Recombinant Human GRB14 Protein: Handling & Usage Protocol

Recombinant Human GRB14 Protein is a growth factor receptor-bound adaptor protein that plays a key regulatory role in insulin and growth factor signaling pathways, making it an essential tool for studies in metabolic disease, receptor tyrosine kinase biology, and signal transduction research.

Product Name

Recombinant Human GRB14 Protein

SKU

rp371614

Protein Family

GRB7 family of adaptor proteins; contains PH, BPS, and SH2 domains

Biological Function

Negative regulator of insulin receptor signaling; binds activated receptor tyrosine kinases via SH2 domain

Recommended Storage

-80°C for long-term storage; avoid repeated freeze-thaw cycles

Primary Applications

Protein interaction studies, ELISA, Western blot, in vitro kinase inhibition assays

Background and Biological Significance of GRB14

GRB14 (Growth Factor Receptor-Bound Protein 14) is a member of the GRB7 family of signal transduction adaptor proteins, which includes GRB7 and GRB10. These proteins share a conserved domain architecture comprising an N-terminal proline-rich region, a Ras-associating (RA)-like domain, a pleckstrin homology (PH) domain, a unique between-PH-and-SH2 (BPS) domain, and a C-terminal SH2 (Src Homology 2) domain.

GRB14 functions primarily as a negative regulator of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) signaling. Upon insulin stimulation, GRB14 binds to the phosphorylated activation loop of the insulin receptor kinase domain via its BPS region, physically occluding substrate access and attenuating downstream signal propagation through pathways such as PI3K/Akt and MAPK/ERK.

Recombinant Human GRB14 Protein is therefore widely utilized in research programs focused on type 2 diabetes, obesity, metabolic syndrome, and cancer biology, where dysregulation of growth factor receptor signaling is a central mechanistic theme.

Reagents, Equipment, and Pre-Use Preparation

Required reagents:

• Recombinant Human GRB14 Protein (rp371614, Aladdin Scientific)
• Sterile, deionized water or appropriate reconstitution buffer (e.g., phosphate-buffered saline, pH 7.4, with 0.1% BSA as carrier protein)
• Low-protein-binding microcentrifuge tubes (1.5 mL)
• Appropriate assay buffers compatible with downstream application

Required equipment:

• Calibrated micropipettes and low-retention tips
• Microcentrifuge (capable of ≥10,000 × g)
• -80°C ultralow-temperature freezer
• Ice bucket with crushed ice for on-bench handling
• UV-Vis spectrophotometer or BCA/Bradford assay kit for concentration verification (optional)

Pre-use checks: Inspect the vial for integrity before opening. Ensure all equipment is clean and RNase/protease-free. Equilibrate the vial briefly on ice before reconstitution to avoid thermal shock to the lyophilized cake.

Step-by-Step Reconstitution and Aliquoting Protocol

Step 1 – Centrifugation before opening: Briefly centrifuge the sealed vial at low speed (≈1,000 × g, 30 seconds) to consolidate lyophilized material at the bottom of the vial and minimize loss on the cap or walls.

Step 2 – Reconstitution: Add the recommended volume of sterile reconstitution buffer slowly down the side of the vial wall (do not pipette directly onto the lyophilized cake). Gently swirl or rotate the vial; do not vortex. Allow the protein to dissolve completely at room temperature for 5–10 minutes, or on ice for 10–15 minutes, with occasional gentle swirling.

Step 3 – Verify solubilization: The solution should appear clear to slightly opalescent with no visible particulates. If particulates remain, centrifuge at 10,000 × g for 5 minutes at 4°C and use the supernatant.

Step 4 – Aliquoting: To avoid repeated freeze-thaw cycles, immediately divide the reconstituted protein into single-use aliquots in low-protein-binding microcentrifuge tubes. Aliquot volumes should be appropriate for your typical assay requirements.

Step 5 – Snap-freeze and store: Snap-freeze each aliquot in liquid nitrogen or a dry-ice/ethanol bath, then transfer to -80°C storage. Label each tube with protein name, concentration, date of reconstitution, and aliquot number.

Note: The specific reconstitution volume and buffer composition should be confirmed from the Certificate of Analysis (CoA) supplied with each lot of rp371614, as these parameters may be lot-specific.

Downstream Application Guidelines

Protein–protein interaction assays (pull-down / co-IP): Recombinant Human GRB14 Protein can be used as bait or prey in binding studies to characterize interactions with the insulin receptor kinase domain or other phosphopeptide ligands. Ensure that the binding buffer is compatible with the SH2 domain (typically near-physiological ionic strength, pH 7.2–7.4).

ELISA and antibody validation: Use as a positive control antigen in sandwich or indirect ELISA formats. Dilute to appropriate concentrations in assay buffer containing a carrier protein (0.1% BSA) to prevent adsorption to microplate surfaces.

In vitro kinase inhibition assays: GRB14 has been characterized as a pseudosubstrate inhibitor of the insulin receptor kinase. Recombinant GRB14 may be incorporated into kinase activity assays to validate inhibitory function or to assess competitive binding with peptide substrates.

Western blot (as standard): Recombinant GRB14 protein can serve as a molecular weight marker and loading control reference in Western blot experiments where endogenous GRB14 expression is being evaluated.

Working concentration guidance: Optimal working concentrations are application-dependent and should be determined empirically. Starting ranges of 0.1–1 µg/mL for ELISA and 1–10 µg per reaction for pull-down assays are commonly reported in the literature; however, researchers should titrate the protein for their specific experimental system.

Troubleshooting and Quality Assurance

Problem: Protein fails to dissolve fully after addition of buffer.
Solution: Allow additional dissolution time on ice with gentle agitation. Ensure the buffer volume is adequate. If a pellet persists after centrifugation, it may reflect aggregated or denatured material; do not use the pellet fraction.

Problem: Low or inconsistent activity in functional assays.
Solution: Verify that the protein has not undergone repeated freeze-thaw cycles. Confirm storage at -80°C without temperature excursions. Check that the assay buffer pH and ionic conditions are appropriate. Consider adding a carrier protein (BSA) or glycerol (5–10%) to stabilize the protein during dilution.

Problem: Unexpected bands on Western blot.
Solution: Confirm protein identity using a validated anti-GRB14 antibody. Multiple bands may reflect post-translational modifications, partial degradation, or disulfide-linked oligomers; run samples under reducing and non-reducing conditions to discriminate.

Problem: High background in ELISA.
Solution: Ensure adequate blocking (1–5% BSA or non-fat dry milk) and include appropriate washing steps. Titrate antigen concentration to determine the optimal signal-to-noise ratio.

Quality assurance: Each lot of Recombinant Human GRB14 Protein (rp371614) should be accompanied by a Certificate of Analysis (CoA) specifying purity (typically assessed by SDS-PAGE or HPLC), endotoxin levels, biological activity data, and recommended storage conditions. Verify lot-specific data before use and retain the CoA for experimental records.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GRB14 Protein?

The reconstitution buffer should be confirmed from the Certificate of Analysis (CoA) provided with each lot of rp371614, as the optimal buffer may be lot-specific. In general, sterile PBS (pH 7.4) supplemented with 0.1% BSA is commonly used for SH2 domain-containing adaptor proteins to maintain solubility and prevent surface adsorption. Always refer to the lot-specific CoA for precise recommendations.

How many freeze-thaw cycles can Recombinant Human GRB14 Protein tolerate?

As a general best practice for recombinant proteins, repeated freeze-thaw cycles should be minimized to preserve protein integrity, activity, and prevent aggregation. It is strongly recommended to prepare single-use aliquots immediately after reconstitution and store them at -80°C. Avoid storing reconstituted protein at 4°C for extended periods. The exact tolerance should be verified against lot-specific stability data from the CoA.

In which assay types can Recombinant Human GRB14 Protein (rp371614) be used?

Recombinant Human GRB14 Protein is suitable for multiple research applications including protein–protein interaction studies (pull-down, co-immunoprecipitation), ELISA (as a positive control antigen), Western blot (as a reference standard), and in vitro kinase inhibition assays where GRB14's role as a pseudosubstrate inhibitor of the insulin receptor kinase is under investigation. Optimal working concentrations should be determined empirically for each application.

What are the main domains of GRB14 and why are they important for its function?

GRB14 contains a PH (pleckstrin homology) domain that mediates membrane localization, a BPS (between PH and SH2) domain that directly contacts and inhibits the insulin receptor kinase activation loop, and an SH2 (Src Homology 2) domain that binds phosphotyrosine motifs on activated receptor tyrosine kinases. Together these domains enable GRB14 to act as a negative regulator of insulin and IGF-1 receptor signaling.

References

1. Daly RJ. Adaptor proteins: directors of the tyrosine kinase signalling orchestra. Cell Signal. 1998;10(9):613-618.
2. Bereziat V, et al. Inhibition of insulin receptor catalytic activity by the molecular adapter Grb14. J Biol Chem. 2002;277(6):4845-4852.
3. Cariou B, et al. The adaptor protein Grb14 is involved in insulin signalling. FEBS Lett. 2003;543(1-3):1-5.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.