irag2 Antibody

What is IRAG2 and what tissues express this protein?

IRAG2, also known as Jaw1 or lymphoid-restricted membrane protein (LRMP), is a type II membrane protein localized to the endoplasmic reticulum. Despite its name suggesting limited expression, IRAG2 has been detected in multiple tissues and cell types. Expression patterns include:

• Immune tissues: B-cell lines, T-cell lines, spleen, and thymus

• Digestive system: Intestinal tuft cells and exocrine pancreas (particularly acinar cells)

• Sensory tissues: Sweet, bitter, and umami taste-responsive cells

• Cardiovascular system: Sinoatrial nodes and platelets

According to the Human Protein Atlas, both protein and mRNA expression of IRAG2 are detectable in human exocrine pancreas, predominantly in exocrine glandular cells with weaker expression in pancreatic duct cells .

How does IRAG2 relate structurally and functionally to IRAG1?

These opposing effects suggest IRAG2 may function as a counterpart to IRAG1 in certain physiological contexts .

What are the optimal conditions for immunoprecipitation of IRAG2?

Successful immunoprecipitation of IRAG2 requires careful optimization of experimental conditions. Based on published protocols , researchers should consider:

Buffer composition:

• Use 2% Lubrol buffer for initial homogenization of tissue/cells

• For co-immunoprecipitation buffer: 50 mM Tris-HCl, 150 mM NaCl, 1% Lubrol, 0.5% sodium deoxycholate, and 0.1% SDS (pH 7.5)

• Include protease inhibitors, PhosSTOP, and 1 mM DTT to prevent protein degradation and preserve phosphorylation status

Antibody incubation parameters:

• Use 1 μg of anti-mouse LRMP (C-terminal) antibody per reaction

• Incubate with 70-1000 μg of protein lysate (depending on expression level)

• Initial incubation: 90 minutes on ice

• After adding Protein-A-Sepharose-Beads (blocked with 3% BSA), continue incubation overnight at 4°C

Washing and elution:

• Perform thorough washes to remove non-specifically bound proteins

• Elute using 2× Laemmli buffer for subsequent SDS-PAGE analysis

• Use pipette rather than vacuum aspiration when removing liquid after centrifugation

This protocol has been validated for detecting IRAG2 interactions with IP₃ receptor subtypes in multiple tissue types, including platelets and pancreatic tissue .

How can I detect IRAG2 phosphorylation in experimental samples?

IRAG2 phosphorylation can be detected through a combination of immunoprecipitation and phospho-specific antibody detection. Two validated approaches include:

In vitro phosphorylation protocol:

• Lyse platelets or target tissue in buffer containing protease and phosphatase inhibitors

• Stimulate lysates with 100 μM 8-Br-cGMP or 100 μM 8-pCPT-cGMP for 20 minutes at 30°C

• Include H₂O as an unstimulated control

• Immunoprecipitate IRAG2 using IRAG2-specific antibody

• Detect phosphorylation using phospho-(Ser/Thr) PKA substrate antibody (1:1000 dilution) that recognizes PKG-, PKA-, or PKC-dependent phosphorylation

Ex vivo phosphorylation protocol:

• Isolate intact platelets (1.0 × 10⁸ platelets)

• Preincubate in buffer B (pH 7.4) for 1 hour at 37°C

• Treat with 100 μM 8-pCPT-cGMP for 20 minutes at 37°C (or H₂O as control)

• Process samples through centrifugation and homogenization

• Perform immunoprecipitation and Western blot as described above

Researchers should note that IRAG2 phosphorylation is detectable only in stimulated samples from wild-type animals, with no signal in unstimulated controls or in samples from IRAG2-knockout animals .

Which IP₃ receptor subtypes interact with IRAG2 and how should these interactions be validated?

IRAG2 has been demonstrated to interact with all three IP₃ receptor subtypes (IP₃R1, IP₃R2, and IP₃R3) in various tissues. To validate these interactions, researchers should:

Experimental approach:

• Perform co-immunoprecipitation using IRAG2-specific antibody

• Analyze interaction partners via Western blot using specific antibodies against each IP₃ receptor subtype

• Include appropriate controls: IRAG2-knockout samples as negative controls, and input samples to verify antibody specificity

Antibody dilutions for Western blot:

• Anti-IP₃R1: 1:1000

• Anti-IP₃R2: 1:200

• Anti-IP₃R3: 1:100

• Anti-IRAG2: 1:1000

Tissue-specific considerations:
Different tissues may show variations in interaction patterns. In pancreatic tissue, all three IP₃ receptor subtypes co-immunoprecipitate with IRAG2 . In platelets, similar interactions are observed, though the molecular weight of IP₃R2 in immunoprecipitated samples may appear lower than in input samples .

How do IRAG2-knockout models affect expression of related signaling proteins?

The absence of IRAG2 can differentially impact the expression of related signaling proteins depending on the tissue type. These effects provide important insights into potential compensatory mechanisms and functional relationships.

In pancreatic tissue:

• IP₃R3 expression: Significantly higher in IRAG2-KO mice compared to wild-type

• IP₃R2 expression: Significantly lower in IRAG2-KO compared to wild-type

• IP₃R1 expression: No significant difference observed

In platelets:

• No significant alterations in expression levels of IP₃R1, IP₃R2, IP₃R3, IRAG1, or PKGIβ between IRAG2-KO and wild-type mice

These differential effects suggest tissue-specific regulatory mechanisms and compensatory responses in the absence of IRAG2, which should be considered when interpreting experimental results from knockout models .

How can I distinguish between IRAG1 and IRAG2 functions in platelet aggregation studies?

Distinguishing between IRAG1 and IRAG2 functions in platelet aggregation requires careful experimental design, as these proteins appear to have opposing effects on platelet function:

Comparative experimental design:

• Isolate platelets from wild-type, IRAG1-KO, and IRAG2-KO mice

• Standardize platelet counts (1.0 × 10⁸ platelets/mL) across all samples

• Perform aggregation assays using varying concentrations of agonists:

  • Thrombin (0.024, 0.05, and 0.1 U/mL)

  • Collagen (5 μg/mL)

• Measure aggregation rate by determining the maximal slope of aggregation curves

• Include NO/cGMP pathway modulators:

  • Sodium nitroprusside (SNP) as NO donor

  • 8-pCPT-cGMP as direct cGMP analog

Expected results:

• IRAG1-KO platelets: Increased aggregation compared to wild-type

• IRAG2-KO platelets: Decreased aggregation compared to wild-type

• IRAG1-KO + cGMP: Minimal inhibition of aggregation

• IRAG2-KO + cGMP: Enhanced inhibition of aggregation

Analysis considerations:

• Calculate fold-change in aggregation relative to unstimulated controls

• Compare basal aggregation between genotypes

• Evaluate NO/cGMP sensitivity across genotypes

• Consider phosphorylation status of both proteins using phospho-specific antibodies

This approach allows researchers to delineate the opposing roles of these related proteins in platelet function and NO/cGMP signaling.

What are the optimal controls for IRAG2 antibody specificity validation?

Validating antibody specificity is critical for reliable IRAG2 research. A comprehensive validation approach should include multiple controls:

Genetic controls:

• IRAG2-knockout tissues/cells as negative controls

• lacZ × IRAG2-KO reporter mice that express β-Galactosidase under control of the IRAG2 promoter (allows confirmation of expression pattern)

Immunological controls:

• Input control: Include whole lysate samples to verify antibody reactivity

• Isotype control: Use matched IgG subclass control antibodies

• Bead-only control: Evaluate non-specific binding to precipitation matrix

Western blot validation protocol:

• Run samples from wild-type and IRAG2-KO tissues in adjacent lanes

• Include multiple tissue types with known IRAG2 expression (spleen, thymus, pancreas) as positive controls

• Normalize to total protein using trichloroethanol-containing hand-casting SDS-gels

• Use standardized antibody dilutions (anti-IRAG2: 1:1000)

• Detect using appropriate secondary antibodies and imaging systems

Expression pattern verification:
Compare antibody staining patterns with established IRAG2 expression profiles from multiple sources (Human Protein Atlas, literature reports) to confirm specificity across different cell types and tissues .

How can IRAG2 phosphorylation status be correlated with functional outcomes in different experimental systems?

Correlating IRAG2 phosphorylation with functional outcomes requires integrated experimental approaches that combine biochemical and functional analyses:

Combined experimental workflow:

• Biochemical characterization:

  • Perform in vitro or ex vivo phosphorylation assays as described in FAQ 2.2

  • Quantify phosphorylation levels using densitometry of Western blots

  • Include time-course analyses to determine phosphorylation kinetics

• Parallel functional assays:

  • For platelets: Perform aggregation studies with varying agonist concentrations

  • For pancreatic tissue: Measure amylase secretion (basal and stimulated)

  • Include appropriate inhibitors to modulate phosphorylation:

    • PKG inhibitors (e.g., KT5823)

    • Guanylyl cyclase inhibitors (e.g., ODQ)

    • Phosphodiesterase inhibitors (e.g., sildenafil)

• Correlation analysis:

  • Plot phosphorylation levels against functional readouts

  • Consider time-dependent relationships

  • Analyze dose-response relationships for both phosphorylation and function

Research findings example (platelets):
IRAG2 phosphorylation correlates with enhanced platelet aggregation in response to thrombin or collagen stimulation. When IRAG2 is phosphorylated by cGMP-dependent protein kinase I (PKGI), it contributes to increased aggregation, contrasting with IRAG1 which inhibits aggregation when phosphorylated in a cGMP-dependent manner .

What are common pitfalls in IRAG2 co-immunoprecipitation experiments and how can they be addressed?

Co-immunoprecipitation studies with IRAG2 can present several challenges. Here are common issues and their solutions:

Problem 1: Non-specific bands in co-immunoprecipitation

• Observed issue: In platelet studies, Western blot images of PKGIβ revealed an unspecific band in co-immunoprecipitated samples from both IRAG2-WT and IRAG2-KO platelets

• Solution: Verify the source of non-specific bands (in this case, derived from the IRAG2 antibody); use proper controls including IRAG2-KO samples to distinguish specific from non-specific signals

Problem 2: Altered molecular weight of co-precipitated proteins

• Observed issue: The molecular weight of IP₃R2 in immunoprecipitated samples appeared lower than in input samples

• Solution: Consider that IRAG2 may interact with specific isoforms or post-translationally modified versions of target proteins; use multiple antibodies recognizing different epitopes to confirm identity

Problem 3: Weak or inconsistent co-precipitation

• Solution:

  • Optimize lysis buffer composition (consider detergent type and concentration)

  • Increase antibody amount (1-5 μg per reaction)

  • Extend incubation times (overnight at 4°C)

  • Use crosslinking approaches for transient interactions

Problem 4: High background in Western blot

• Solution:

  • Increase blocking time (2 hours) with 5% non-fat dry milk or 3% BSA

  • Use more stringent washing conditions

  • Optimize antibody dilutions

  • Consider using TBS-T with 0.1% Tween-20 for washing and antibody dilution

Problem 5: Inconsistent immunoprecipitation efficiency

• Solution:

  • Standardize input protein amounts (70-1000 μg recommended)

  • Use fresh tissue/cell lysates

  • Include protease and phosphatase inhibitors

  • Maintain cold temperature throughout the procedure

  • Pre-clear lysates before immunoprecipitation

How should IRAG2 antibodies be validated in the context of phosphorylation studies?

Validating IRAG2 antibodies for phosphorylation studies requires additional considerations beyond standard specificity testing:

Comprehensive validation strategy:

• Phosphorylation-state specificity:

  • Compare phospho-(Ser/Thr) PKA substrate antibody detection before and after phosphatase treatment

  • Include both cGMP-stimulated and unstimulated samples

  • Verify that phosphorylation signal is absent in IRAG2-KO samples

• Phosphorylation site mapping:

  • Consider using mass spectrometry to identify specific phosphorylation sites

  • Generate phospho-site specific antibodies for more precise analyses

  • Use site-directed mutagenesis of potential phosphorylation sites to confirm antibody specificity

• Cross-reactivity assessment:

  • Test for cross-reactivity with related proteins (especially IRAG1)

  • Evaluate detection in tissues with different IRAG1/IRAG2 expression ratios

  • Include appropriate controls (IRAG1-KO, IRAG2-KO, and double-KO if available)

• Kinase specificity validation:

  • Use selective PKG inhibitors to confirm kinase specificity

  • Include PKA inhibitors to rule out cross-reactivity with related phosphorylation events

  • Test alternative cGMP analogs with different PKG activation properties

• Quantification standards:

  • Develop phosphorylation standards for quantitative Western blot

  • Ensure linear detection range for accurate quantification

  • Use total protein normalization rather than single housekeeping protein references

This comprehensive validation approach ensures that phosphorylation-specific signals are truly attributable to IRAG2 and provides confidence in subsequent functional correlation analyses.