SH2D1A Antibody

What is SH2D1A and why is it significant for immunological research?

SH2D1A (also known as SAP or SLAM-Associated Protein) is a small adaptor protein consisting of 128 amino acids with a characteristic SH2 domain. It plays a crucial role in immune signaling by modulating the activity of signaling lymphocyte activation molecules (SLAM) at the interface between T and B cells. SH2D1A is predominantly expressed in T cells and NK cells, where it functions to regulate signal transduction pathways downstream of the SLAM family of surface receptors .

The significance of SH2D1A stems from its central role in:

• T-dependent humoral immune responses

• Germinal center formation

• Regulation of B cell activities resulting in antigen-specific IgG production

• Signal transduction events in immune cell communication

Mutations in the SH2D1A gene are associated with X-linked lymphoproliferative disease (XLP), making it an important target for understanding immunodeficiency disorders .

How do I select the appropriate SH2D1A antibody for my experiment?

Selection of the appropriate SH2D1A antibody depends on several factors:

For applications requiring high specificity, such as investigating specific domains or interacting partners, monoclonal antibodies like clone 1A9 are recommended . For broader detection of potentially modified forms, polyclonal antibodies targeting multiple epitopes may be advantageous .

What are the optimal conditions for SH2D1A antibody validation?

Thorough validation of SH2D1A antibodies is essential for reliable research results:

• Positive and negative controls:

  • Positive: Cell lines with known SH2D1A expression (T cells, NK cells)

  • Negative: B cell lines (SH2D1A is absent in most B cells except germinal center B cells)

  • Knockout/knockdown validation: Compare wild-type vs. SH2D1A-/- samples

• Validation across multiple applications:

  • Western blot: Confirm 14 kDa band with proper controls

  • Immunoprecipitation: Verify pull-down of known interaction partners

  • Flow cytometry: Compare with isotype controls and known expression patterns

• Cross-reactivity assessment:

  • Test against related SH2-domain containing proteins to ensure specificity

  • Verify reactivity against recombinant protein standards

Validation should always include concentration optimization. For Western blotting, the recommended starting dilution is 1:1000, but this should be titrated for your specific experimental system .

How can SH2D1A antibodies be used to investigate T-dependent humoral immune responses?

SH2D1A plays a critical role in T-dependent humoral immune responses as demonstrated by research showing that SH2D1A-deficient mice have severely impaired primary and secondary responses of all Ig subclasses . When designing experiments to investigate this:

• Experimental approach:

  • Immunohistochemistry of lymphoid tissues to track germinal center formation using anti-SH2D1A antibodies (dilution 1:50-1:200)

  • Flow cytometry to identify SH2D1A expression in different lymphocyte populations during immune responses

  • Co-immunoprecipitation to study SH2D1A interactions with SLAM family receptors during T-B cell collaboration

• Key considerations:

  • Time course experiments are essential as SH2D1A's role changes during primary versus memory responses

  • Combine SH2D1A antibodies with markers for germinal centers (e.g., GL7, PNA)

  • Include analysis of both early (IgM) and late (class-switched IgG) antibody responses

Research by Crotty et al. demonstrated that "both primary and secondary responses of all Ig subclasses are severely impaired in SH2D1A-/- mice" in response to specific antigens . This phenotype is associated with defective germinal center formation, suggesting that SH2D1A antibodies can be valuable tools for studying the mechanisms of T-dependent antibody responses and germinal center dynamics.

What are the technical challenges in using SH2D1A antibodies for detection in tissue sections?

Detection of SH2D1A in tissue sections presents several technical challenges:

• Low expression levels:

  • SH2D1A is expressed at relatively low levels in physiological conditions

  • Solution: Use signal amplification methods such as tyramide signal amplification

  • Recommended antibody concentration: 2-5 µg/ml for immunohistochemistry with amplification

• Cell-type specificity:

  • SH2D1A expression is limited to specific immune cell subsets (T cells, NK cells, some germinal center B cells)

  • Solution: Use dual or triple immunofluorescence with lineage markers (CD3, CD56, CD19)

  • Critical control: Include SH2D1A-deficient tissues to confirm specificity

• Fixation sensitivity:

  • The SH2D1A epitope can be sensitive to overfixation

  • Recommended protocol: 4% paraformaldehyde fixation for 12-24 hours, followed by antigen retrieval (citrate buffer pH 6.0)

  • Test multiple antibody clones targeting different epitopes if signal is weak

• Validation strategy:

  • Compare staining patterns with in situ hybridization for SH2D1A mRNA

  • Confirm cellular localization (primarily cytoplasmic with some nuclear localization)

  • Use SH2D1A knockout tissues or siRNA knockdown samples as negative controls

Studies have shown that SH2D1A is detectable in germinal center B cells, which was unexpected based on earlier research that suggested B cells lacked SH2D1A expression. This finding highlights the importance of rigorous controls when studying tissue-specific expression patterns .

How do post-translational modifications affect SH2D1A antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of SH2D1A:

• Phosphorylation effects:

  • SH2D1A contains serine/threonine phosphorylation sites that can affect antibody binding

  • Antibodies targeting regions containing phosphorylation sites may show differential binding depending on the phosphorylation state

  • For phosphorylation-independent detection, select antibodies targeting non-modified regions

• Epitope-specific considerations:

  • C-terminal antibodies (AA 85-114) may be less affected by N-terminal modifications

  • Antibodies targeting the SH2 domain may be sensitive to conformational changes induced by protein-protein interactions

• Experimental recommendations:

  • When studying PTMs, use multiple antibodies targeting different epitopes

  • Include phosphatase treatment controls when appropriate

  • Consider using specialized PTM-specific antibodies if studying particular modifications

• Sample preparation protocol:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation state

  • Avoid freeze-thaw cycles that may affect protein conformation and epitope accessibility

  • Use reducing conditions for Western blotting to maximize epitope exposure

Research indicates that the interaction between SH2D1A and SLAM family receptors can be regulated by phosphorylation events, highlighting the importance of considering PTMs when selecting antibodies for studying these interactions .

How can I optimize Western blotting protocols for detecting endogenous SH2D1A?

Optimizing Western blotting for endogenous SH2D1A detection requires addressing several technical aspects:

• Sample preparation optimizations:

  • Use T cell or NK cell-rich samples (PBMCs, thymus, spleen)

  • Lysis buffer recommendation: RIPA buffer with protease inhibitors

  • Protein concentration: Load 30-50 μg of total protein per lane for optimal detection

• Gel/blotting conditions:

  • Use 15% or gradient (4-20%) polyacrylamide gels to effectively resolve the small 14 kDa protein

  • Transfer conditions: 100V for 1 hour or 30V overnight using 0.2 μm PVDF membrane (preferred over nitrocellulose for small proteins)

• Antibody incubation:

  • Primary antibody dilution: Start with 1:1000 and optimize as needed

  • Incubation: Overnight at 4°C with gentle agitation

  • Blocking: 5% non-fat dry milk or BSA in TBST (1 hour at room temperature)

• Detection considerations:

  • Enhanced chemiluminescence (ECL) with longer exposure times may be necessary

  • Consider using signal enhancers for low abundance detection

  • Secondary antibody recommendation: HRP-conjugated, highly cross-adsorbed at 1:5000 dilution

• Critical controls:

  • Positive control: Jurkat cell lysate (T cell line with known SH2D1A expression)

  • Negative control: SH2D1A-knockout samples or B cell lines (most B cells don't express SH2D1A)

  • Size verification: Recombinant SH2D1A protein run alongside samples

Based on published protocols, most optimized Western blotting methods can detect endogenous SH2D1A when the protein is run under reducing conditions and transferred to PVDF membranes .

What are the best approaches for using SH2D1A antibodies in immunoprecipitation studies?

Immunoprecipitation (IP) of SH2D1A presents unique challenges due to its small size and involvement in multiple protein complexes:

• Antibody selection criteria:

  • Use antibodies specifically validated for IP applications

  • Recommended dilution: 1:50 or approximately 2-4 μg antibody per 500 μg total protein

  • Consider using agarose-conjugated antibodies for direct precipitation

• Lysis conditions optimization:

  • Gentler lysis buffers (1% NP-40 or 0.5% Triton X-100) better preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation-dependent interactions

  • Pre-clear lysates thoroughly to reduce background

• Co-IP considerations:

  • When studying SH2D1A interactions with SLAM family receptors, crosslinking may be necessary

  • Use DSP (dithiobis(succinimidyl propionate)) at 1-2 mM for reversible crosslinking

  • Analyze both SH2D1A and interacting proteins by Western blot

• Elution strategies:

  • For SH2D1A protein complexes: Gentle elution with low pH glycine buffer (0.1 M, pH 2.5)

  • For subsequent mass spectrometry: Elution with SDS sample buffer without reducing agent

  • For phosphorylation studies: Include phosphatase inhibitors throughout procedure

• Validation approaches:

  • Reciprocal IP (pull down the partner and probe for SH2D1A)

  • Sequential IP to verify multi-protein complexes

  • Compare results from different antibody clones to confirm specificity

Recent research has successfully used anti-SH2D1A antibodies to immunoprecipitate complexes containing SLAM family receptors, demonstrating the utility of this approach for studying SH2D1A-mediated signaling networks .

Why might I observe discrepancies in SH2D1A detection between different experimental platforms?

Researchers frequently encounter discrepancies in SH2D1A detection across different experimental platforms. Understanding these variations is critical for accurate data interpretation:

• Epitope accessibility variations:

  • In flow cytometry: Fixation and permeabilization methods can differentially expose epitopes

  • In Western blotting: Reducing vs. non-reducing conditions affect protein conformation

  • In immunohistochemistry: Different fixatives and antigen retrieval methods expose different epitopes

• Expression level thresholds:

  • Flow cytometry may detect cellular SH2D1A that is below Western blot detection limits

  • Western blotting provides population averages, whereas flow cytometry reveals cell-to-cell variability

  • RT-PCR may detect mRNA in cells where protein is below detection threshold

• Methodological resolution differences:

  Method	Sensitivity	Cell-level Resolution	Protein Modification Info
  Western Blot	Moderate	No (population average)	Size-based information
  Flow Cytometry	High	Yes (single-cell)	Limited
  Immunohistochemistry	Moderate	Yes (in tissue context)	Limited
  Mass Spectrometry	Very High	No	Comprehensive

• Technical recommendations:

  • Always validate findings across multiple platforms

  • Use multiple antibodies targeting different epitopes

  • Include appropriate positive and negative controls for each method

  • Consider that true biological variation may exist between intact cells vs. lysates

Studies have shown that SH2D1A expression in germinal center B cells was initially missed using flow cytometry but was later detected using more sensitive immunohistochemistry techniques with optimized antibodies . This highlights how methodological differences can lead to apparently contradictory results.

How can SH2D1A antibodies be utilized to study X-linked lymphoproliferative disease mechanisms?

X-linked lymphoproliferative disease (XLP) is caused by mutations in the SH2D1A gene. SH2D1A antibodies offer powerful tools for investigating disease mechanisms:

• Patient sample analysis approaches:

  • Western blotting to assess SH2D1A protein expression levels in patient vs. control samples

  • Flow cytometry to analyze cellular distribution and expression patterns

  • Immunohistochemistry to examine lymphoid tissue architecture and germinal center formation

• Mutation impact assessment:

  • Use multiple antibodies targeting different epitopes to determine if mutations affect protein expression or just function

  • Co-immunoprecipitation studies to determine how mutations alter interactions with SLAM family receptors

  • Combine with functional assays to correlate protein expression with cellular defects

• Experimental models:

  • Compare antibody staining patterns between wild-type and SH2D1A-deficient mouse models

  • Use antibodies to validate gene-edited cell lines modeling specific patient mutations

  • Track SH2D1A expression during EBV infection in relevant cell types

• Clinical research applications:

  • Diagnostic immunophenotyping using flow cytometry (1:400 dilution recommended)

  • Monitoring response to experimental therapies targeting SH2D1A-related pathways

  • Correlating SH2D1A expression patterns with clinical outcomes

Research has shown that "more than half of patients with X-linked lymphoproliferative disease suffer from an extreme susceptibility to Epstein-Barr virus," while "one-third of these patients develop dysgammaglobulinemia without an episode of severe mononucleosis" . These different disease presentations can be studied using SH2D1A antibodies to characterize specific cellular and molecular defects.

What are the considerations when using SH2D1A antibodies to study germinal center formation?

SH2D1A plays a critical role in germinal center formation, and antibodies against this protein are valuable tools for studying this process:

• Experimental design considerations:

  • Time course analysis is critical: examine days 7, 14, and 21 post-immunization

  • Use multicolor approaches combining SH2D1A with germinal center markers (GL7, PNA)

  • Compare primary vs. secondary immune responses to assess memory formation

• Technical approach:

  • Immunohistochemistry protocol: 5 μm frozen sections, acetone fixation (10 minutes), blocking with 5% serum

  • Flow cytometry: Fix with 2% paraformaldehyde, permeabilize with 0.1% saponin

  • Confocal microscopy: Use Z-stack imaging to visualize the 3D organization of germinal centers

• Data interpretation challenges:

  • SH2D1A expression varies between different germinal center zones (light vs. dark)

  • Expression levels change during germinal center maturation

  • Both T cells and a subset of B cells express SH2D1A within germinal centers

• Controls and validation:

  • Include germinal center B cell identification (CD19+GL7+)

  • Compare SH2D1A-deficient and wild-type tissues

  • Use isotype controls for background determination

Research demonstrates that "germinal centers were absent in SH2D1A-/- mice upon primary immunization," and "SH2D1A was detectable in wild-type germinal center B cells" . These findings highlight the importance of SH2D1A in germinal center formation and provide a foundation for experimental approaches using SH2D1A antibodies to study this process.

How can SH2D1A antibodies be used in studies of autoimmune diseases?

SH2D1A has been implicated in autoimmune disease pathogenesis, particularly in antibody-mediated conditions:

• Experimental approaches for autoimmunity research:

  • Flow cytometric analysis of SH2D1A expression in patient vs. healthy immune cell subsets

  • Immunohistochemistry of autoimmune target tissues and associated lymphoid structures

  • Correlation of SH2D1A expression with autoantibody levels and disease severity

• Specific autoimmune disease applications:

  • Systemic lupus erythematosus (SLE): "SH2D1A deficiency protects mice from an experimental model of lupus"

  • Study SLAM-SH2D1A pathway in germinal center responses during autoimmunity

  • Investigate SH2D1A's role in breaking B cell tolerance

• Technical protocol recommendations:

  • For SLE studies: Compare SH2D1A expression in germinal centers from lupus models vs. controls

  • Flow cytometry panel: Include SH2D1A (1:400 dilution) with markers for T follicular helper cells (CXCR5, PD-1) and germinal center B cells (GL7, Fas)

  • Western blotting: Compare SH2D1A levels in B cells from autoimmune vs. healthy subjects

• Translational research considerations:

  • Target validation: Use anti-SH2D1A antibodies to monitor pathway activity during experimental treatments

  • Biomarker potential: Evaluate whether SH2D1A expression patterns correlate with disease activity or treatment response

  • Therapeutic development: Study SH2D1A-SLAM interactions as potential intervention targets

Research has demonstrated that "deficiency in SH2D1A protects mice from an experimental model of lupus, including the development of hypergammaglobulinemia, autoantibodies including anti-double stranded DNA, and renal disease" . These findings highlight the potential importance of SH2D1A as a target in autoimmune disease research.

What emerging technologies might enhance SH2D1A antibody applications in research?

Several emerging technologies are poised to revolutionize how SH2D1A antibodies are used in research:

• Single-cell approaches:

  • Single-cell Western blotting for heterogeneity analysis of SH2D1A expression

  • Mass cytometry (CyTOF) using metal-conjugated SH2D1A antibodies for high-dimensional analysis

  • Spatial transcriptomics combined with SH2D1A immunostaining for tissue context understanding

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, STED) to visualize nanoscale SH2D1A-SLAM interactions

  • Intravital multiphoton microscopy with fluorescently labeled SH2D1A antibodies to track dynamics in vivo

  • Expansion microscopy to better resolve SH2D1A distribution in complex tissues

• Proximity labeling approaches:

  • BioID or APEX2 fusions with SH2D1A to map the complete interactome

  • Proximity ligation assays using SH2D1A antibodies to visualize protein-protein interactions in situ

  • Split-protein complementation assays to study dynamic interaction partners

• Therapeutic antibody development:

  • Modified anti-SH2D1A antibodies that can modulate SLAM-SH2D1A interactions

  • Antibody-drug conjugates for targeting SH2D1A-expressing cells in lymphoproliferative disorders

  • Intrabodies to modify SH2D1A function in specific cellular compartments

These technologies will enable researchers to move beyond merely detecting SH2D1A to understanding its dynamic functions in complex immune responses and disease processes.

How can SH2D1A antibodies contribute to understanding the interplay between innate and adaptive immunity?

SH2D1A functions at the intersection of innate and adaptive immunity, making antibodies against this protein valuable tools for studying this interface:

• Research approaches:

  • Multi-color flow cytometry to examine SH2D1A expression in innate lymphoid cells vs. adaptive T cells

  • Sequential immunoprecipitation to identify novel SH2D1A-containing complexes in different immune cell types

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify transcriptional networks regulated by SH2D1A-dependent signaling

• Key biological questions addressable with SH2D1A antibodies:

  • How does SH2D1A regulate NK cell education vs. T cell activation?

  • What role does SH2D1A play in innate-like T cells (NKT, MAIT, γδ T cells)?

  • How do SLAM family receptors differentially engage SH2D1A in innate vs. adaptive cells?

• Experimental systems:

  • Human and mouse comparative studies using cross-reactive antibodies

  • Ex vivo infection models to track SH2D1A during pathogen encounters

  • In vitro co-culture systems to study SH2D1A-dependent cell-cell communication

Research has shown that SH2D1A is expressed in both T cells and NK cells, indicating its importance at the innate-adaptive interface . Further studies using SH2D1A antibodies could reveal how this adaptor protein coordinates responses between different arms of the immune system, particularly in the context of infections such as EBV where both innate and adaptive responses are critical.