyze by multiparameter flow cytometry to assess phosphorylation in specific cell subsets

Considerations for Patient Samples:

• Process samples immediately after collection to preserve phosphorylation status

• Include age-matched and gender-matched healthy controls

• Document patient medications that might affect IL-2 signaling

• Consider disease state when interpreting phosphorylation levels

This approach allows investigation of IL-2 signaling differences between healthy individuals and patients with immune disorders, potentially revealing disease-specific alterations in IL-2Rβ phosphorylation.

What troubleshooting approaches can resolve common issues with Phospho-IL2RB (Tyr364) Antibody experiments?

When working with phospho-specific antibodies like Phospho-IL2RB (Tyr364), researchers may encounter several challenges:

Advanced troubleshooting may require optimizing:

• Primary antibody incubation temperature (4°C vs. room temperature)

• Incubation time (1 hour vs. overnight)

• Membrane blocking conditions

• Detection method sensitivity (standard ECL vs. enhanced sensitivity reagents)

Human primary samples often give weaker signals than cell lines due to lower receptor expression. In these cases, increasing protein loading and using more sensitive detection reagents may be necessary.

How can Phospho-IL2RB (Tyr364) Antibody be used to study IL-2 receptor signaling in immunological disorders?

Phospho-IL2RB (Tyr364) Antibody provides valuable insights into immunological disorders where IL-2 signaling may be dysregulated:

Autoimmune Disease Research:

• Compare IL-2Rβ phosphorylation patterns between healthy controls and patients with autoimmune conditions

• Correlate phosphorylation levels with disease severity markers

• Monitor changes in phosphorylation before and after immunosuppressive therapy

• Investigate how genetic variations in IL2RB affect phosphorylation patterns

Primary Immunodeficiency Studies:
The IL2RB gene is associated with Immunodeficiency 63 with Lymphoproliferation and Autoimmunity . Using Phospho-IL2RB (Tyr364) Antibody:

• Assess phosphorylation in patient-derived cells with IL2RB mutations

• Determine if specific mutations affect Tyr364 phosphorylation versus other sites

• Correlate phosphorylation defects with clinical features

Therapeutic Response Monitoring:

• Measure IL-2Rβ phosphorylation in patients receiving IL-2 therapy or IL-2 pathway modulators

• Use phosphorylation status as a pharmacodynamic biomarker

• Correlate phosphorylation levels with clinical response

Emerging research suggests that IL-2 pathway modulation through low-dose IL-2 therapy may restore regulatory T cell function in autoimmune diseases. Phospho-IL2RB (Tyr364) Antibody could help optimize dosing regimens by providing direct measurement of target engagement.

What emerging technologies can enhance the utility of Phospho-IL2RB (Tyr364) Antibody in signaling research?

Recent technological advances offer new opportunities for studying IL-2Rβ phosphorylation:

Single-Cell Phosphoprotein Analysis:

• Combine Phospho-IL2RB (Tyr364) Antibody with mass cytometry (CyTOF)

• Simultaneously measure multiple phosphorylation sites on IL-2Rβ and downstream proteins

• Correlate with cell surface markers to identify responsive cell subpopulations

• Reveal heterogeneity in signaling responses within seemingly homogeneous cell populations

Proximity Ligation Assays (PLA):

• Use Phospho-IL2RB (Tyr364) Antibody with antibodies against potential interaction partners

• Detect protein-protein interactions that depend on Tyr364 phosphorylation

• Visualize subcellular localization of signaling complexes

• Quantify interaction dynamics following IL-2 stimulation

CRISPR/Cas9 Gene Editing:

• Generate precise point mutations at Tyr364 (Y364F)

• Create reporter cell lines with fluorescent tags on IL-2Rβ

• Combine with live-cell imaging to track receptor dynamics

• Correlate phosphorylation with receptor trafficking and signaling outcomes

Phosphoproteomic Integration:

• Use Phospho-IL2RB (Tyr364) Antibody for immunoprecipitation

• Couple with mass spectrometry to identify the complete interactome of phosphorylated IL-2Rβ

• Map temporal changes in protein interactions following IL-2 stimulation

• Build comprehensive signaling networks that link Tyr364 phosphorylation to downstream effects