GRAP2 Antibody

What is GRAP2 and why is it important in immunological research?

GRAP2 (GRB2-related adaptor protein 2) is a member of the GRB2/Sem5/Drk family functioning as an adaptor protein involved in leukocyte-specific protein-tyrosine kinase signaling. Its structural organization includes a central SH2 domain flanked by two SH3 domains, with a unique 120-amino acid glutamine/proline rich sequence not found in GRB2 or GRAP .

GRAP2 is primarily expressed in lymphoid tissues and hematopoietic cells, particularly T cells, where it plays a pivotal role during early events of T cell signal transduction . Mechanistically, GRAP2:

• Interacts with SLP-76 to regulate NF-AT (nuclear factor of activated T cells) activation

• Forms signaling complexes with HPK1 to mediate the JNK signaling pathway

• Recruits adaptor protein SLP-76 and its associated molecules (Vav, Nck, Itk, ADAP) to the transmembrane adaptor protein LAT

• Interacts with M-CSF receptor and the activated T cell co-stimulatory receptor CD28

• Binds to tyrosine-phosphorylated shc

• Associates with other signaling proteins including Gab2, HPK1, and Cbl

Recent studies have identified GRAP2 as a potential prognostic biomarker in certain cancers, making it increasingly relevant to both immunological and oncological research .

What applications are most effective for GRAP2 antibody detection in research protocols?

GRAP2 antibodies have been validated for multiple applications, with varying effectiveness depending on the specific antibody clone and experimental conditions:

For optimal results:

• Western blotting is most widely validated and shows consistent results across different antibodies

• Flow cytometry requires specific conjugated antibodies for best results

• IHC applications benefit from antigen retrieval optimization

• Cross-validate findings using at least two different detection methods

How can researchers validate the specificity of GRAP2 antibodies for experimental design?

Validating antibody specificity is crucial for obtaining reliable results. For GRAP2 antibodies, several validation strategies are recommended:

Orthogonal Validation

Compare protein expression using antibody-based and antibody-independent methods:

• Correlate antibody detection with mRNA expression data from RT-PCR or RNA-seq

• Compare results with mass spectrometry-based protein detection

Independent Antibody Validation

• Use multiple antibodies targeting different epitopes of GRAP2

• Compare staining patterns between antibodies recognizing different regions (e.g., N-terminal vs. C-terminal)

• Antibodies targeting different domains (SH2 vs. SH3) should show similar expression patterns

Genetic Validation

• Use CRISPR/Cas9 knockout or siRNA knockdown of GRAP2

• Absence of signal in knockout samples confirms specificity

Recombinant Expression Validation

• Overexpress tagged GRAP2 in cell lines with low endogenous expression

• Confirm co-localization of anti-GRAP2 antibody with tag-specific antibodies

RNA Similarity Scoring

As described in the Enhanced Validation methodology, compare IHC staining pattern with RNA expression levels to determine consistency scores :

• High consistency: Strong correlation between antibody staining and RNA expression

• Medium consistency: General agreement with some discrepancies

• Low consistency: Limited correlation requiring additional validation

What are the optimal protocols for detecting GRAP2 in different cell and tissue types?

The detection of GRAP2 requires tailored approaches for different biological samples:

Hematopoietic Cells and Lymphoid Tissues

• Flow Cytometry (Preferred): Use PE or APC-conjugated anti-GRAP2 antibodies for intracellular staining

  • Sample: Human peripheral whole blood

  • Protocol: Fix with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • Dilution: 1.7 μg/ml (for specific antibodies like ab278056)

  • Results: Successfully detects GRAP2 in T cells and monocytes

Cancer Cell Lines

• Western Blot:

  • Validated cell lines: K-562, MOLT-4 (human), CTLL-2 (mouse)

  • Lysis buffer: RIPA with protease/phosphatase inhibitors

  • Expected band: 38-39 kDa

  • Reducing conditions recommended

Solid Tumor Tissues

• IHC on Paraffin Sections:

  • Pre-treatment: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Primary antibody incubation: Overnight at 4°C

  • Detection: HRP-polymer and DAB visualization

  • Counterstain: Hematoxylin

Non-lymphoid Tissues

GRAP2 expression is generally lower in non-lymphoid tissues, requiring:

• Enhanced signal amplification techniques

• Longer primary antibody incubation times

• More sensitive detection systems (e.g., TSA amplification)

For lung adenocarcinoma specifically, IHC has confirmed lower GRAP2 protein expression in tumor tissues compared to adjacent normal tissues .

How does GRAP2 expression correlate with immune infiltration in cancer?

Research has revealed significant relationships between GRAP2 expression and immune infiltration in cancer:

Immune Infiltration Correlation

• GRAP2 expression positively correlates with infiltration levels of multiple immune cell types in tumors

• A hub gene set of 91 genes coexpressed with GRAP2 was found to be closely related to immune response in LUAD

• GRAP2 expression positively correlates with multiple immune markers, chemokines, chemokine receptors, and MHC molecules

Methodological Approach for Studying This Correlation

• Analyze GRAP2 expression using RNA-seq or microarray data from cancer databases (TCGA, GEO)

• Quantify immune cell infiltration using computational approaches (TIMER, CIBERSORT)

• Perform correlation analysis between GRAP2 expression and immune infiltration metrics

• Validate findings using multiplex immunofluorescence or immunohistochemistry

• Conduct functional studies to elucidate mechanistic relationships

These findings suggest GRAP2 could serve as a biomarker for assessing both prognosis and immune infiltration levels in certain cancers .

What are the challenges in detecting post-translational modifications of GRAP2?

Detecting post-translational modifications (PTMs) of GRAP2 presents several technical challenges:

Known PTMs and Their Significance

• GRAP2 can undergo phosphorylation at specific tyrosine residues

• PTMs may alter protein-protein interactions, particularly with binding partners like SLP-76 and LAT

• Modified GRAP2 can generate autoantibodies in certain conditions

Technical Challenges

• Limited PTM-specific antibodies: Few commercial antibodies specifically target modified forms of GRAP2

• Low abundance of modified forms: PTM-bearing GRAP2 may represent a small fraction of total GRAP2

• Labile modifications: Some PTMs may be lost during sample preparation

• Background interference: Cross-reactivity with other modified proteins may occur

Recommended Detection Approaches

• Phospho-specific antibodies: When available, use antibodies specifically targeting phosphorylated residues

• Mass spectrometry:

  • Immunoprecipitate GRAP2 followed by LC-MS/MS analysis

  • Use neutral loss scanning to detect phosphorylation events

  • Apply electron transfer dissociation (ETD) for improved PTM site identification

• Phos-tag SDS-PAGE: For detecting phosphorylated GRAP2 variants with mobility shift

• 2D gel electrophoresis: To separate GRAP2 isoforms with different PTMs

Validation Strategies

• Use phosphatase treatment as negative control for phosphorylation

• Compare stimulated vs. unstimulated cells to identify inducible modifications

• Express mutant GRAP2 (with modified PTM sites) as controls

What are the recommended storage and handling conditions for maximizing GRAP2 antibody stability?

Proper storage and handling of GRAP2 antibodies is critical for maintaining their activity and specificity:

Storage Conditions

Buffer Composition

Most GRAP2 antibodies are supplied in:

• PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• PBS without Mg²⁺ and Ca²⁺, pH 7.4, with 150 mM NaCl, 0.02% sodium azide, 50% glycerol

Handling Recommendations

• Avoid repeated freeze/thaw cycles (more than 3-5 cycles significantly reduces activity)

• For -20°C storage of larger volumes (>20μL), aliquoting is recommended

• Small volume antibodies (20μL) containing 0.1% BSA can be stored without aliquoting

• Allow antibodies to equilibrate to room temperature before opening

• Briefly centrifuge before use to collect solution at the bottom of the tube

• For conjugated antibodies, protect from light during all handling steps

Dilution and Working Solution Stability

• Diluted antibody working solutions are typically stable for up to 7 days at 2-8°C

• For longer-term storage of working dilutions, add carrier protein (e.g., 1% BSA)

• Sodium azide (0.02%) can be added to prevent microbial growth, but may interfere with some applications (e.g., HRP detection systems)

How do different GRAP2 antibody clones compare in research applications?

Various GRAP2 antibody clones show different performance characteristics across applications:

Monoclonal Antibodies

Polyclonal Antibodies

Performance Comparison

• Specificity: Monoclonal antibodies generally show higher specificity but recognize single epitopes

• Sensitivity: Polyclonal antibodies often provide higher sensitivity by binding multiple epitopes

• Background: Monoclonal antibodies typically generate lower background in immunohistochemistry

• Batch consistency: Monoclonal antibodies offer better lot-to-lot consistency

• Species cross-reactivity: Polyclonal antibodies often have broader species cross-reactivity

For critical experiments, validation using more than one antibody clone is recommended to confirm findings .

How can GRAP2 antibodies be employed in cancer biomarker studies?

GRAP2 has emerging potential as a cancer biomarker, with several methodological approaches for investigation:

Prognostic Value Assessment

• GRAP2 has been identified as a prognostic biomarker in lung adenocarcinoma and cervical cancer

• Lower GRAP2 expression correlates with poorer prognosis in LUAD patients

• Similar findings were confirmed in cervical cancer patients

Methodological Approach

• Tissue Microarray (TMA) Analysis:

  • Collect tumor samples and matched normal tissues

  • Prepare TMA blocks and perform IHC using validated GRAP2 antibodies

  • Score expression levels based on staining intensity and percentage of positive cells

  • Correlate with clinical parameters and survival data

• Transcriptomic Analysis:

  • Analyze GRAP2 mRNA expression from public databases (TCGA, GEO)

  • Correlate expression with clinical parameters using Kaplan-Meier analysis

  • Identify gene sets co-expressed with GRAP2

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation using GRAP2 antibodies

  • Identify interaction partners in normal vs. cancer tissues

  • Investigate altered signaling pathways in cancer context

• Immune Infiltration Correlation:

  • Use multiplexed IHC to simultaneously detect GRAP2 and immune cell markers

  • Quantify immune cell types in relation to GRAP2 expression levels

  • Correlate findings with response to immunotherapy where applicable

Practical Considerations

• Use antibodies with demonstrated specificity in cancer tissues

• Include positive controls (lymphoid tissues) and negative controls

• Consider heterogeneity within tumors by analyzing multiple regions

• Validate findings across independent patient cohorts

• Combine with other established biomarkers for improved prognostic value

What are the methodological considerations when studying GRAP2 in RET signaling pathways?

GRAP2 has been identified as a binding partner of RET receptor tyrosine kinase, with implications for neuroendocrine tumors:

Experimental Design for Studying GRAP2-RET Interactions

• Co-immunoprecipitation:

  • Lysates from neuroendocrine tumor cells or medullary thyroid carcinoma lines

  • Immunoprecipitate with anti-RET or anti-GRAP2 antibodies

  • Western blot to detect interacting proteins

• In vitro Binding Assays:

  • Pull-down experiments using in vitro translated proteins

  • GST-fusion proteins to map interaction domains

• Functional Studies:

  • Overexpression of GRAP2 in RET-positive cell lines

  • Monitoring of RET-induced NF-κB activation

  • Focus formation assays in NIH 3T3 cells expressing oncogenic RET

Key Findings and Methodological Insights

• GRAP2 is expressed in neuroendocrine tumors and cell lines bearing mutated RET

• Endogenous RET and GRAP2 co-immunoprecipitate from medullary thyroid carcinoma cell lines

• GRAP2 inhibits RET-induced NF-κB activation

• GRAP2 overexpression reduces focus formation induced by oncogenic RET

Technical Considerations

• Include proper controls for immunoprecipitation (isotype control antibodies)

• Validate interactions using multiple antibodies targeting different epitopes

• Consider the cell type-specific context, as GRAP2 is predominantly expressed in hematopoietic cells

• Examine the effects of RET activation (ligand stimulation or oncogenic mutations) on GRAP2 binding

• Investigate the role of GRAP2 phosphorylation in mediating RET interactions

These findings suggest GRAP2 may play a tissue-specific role as an inhibitor of RET mitogenic signaling, extending its known functions beyond hematopoietic cell signaling .