SH2D3A Antibody

What is SH2D3A and what functional roles does it play in cellular signaling pathways?

SH2D3A (SH2 domain containing 3A), also known as Novel SH2-containing protein 1 (NSP1), is a 576 amino acid protein that plays a significant role in JNK activation within cellular signaling networks. The protein contains one Src homology 2 (SH2) domain, which binds to tyrosine-phosphorylated regions of target proteins, frequently linking activated growth factors to putative signal transduction proteins .

SH2D3A interacts with p130 Cas and is expressed at low levels in fetal kidney, fetal lung, placenta, adult pancreas, kidney and lung . It undergoes post-translational phosphorylation on multiple tyrosine residues, which is crucial for its function . Recent research has also implicated SH2D3A in the JAK1/STAT3 signaling pathway, as knockdown experiments demonstrated decreased levels of JAK1 and STAT3 proteins .

The predicted molecular weight of SH2D3A is 63.1 kDa, though it typically appears around 60 kDa on western blots . This information is essential for researchers to accurately identify the protein in experimental samples.

What applications are most suitable for SH2D3A antibody usage in experimental research?

SH2D3A antibodies have been validated for multiple applications in molecular and cellular research:

The choice of application should be guided by the specific research question, with consideration for the advantages and limitations of each technique. For protein quantification, western blotting or ELISA are preferred, while localization studies benefit from immunohistochemistry or immunofluorescence approaches .

How should researchers select the appropriate SH2D3A antibody for their specific experimental needs?

When selecting an SH2D3A antibody, researchers should consider several critical factors to ensure optimal experimental outcomes:

• Target epitope location: Different antibodies recognize different regions of SH2D3A:

  • Internal region near C-terminus (e.g., peptide sequence APRAERFEKFQR)

  • N-terminal regions

  • Specific amino acid stretches (AA 460-575, AA 1-576, AA 211-231, etc.)

• Host species and clonality:

  • Goat polyclonal antibodies

  • Rabbit polyclonal antibodies

  • Mouse monoclonal antibodies

  The choice affects compatibility with other antibodies in multi-labeling experiments and available secondary detection systems.

• Validated applications: Ensure the antibody has been specifically validated for your intended application (WB, IHC, IF, ELISA) .

• Purification method: Many quality SH2D3A antibodies are purified by ammonium sulfate precipitation followed by antigen affinity chromatography using the immunizing peptide .

• Storage conditions: Most SH2D3A antibodies should be stored at -20°C, with recommendations to avoid freeze/thaw cycles .

For critical experiments, it may be advisable to test multiple antibodies targeting different epitopes to confirm results and ensure specificity.

What is the role of SH2D3A in cancer development, particularly in relation to HPV infection?

Recent research published in 2024 has revealed significant insights into SH2D3A's role in cervical cancer and its relationship with human papillomavirus (HPV) infection :

• Expression profile: SH2D3A expression is significantly elevated in cervical cancer tissues compared to normal tissues, suggesting a potential oncogenic role .

• Functional significance:

  • Silencing SH2D3A in cervical cancer cell lines (SiHa and HeLa) inhibited cell proliferation and invasion

  • SH2D3A knockdown induced apoptosis

  • In vivo studies showed reduced tumorigenesis in nude mice with SH2D3A-silenced cells

• Regulatory mechanism - HPV E7/miR-143-3p/SH2D3A pathway:

  • SH2D3A was found to be regulated by miR-143-3p, confirmed through bioinformatics prediction and luciferase reporter assays

  • HPV E7 silencing led to decreased SH2D3A mRNA levels and increased miR-143-3p levels

  • This suggests a pathway where HPV E7 suppresses miR-143-3p, which normally inhibits SH2D3A

  • In HPV-infected cells, this leads to elevated SH2D3A levels

• Downstream signaling: SH2D3A knockdown decreased levels of JAK1 and STAT3 proteins, suggesting that SH2D3A promotes cervical cancer progression partly through the JAK/STAT pathway .

These findings establish a novel HPV E7/miR-143-3p/SH2D3A/JAK-STAT regulatory axis in cervical cancer, highlighting SH2D3A as both a potential biomarker and therapeutic target for HPV-associated cancers .

How can researchers effectively study the interaction between SH2D3A and p130 Cas using antibody-based approaches?

The interaction between SH2D3A and p130 Cas is a critical aspect of SH2D3A function . Here are methodological approaches for studying this interaction:

• Co-immunoprecipitation (Co-IP):

  • Use anti-SH2D3A antibodies to pull down the complex and detect p130 Cas, or vice versa

  • Include appropriate controls (IgG control, lysate input)

  • Buffer considerations: Include phosphatase inhibitors due to SH2D3A's tyrosine phosphorylation sites

• Proximity Ligation Assay (PLA):

  • Use primary antibodies against SH2D3A and p130 Cas from different host species

  • PLA generates fluorescent signals only when the two proteins are in close proximity (<40 nm)

  • Allows visualization of the interaction in situ with subcellular resolution

• Antibody selection considerations:

  • Choose SH2D3A antibodies that recognize epitopes away from the p130 Cas binding site

  • For co-staining experiments, select antibodies from different host species

  • Consider using multiple antibodies targeting different regions of SH2D3A to confirm results

• Experimental variables to test:

  • Tyrosine phosphorylation status (treat with phosphatase inhibitors or kinase activators)

  • Growth factor stimulation (which may modulate the interaction)

  • Cell adhesion status (as p130 Cas is involved in adhesion signaling)

Understanding this interaction may provide insights into how SH2D3A contributes to cellular signaling networks and potentially to disease states such as cancer .

What experimental designs are most effective for analyzing the functional consequences of SH2D3A knockdown or overexpression?

Based on recent research, the following experimental designs are effective for analyzing SH2D3A functional roles:

Knockdown Approaches:

• siRNA/shRNA-mediated silencing:

  • Used successfully in SiHa and HeLa cervical cancer cells

  • Verify knockdown efficiency by western blot using specific SH2D3A antibodies

  • Optimal antibody dilutions: 0.04-0.4 μg/mL or 0.1-0.3 μg/mL

• CRISPR-Cas9 gene editing:

  • For stable knockout models

  • Verify by genomic sequencing and protein absence via western blot

Functional Assays Following Manipulation:

• Proliferation: Cell Counting Kit-8 assay demonstrated reduced proliferation after SH2D3A knockdown in cervical cancer cells

• Apoptosis: Flow cytometry with Annexin V/PI staining showed increased apoptosis following SH2D3A silencing

• Invasion: Transwell assay revealed decreased invasive capacity in SH2D3A-silenced cells

• In vivo tumorigenesis: A transplantation tumor model in nude mice demonstrated reduced tumor growth with SH2D3A-silenced cells

• Signaling pathway analysis:

  • Western blot analysis of JAK1 and STAT3 proteins showed decreased levels after SH2D3A knockdown

  • Recommended antibody dilutions for these downstream targets should be optimized

Control Experiments:

• Rescue experiments (re-expressing SH2D3A to restore phenotype)

• Dose-response relationships with partial knockdowns

• Time-course analysis to distinguish primary from secondary effects

This comprehensive approach allows researchers to establish both the phenotypic consequences of SH2D3A manipulation and the underlying molecular mechanisms .

What is the optimal western blotting protocol for SH2D3A detection?

Based on available research data, here is an optimized western blotting protocol for SH2D3A detection:

Sample Preparation:

• Positive control: Human tonsil lysate (validated control)

• Expected molecular weight: 60-63.1 kDa

• Include phosphatase inhibitors in lysis buffer (SH2D3A is phosphorylated on multiple tyrosine residues)

Protocol Steps:

• Gel selection and protein loading:

  • 8-10% SDS-PAGE gels (optimal for 60-63 kDa proteins)

  • Load 20-50 μg total protein per lane

• Transfer conditions:

  • Transfer to PVDF or nitrocellulose membrane

  • Wet transfer at 100V for 1 hour or 30V overnight

• Blocking:

  • 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute SH2D3A antibody according to manufacturer's recommendation:

    • 0.04-0.4 μg/mL for rabbit polyclonal antibodies

    • 0.1-0.3 μg/mL for goat polyclonal antibodies

  • Incubate overnight at 4°C

• Washing and secondary antibody:

  • Wash 3x for 10 minutes each with TBST

  • Use appropriate species-specific secondary antibody (anti-goat or anti-rabbit)

  • Incubate for 1 hour at room temperature

• Detection:

  • ECL substrate for HRP-conjugated secondaries

  • Digital imaging systems preferred over film for quantification

Troubleshooting Tips:

• For weak signal: Try longer exposure times or increase antibody concentration

• For high background: Increase washing steps or decrease antibody concentration

• For multiple bands: Confirm specificity with positive controls and consider using antibodies targeting different epitopes

What controls are essential when validating SH2D3A antibody specificity?

Rigorous validation of SH2D3A antibody specificity requires the following controls:

Positive Controls:

• Recommended tissue/cell samples:

  • Human tonsil (validated for western blotting)

  • Human brain (validated for immunohistochemistry)

  • Tissues known to express SH2D3A: fetal kidney, fetal lung, placenta, adult pancreas, kidney and lung

• Recombinant protein controls:

  • Full-length SH2D3A protein

  • Peptide fragments containing the specific epitope

Negative Controls:

• Technical negative controls:

  • Omission of primary antibody

  • Isotype control (matched immunoglobulin at same concentration)

  • Pre-adsorption control (antibody pre-incubated with immunizing peptide)

• Biological negative controls:

  • Tissues with very low/no SH2D3A expression

  • SH2D3A knockdown/knockout samples

Multiple Antibody Validation:
Comparing results from antibodies targeting different SH2D3A epitopes:

• Internal region near C-terminus (APRAERFEKFQR)

• Various amino acid stretches (AA 460-575, AA 1-576, AA 211-231)

Western Blot Validation Criteria:

• Single band at expected molecular weight (60-63.1 kDa)

• Band disappearance in knockdown/knockout samples

• Consistent results across multiple antibodies

Prestige Antibodies Additional Validation:
The Prestige Antibodies in search results and have undergone extensive validation:

• IHC tissue array testing on 44 normal human tissues and 20 cancer type tissues

• Protein array testing against 364 human recombinant protein fragments

These comprehensive controls ensure reliable and specific detection of SH2D3A in research applications .

What are effective troubleshooting approaches for optimizing immunohistochemistry with SH2D3A antibodies?

When optimizing immunohistochemistry with SH2D3A antibodies, researchers may encounter several challenges. Here are effective troubleshooting approaches:

Issue: No Signal or Weak Signal

Issue: High Background or Non-specific Staining

Optimization Strategy:

• Antibody titration matrix:

  • Test 3-4 different antibody dilutions

  • Try both overnight 4°C and 1-2 hour room temperature incubations

  • Document results systematically

• Antigen retrieval comparison:

  • Test no retrieval, citrate buffer, and EDTA buffer

  • Compare microwave, pressure cooker, and water bath methods

• Detection system optimization:

  • Compare standard ABC method vs. polymer detection systems

  • Consider signal amplification for weak signals

• Positive control validation:

  • Human brain tissue is recommended as a positive control

  • Expected staining pattern is cytoplasmic

By systematically addressing these factors, researchers can develop a robust protocol for SH2D3A immunohistochemistry .

How can researchers reliably quantify SH2D3A expression levels across different experimental conditions?

Reliable quantification of SH2D3A requires careful methodological considerations across different experimental platforms:

Western Blot Quantification:

• Standardization approach:

  • Use consistent loading controls (β-actin, GAPDH)

  • Include standard curve of positive control lysate (human tonsil recommended)

  • Process experimental and control samples simultaneously

• Imaging and analysis guidelines:

  • Use digital imaging systems with linear dynamic range

  • Avoid saturated signals (verify with exposure series)

  • Normalize SH2D3A band intensity (60-63.1 kDa) to loading controls

  • Analyze with software like ImageJ using consistent measurement parameters

Immunohistochemistry Quantification:

• Scoring parameters:

  • Staining intensity (0-3 scale)

  • Percentage of positive cells

  • H-score calculation: Σ(intensity × % positive cells)

  • Document representative images at consistent magnification

• Automation considerations:

  • Use digital pathology software for unbiased assessment

  • Maintain consistent thresholds across samples

  • Validate automated scoring against expert pathologist evaluation

ELISA Quantification:

• Optimal conditions:

  • Use antibodies specifically validated for ELISA

  • Recommended dilution: 1:16,000

  • Include standard curve and run samples in triplicate

Comparative Analysis Table:

Statistical Considerations:

• Perform at least three independent biological replicates

• Apply appropriate statistical tests (t-test, ANOVA)

• Report both fold-change and p-values

• Consider power analysis for sample size determination

By implementing these rigorous quantification approaches, researchers can generate reliable and reproducible data on SH2D3A expression across different experimental conditions .

What is known about the HPV E7/miR-143-3p/SH2D3A regulatory pathway in cervical cancer?

Recent research has uncovered a novel regulatory pathway involving HPV E7, miR-143-3p, and SH2D3A in cervical cancer development . This pathway represents a significant advance in understanding how HPV infection contributes to oncogenesis.

Key Components of the Regulatory Axis:

• HPV E7 oncoprotein:

  • Silencing HPV E7 leads to decreased SH2D3A mRNA levels

  • HPV E7 silencing results in significantly increased miR-143-3p levels

• miR-143-3p:

  • Bioinformatics analysis predicted binding between miR-143-3p and SH2D3A

  • This binding relationship was confirmed by luciferase reporter assays

  • Functions as a tumor suppressor by negatively regulating SH2D3A expression

• SH2D3A:

  • Expression is significantly elevated in cervical cancer tissues

  • Acts as an oncogene promoting proliferation and invasion

  • Silencing induces apoptosis in cervical cancer cells

Downstream Consequences:
Western blot analysis revealed that SH2D3A knockdown led to decreased levels of JAK1 and STAT3 proteins, suggesting that SH2D3A promotes cervical cancer progression partly through the JAK/STAT pathway .

Experimental Validation Methods:

• qRT-PCR and immunohistochemistry to compare SH2D3A expression in tissues

• SH2D3A knockdown in SiHa and HeLa cells followed by functional assays

• Transplantation tumor model in nude mice

• Luciferase reporter assays to verify miRNA binding

• Western blot analysis of downstream targets

This regulatory axis provides new insights into HPV-mediated carcinogenesis and identifies SH2D3A as a potential therapeutic target in HPV-positive cervical cancers .

How can researchers effectively analyze post-translational modifications of SH2D3A?

SH2D3A undergoes post-translational phosphorylation on multiple tyrosine residues , which likely affects its function in signaling pathways. Here are methodological approaches for analyzing these modifications:

Experimental Approaches:

• Phosphorylation-specific detection:

  • Immunoprecipitation with SH2D3A antibodies followed by anti-phosphotyrosine western blotting

  • Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Lambda phosphatase treatment comparison to confirm phosphorylation

• Mass spectrometry analysis:

  • Immunoprecipitate SH2D3A using validated antibodies (0.1-0.3 μg/mL recommended for IP)

  • Perform tryptic digestion and phosphopeptide enrichment

  • LC-MS/MS analysis to identify specific phosphorylation sites

• Functional analysis of phosphorylation sites:

  • Site-directed mutagenesis of tyrosine residues to phenylalanine

  • Compare wild-type and phospho-mutant SH2D3A effects on downstream signaling

  • Western blotting of JAK1 and STAT3 activation, as these have been identified as downstream targets

Experimental Stimulation Protocols:

• Serum stimulation time-course (0-60 minutes)

• Growth factor treatment (EGF, PDGF, etc.)

• Phosphatase inhibitor treatment

• Tyrosine kinase inhibitors to identify responsible kinases

Sample Preparation Considerations:

• Include phosphatase inhibitors in all buffers

• Process samples rapidly at 4°C

• Consider crosslinking before lysis to preserve transient interactions

By combining these approaches, researchers can generate a comprehensive understanding of how post-translational modifications regulate SH2D3A function in normal physiology and disease contexts .

What are the emerging applications of SH2D3A antibodies in cancer research and diagnostics?

Based on recent findings about SH2D3A's role in cancer, particularly its elevated expression in cervical cancer and its position in the HPV E7/miR-143-3p/SH2D3A/JAK-STAT pathway , several emerging applications for SH2D3A antibodies in cancer research and diagnostics can be identified:

Diagnostic Applications:

• Tissue biomarker development:

  • IHC analysis of tumor biopsies using optimized antibody dilutions (1:20-1:50 or 5 μg/ml )

  • Development of quantitative scoring systems correlating SH2D3A levels with disease progression

  • Integration into multi-marker panels for improved diagnostic accuracy

• Liquid biopsy approaches:

  • Detection of circulating tumor cells expressing SH2D3A

  • ELISA-based quantification of SH2D3A in serum/plasma (using 1:16,000 dilution )

Therapeutic Research Applications:

• Target validation studies:

  • Antibody-based confirmation of SH2D3A knockdown efficiency in preclinical models

  • Correlation of SH2D3A reduction with therapeutic outcomes

• Combination therapy research:

  • Monitoring SH2D3A and JAK/STAT pathway components during treatment

  • Identifying synergistic approaches targeting multiple pathway components

Mechanistic Investigation Approaches:

• Interaction proteomics:

  • Immunoprecipitation with SH2D3A antibodies followed by mass spectrometry

  • Identification of novel binding partners in different cancer contexts

• Pathway analysis:

  • Multi-antibody panels targeting SH2D3A alongside JAK1, STAT3, and other pathway components

  • Correlative analysis of pathway activation in patient samples

As research continues to uncover SH2D3A's roles in different cancer types and signaling pathways, antibody-based detection methods will remain essential tools for both basic research and translational applications .

What methodological advances are needed to improve SH2D3A research tools and techniques?

Despite the availability of several SH2D3A antibodies and research tools, several methodological advances would significantly enhance SH2D3A research:

Antibody Technology Improvements:

• Phospho-specific antibodies:

  • Development of antibodies targeting specific phosphorylated tyrosine residues on SH2D3A

  • These would enable direct monitoring of SH2D3A activation status

  • Current antibodies detect total SH2D3A protein but not specific modifications

• Highly selective monoclonal antibodies:

  • Further development of monoclonal antibodies with defined epitopes

  • Rigorous cross-reactivity testing against related SH2 domain-containing proteins

  • Standardization across research laboratories

Advanced Research Tools:

• Genome editing validation resources:

  • Well-characterized CRISPR knockout cell lines for antibody validation

  • Isogenic cell line panels with controlled SH2D3A expression levels

• Live-cell imaging tools:

  • SH2D3A biosensors for real-time activation monitoring

  • FRET-based approaches to study protein-protein interactions

Standardization Requirements:

• Quantification protocols:

  • Standardized scoring systems for SH2D3A immunohistochemistry

  • Reference materials with defined SH2D3A levels for assay calibration

• Reporting standards:

  • Detailed documentation of antibody validation methods

  • Comprehensive experimental protocols for reproducibility

Research Platform Integration:

• Multi-omics approaches:

  • Integration of antibody-based protein detection with transcriptomics and genomics

  • Systems biology frameworks for contextualizing SH2D3A function