SH2D2A Antibody

What is SH2D2A and why is it a significant target for immunological research?

SH2D2A (SH2 Domain Protein 2A), also known as TSAd (T cell-specific adapter protein), VRAP (VEGF receptor-associated protein), or SCAP, is a 43 kDa (calculated) cytoplasmic adapter protein that functions in T cell signaling pathways. Its significance lies in its role as a modulator of T cell activation through interaction with critical kinases.

SH2D2A contains an SH2 domain that enables protein-protein interactions with phosphorylated tyrosine residues. Research has established that SH2D2A interacts with and modulates Lck activity in T cells . As an adaptor protein lacking catalytic activity, it mediates protein-protein interactions through conserved binding domains .

SH2D2A has been identified as a binding partner for multiple signaling molecules, including:

• Lck, a key T cell tyrosine kinase

• Itk/Rlk, important in T cell activation

• MEKK2, involved in MAP kinase signaling

• Grb2, an adapter in multiple signaling pathways

• VEGFR-2, in vascular endothelial cells

Recent research indicates SH2D2A may serve as a favorable prognostic marker in bladder cancer, specifically in a population of SH2D2A+FOXP3+IL2RA high activated Tregs .

How do the different SH2D2A transcript variants impact antibody selection strategy?

Multiple SH2D2A transcript variants exist, necessitating careful antibody selection based on research objectives. Studies have identified at least five SH2D2A transcript variants in CD3-stimulated CD4+ T cells, with SH2D2A-2 and SH2D2A-3 constituting 10-15% each, and SH2D2A-5 comprising 5-10% of total TSAd transcripts .

When selecting antibodies:

• Consider which domain or epitope you need to target. SH2D2A aa239-334 (encoded by exon 7) contains critical regions for protein interactions with Lck and nuclear translocation

• Determine whether you need to detect all isoforms or specific variants

• Examine the antibody's immunogen information (e.g., antibody AF6020 targets Ile279-Gln389 )

Structural-functional analysis reveals three interaction modes between TSAd and Lck:

• TSAd aa239-256 binds to Lck-SH3 domain

• One or more tyrosines within aa239-334 interact with Lck-SH2 domain

• The TSAd-SH2 domain interacts with Lck

Alternative splicing of exon 7 (aa239-334) results in loss of protein interaction motifs and produces truncated TSAd molecules with altered ability to modulate Lck activity .

What are the optimal protocols for detecting SH2D2A in Western blot experiments?

Sample Preparation:

• Prepare cell lysates under reducing conditions using Immunoblot Buffer Group 1

• For detection in T cells, consider activation with anti-CD3/CD28 to upregulate expression

Protocol Parameters:

• Expected molecular weight: The calculated weight is 43 kDa, but observed weights typically range from 45-52 kDa

• Recommended dilutions: 1:500-1:2000 (antibody-dependent)

• Secondary antibody: Use appropriate HRP-conjugated antibody (e.g., HAF016 for sheep primary)

Cell Lines for Positive Controls:

• Jurkat (human acute T cell leukemia cell line)

• K-562 cells

• Primary differentiated Th2 and Th17 cells

Detection Technique Variations:
Western blot detection can be performed using standard methods or capillary electrophoresis (Simple Western). For Simple Western, load samples at 0.2 mg/mL and use a 12-230 kDa separation system .

How should researchers optimize immunohistochemistry protocols for SH2D2A detection in tissue samples?

Sample Preparation:

• For paraffin-embedded tissues, antigen retrieval with TE buffer pH 9.0 is suggested

• Alternatively, citrate buffer pH 6.0 may be used for antigen retrieval

Protocol Parameters:

• Recommended dilutions: 1:20-1:200 for IHC applications

• Positive control tissues: Human heart tissue has shown positive detection

Optimization Steps:

• Test multiple antigen retrieval methods if initial results are weak

• Optimize primary antibody concentration through titration

• Consider signal amplification methods for low-expressing tissues

• Include appropriate negative controls (isotype control or secondary antibody only)

Special Considerations:

• Expression levels may vary between tissues; SH2D2A is primarily expressed in T cells but has been detected in other tissues including heart and bladder cancer specimens

• For cancer tissue studies, consider correlating SH2D2A with other T cell markers (e.g., FOXP3, IL2RA) to identify specific cell populations

How can researchers use SH2D2A antibodies to investigate protein-protein interactions in T cell signaling?

Co-immunoprecipitation (Co-IP) Strategies:
SH2D2A antibodies can be used to pull down protein complexes and analyze binding partners:

• Cell Preparation:

  • Use primary T cells or Jurkat cells

  • Consider both resting and activated states (anti-CD3/CD28 stimulation enhances some interactions)

• Co-IP Protocol:

  • Use approximately 2mg protein for immunoprecipitation

  • Perform Western blot using capillary electrophoresis for high resolution

  • Include cleared lysate as loading control

• Key Interactions to Analyze:

  • Lck: Multiple interaction domains (SH2 and SH3 domains)

  • TIM-3: Demonstrated association in CD8+/MART-1+ T cells

  • VEGFR2: Important for vascular permeability and angiogenesis

Domain-Specific Interaction Analysis:
Research has identified specific regions of SH2D2A critical for protein interactions:

• aa239-256 mediates binding to Lck-SH3 domain

• One or more tyrosines within aa239-334 mediate interaction with Lck-SH2 domain

• The SH2D2A-SH2 domain interacts with Lck

To investigate these interactions, researchers can employ:

• Domain-specific antibodies

• Mutant constructs (e.g., TSAd-4YF, TSAd d239-256)

• Pull-down experiments with GST-fusion proteins

What approaches can researchers use to investigate SH2D2A's role in multiple sclerosis and other autoimmune conditions?

Genetic Association Studies:

• Analyze the polymorphic GA repeat (GA₁₃-GA₃₃) within the SH2D2A promoter region:

  • Short alleles (GA₁₃-₁₆) show increased frequency in MS patients (OR 1.5)

  • The GA₁₆ allele shows increased transmission to MS patients in family studies

• Expression Analysis Protocol:

  • Isolate CD4+ T cells from individuals with different SH2D2A genotypes

  • Activate T cells and measure SH2D2A expression levels

  • T cells homozygous for MS-associated short alleles display lower TSAd levels after activation compared to cells with at least one long allele

Functional Studies:

• T Cell Activation Analysis:

  • Compare SH2D2A-deficient to wild-type T cells

  • Analyze TCR signaling pathways and T cell activation markers

  • Examine cytokine production profiles

• Animal Models:

  • Studies with SH2D2A-deficient mice show they have increased resistance to tumor development in a TCR-transgenic model, suggesting immunomodulatory roles

  • Consider using SH2D2A knockout mice in experimental autoimmune encephalomyelitis (EAE) models to directly test its role in MS-like disease

Biomarker Development:

• Develop flow cytometry panels including SH2D2A to characterize T cell subsets in MS patients

• Correlate SH2D2A expression with disease activity and progression

• Investigate potential therapeutic targeting of SH2D2A pathways

Why might researchers observe variable molecular weights when detecting SH2D2A by Western blot?

SH2D2A has a calculated molecular weight of 43 kDa, but is commonly observed at 45-52 kDa in Western blot experiments . This discrepancy can arise from several factors:

Isoform Variation:

• Multiple transcript variants exist with different molecular weights

• SH2D2A-2 and SH2D2A-3 variants constitute 10-15% each of total TSAd transcripts in stimulated CD4+ T cells

Post-translational Modifications:

• Phosphorylation: SH2D2A undergoes tyrosine phosphorylation during T cell activation

• Different phosphorylation states may cause migration shifts

Technical Factors Affecting Migration:

• Gel percentage and type (gradient vs. fixed)

• Running buffer composition

• Sample preparation conditions (reducing vs. non-reducing)

• Reference protein markers used

Troubleshooting Strategies:

• Use positive control lysates from cells known to express SH2D2A (e.g., Jurkat, K-562, activated T cells)

• Compare results using different antibodies targeting distinct epitopes

• Consider phosphatase treatment to eliminate phosphorylation-induced shifts

• Include careful molecular weight standards

Published observations report:

• ~52 kDa in Jurkat cells (Western blot)

• ~58 kDa in Th2 cells (Simple Western)

• 45-50 kDa observed molecular weight range

What controls and validation methods are essential when using SH2D2A antibodies?

Positive Controls:

• Cell lines: Jurkat, K-562, human primary differentiated Th2/Th17 cells

• Activation conditions: Treat T cells with anti-CD3 (1 μg/mL) and anti-CD28 (5 μg/mL) for 120 minutes to enhance expression

• Tissues: Human heart tissue has shown positive staining in IHC

Negative Controls:

• Isotype controls matching the primary antibody host species

• Secondary antibody-only controls

• Non-expressing cell lines or tissues

• SH2D2A knockout or knockdown samples when available

Antibody Validation Methods:

• Knockdown/Knockout Validation:

  • RNAi knockdown of SH2D2A in human cells

  • Use of SH2D2A-deficient mouse models

• Immunogen Blocking:

  • Pre-incubate antibody with immunizing peptide/protein

  • Observe elimination of specific signal

• Multi-antibody Verification:

  • Compare results using antibodies targeting different epitopes

  • Confirm consistent expression patterns across antibodies

• Application-specific Validations:

  • For WB: Verify correct molecular weight

  • For IHC/ICC: Include proper tissue/cell controls with known expression

  • For Flow Cytometry: Compare with transcript data in sorted populations

Recommended Dilution Optimization:
Each laboratory should determine optimal dilutions for specific applications:

• WB: 1:500-1:2000

• IHC: 1:20-1:200

• Flow Cytometry: 0.40 μg per 10^6 cells in 100 μl suspension

How can SH2D2A antibodies be utilized in cancer immunology research?

Recent research has identified SH2D2A as a favorable prognostic marker in bladder cancer (BLCA), opening new avenues for cancer immunology research :

Methodology for Cancer Tissue Analysis:

• Multiplex Immunohistochemistry:

  • Co-stain for SH2D2A together with immune cell markers (FOXP3, IL2RA)

  • Quantify SH2D2A+ cells in tumor microenvironment

  • Correlate with clinical outcomes

• Single-cell RNA-seq Integration:

  • Identify SH2D2A-expressing cell populations in tumor tissues

  • Compare with antibody-based detection methods

  • Characterize transcriptional programs in SH2D2A+ cells

Key Research Questions to Address:

• How does SH2D2A expression in Tregs influence their function in the tumor microenvironment?

• Does SH2D2A expression correlate with response to immune checkpoint inhibitors?

• Can SH2D2A serve as a biomarker for patient stratification in immunotherapy?

Experimental Approaches:

• Patient Sample Analysis:

  • Compare SH2D2A expression in responders vs. non-responders to immunotherapy

  • Correlate with other known immune markers and checkpoint molecules

• Functional Studies:

  • Isolate SH2D2A+ Tregs from tumor samples using antibody-based methods

  • Characterize their suppressive capacity compared to SH2D2A- Tregs

  • Investigate mechanisms behind the favorable prognostic effect

What considerations should researchers take when using SH2D2A antibodies to study its role in vascular biology?

SH2D2A (also known as VRAP - VEGF receptor-associated protein) plays important roles in vascular biology through interaction with VEGFR2 . When investigating these processes:

Experimental Design Considerations:

• Cell Type Selection:

  • Endothelial cells (primary HUVECs or cell lines)

  • Co-culture systems with T cells and endothelial cells

• Stimulation Conditions:

  • VEGF treatment to trigger VEGFR2 signaling

  • Time course analysis of SH2D2A phosphorylation and localization

• Functional Readouts:

  • Actin polymerization assays

  • Cell migration assays

  • Vascular permeability measurements

  • Angiogenesis assays (tube formation)

Interaction Analysis Methods:

• Co-immunoprecipitation Studies:

  • Precipitate with anti-VEGFR2 antibodies and probe for SH2D2A

  • Precipitate with anti-SH2D2A antibodies and probe for VEGFR2

  • Analyze phosphorylation status using phospho-specific antibodies

• Signaling Pathway Investigation:

  • Focus on PI3K/Akt pathway activation, which requires SH2D2A for VEGF-A-dependent activation

  • Analyze c-Src signaling, which is induced by VEGFR2 via the SH2D2A adaptor protein

Published Research Findings:

• SH2D2A (TSAd) mediates VEGFR2-induced c-Src signaling and vascular permeability in vivo

• SH2D2A is essential for VEGF-A-dependent activation of PI3K/Akt

• SH2D2A promotes actin polymerization and migration in endothelial cells