Recombinant Human GRAP Protein: Research Guide & Applications
Recombinant Human GRAP Protein: Research Guide & Applications
Recombinant Human GRAP Protein is a bacterially or recombinantly expressed form of the GRB2-related adaptor protein that plays a critical role in T-cell receptor signaling by coupling activated receptors to downstream RAS pathway components via its SH2 and SH3 domains.
- Product Name
- Recombinant Human GRAP Protein
- SKU
- rp373847
- Protein Family
- GRB2-related adaptor protein (GRAP); contains SH2 and SH3 domains
- Primary Application
- T-cell receptor signaling research, RAS pathway studies, protein–protein interaction assays
- Recommended Storage
- -20 °C or -80 °C; avoid repeated freeze–thaw cycles
- Purity
- >85% as determined by SDS-PAGE (verify with CoA)
Biological Background of GRAP
GRAP (GRB2-Related Adaptor Protein) is a member of the GRB2 family of adaptor proteins and shares a conserved domain architecture consisting of one central SH2 (Src Homology 2) domain flanked by two SH3 (Src Homology 3) domains. This structural arrangement enables GRAP to bridge phosphotyrosine-containing receptors and downstream effectors.
GRAP is predominantly expressed in hematopoietic tissues and plays a key role in T-cell receptor (TCR) and B-cell receptor (BCR) signaling cascades. Upon receptor activation, GRAP couples phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) and scaffold proteins such as SLP-76 and LAT to the RAS–MAPK pathway, promoting downstream gene expression and lymphocyte activation.
Due to its selective expression pattern and role in immune-cell signal transduction, GRAP is a relevant target for studies of lymphocyte development, immune disorders, and potential therapeutic intervention in hematologic malignancies.
Product Specifications & Quality Assurance
Recombinant Human GRAP Protein (SKU: rp373847) is produced using recombinant expression technology to ensure batch-to-batch consistency and high biological relevance. Key specification parameters to verify on the Certificate of Analysis (CoA) include:
- Purity: Typically >85% by SDS-PAGE; confirm with the lot-specific CoA.
- Formulation: Supplied in a carrier buffer optimized for protein stability; refer to the product datasheet for exact composition.
- Endotoxin Level: Determined by LAL assay; confirm acceptable limits with the CoA for cell-based assays.
- Molecular Weight: Verify the expected apparent molecular weight by SDS-PAGE, noting that recombinant tags or glycosylation status may shift apparent mass.
Always request the lot-specific CoA and datasheet upon receipt to confirm that the product meets specifications required for your particular assay format.
Handling, Reconstitution & Storage Guidelines
Proper handling of Recombinant Human GRAP Protein is essential to preserve activity and ensure reproducible results. Follow these best-practice guidelines:
- Upon Receipt: Inspect the vial for integrity. If lyophilized, briefly centrifuge before opening to collect powder at the bottom of the vial.
- Reconstitution: Reconstitute lyophilized protein with the volume of sterile, ultrapure water or reconstitution buffer specified on the datasheet. Allow the solution to equilibrate for 10–15 minutes at room temperature without vortexing; mix gently by pipetting.
- Aliquoting: Prepare single-use aliquots immediately after reconstitution to minimize repeated freeze–thaw cycles, which can denature the protein and reduce activity.
- Storage: Store working aliquots at -20 °C for short-term use (up to 1 month) or at -80 °C for long-term storage. Avoid storing at 4 °C for extended periods.
- Carrier Protein: For dilute solutions (<10 µg/mL), consider adding a carrier protein such as 0.1% BSA to prevent adsorptive losses on tube surfaces.
Always maintain a cold chain during any transfer step and record lot numbers for traceability.
Research Applications & Experimental Protocols
Recombinant Human GRAP Protein is suitable for a range of biochemical and cell-biology experiments. Validated and suggested applications include:
- Protein–Protein Interaction Studies: Use GRAP as bait or prey in pull-down assays, co-immunoprecipitation (Co-IP), or surface plasmon resonance (SPR) experiments to characterize binding partners via its SH2 and SH3 domains.
- Competitive Inhibition Assays: Employ recombinant GRAP to competitively displace endogenous adaptor proteins from signaling complexes and evaluate the functional consequences on downstream MAPK activation.
- Western Blot Standard: Use as a positive control or molecular-weight standard when probing for GRAP expression in cell lysates.
- ELISA Development: Serve as a calibration standard or coating antigen for the development of sandwich ELISA kits targeting GRAP.
- Structural & Biophysical Studies: Suitable for circular dichroism (CD) spectroscopy, thermal shift assays, or crystallography studies aimed at characterizing GRAP domain structure.
For each application, perform a titration experiment to determine the optimal protein concentration. Include appropriate negative controls (e.g., denatured GRAP or irrelevant recombinant protein) to validate specificity.
Troubleshooting Common Issues
If unexpected results are obtained when working with Recombinant Human GRAP Protein, consider the following troubleshooting strategies:
| Observed Issue | Likely Cause | Recommended Action |
|---|---|---|
| Low or absent signal in pull-down | Protein aggregation or denaturation | Check storage conditions; prepare fresh aliquots; reduce freeze–thaw cycles |
| Multiple bands on SDS-PAGE | Protein degradation or incomplete purity | Use fresh protease inhibitors; verify lot CoA purity data |
| Inconsistent activity between lots | Batch variability | Request lot-specific activity data; run parallel internal controls |
| Precipitation upon reconstitution | Incorrect buffer or rapid temperature change | Use recommended buffer; equilibrate at room temperature gradually |
Contact Aladdin Scientific technical support with your lot number and CoA if issues persist after applying these corrective measures.
Frequently asked questions
What is the recommended reconstitution buffer for Recombinant Human GRAP Protein?
The reconstitution buffer and volume are specified on the lot-specific datasheet accompanying the product. In general, sterile ultrapure water or a phosphate-buffered saline (PBS) solution containing a carrier protein such as 0.1% BSA is suitable. Always refer to the product datasheet for exact formulation guidance.
How should Recombinant Human GRAP Protein be stored to maintain maximum activity?
After reconstitution, divide the protein into single-use aliquots and store at -80 °C for long-term preservation, or at -20 °C for short-term use up to one month. Avoid repeated freeze–thaw cycles, as these can lead to protein denaturation and significant loss of biological activity.
What assays are validated for Recombinant Human GRAP Protein (rp373847)?
Recombinant Human GRAP Protein is suitable for protein–protein interaction assays (pull-down, Co-IP, SPR), Western blot as a positive control, ELISA standard, and biophysical characterization studies. Confirm validated applications on the lot-specific datasheet or by contacting Aladdin Scientific technical support.
Is Recombinant Human GRAP Protein suitable for use in cell-based assays?
If using GRAP in cell-based functional assays, the endotoxin level of the preparation is a critical consideration. Verify the endotoxin content on the CoA using the LAL assay result, and ensure levels are below thresholds required for your cell line or primary cell system. Contact Aladdin Scientific for endotoxin-tested lots if needed.
How can I confirm the identity and purity of the received Recombinant Human GRAP Protein?
Upon receipt, request or locate the lot-specific Certificate of Analysis (CoA), which documents SDS-PAGE purity, identity confirmation (e.g., by Western blot using an anti-GRAP antibody), endotoxin levels, and concentration. If you require additional identity confirmation, mass spectrometry or N-terminal sequencing can be performed independently.
References
- Feng GS, Ouyang YB, Hu DP, Shi ZQ, Gentz R, Ni J. GRAP is a novel SH3-SH2-SH3 adaptor protein that couples tyrosine kinases to the Ras pathway. J Biol Chem. 1996;271(21):12129–12132. PMID:8647810
- Trüb T, Frantz JD, Miyazaki M, Band H, Bhatt S. The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling. J Biol Chem. 1997;272(2):894–902. PMID:8995381
- Aladdin Scientific. Recombinant Human GRAP Protein (rp373847) Product Datasheet. Aladdin Scientific; 2024.
Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.
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