Recombinant Human GRWD1 Protein: Expression & Usage Protocol

Recombinant Human GRWD1 Protein is a research-grade recombinant protein designed to support in vitro studies of chromatin remodeling, ribosome biogenesis, and cell-cycle checkpoint regulation in human cell biology.

Product Name

Recombinant Human GRWD1 Protein

SKU

rp368073

Species

Human (Homo sapiens)

Protein Family

WD repeat-containing protein family

Primary Research Applications

Chromatin remodeling, ribosome biogenesis, cell-cycle regulation

Format

Recombinant protein (lyophilized or liquid; verify with datasheet)

Storage Recommendation

Store at -20°C or -80°C; avoid repeated freeze-thaw cycles

Background and Biological Function of GRWD1

GRWD1 (Glutamate-Rich WD Repeat-Containing Protein 1), also known as CDT2-binding protein or GRWD1, is a member of the WD repeat protein superfamily. WD repeat proteins serve as scaffolding platforms that coordinate multi-protein complex assembly. GRWD1 has been implicated in multiple cellular processes, including ribosome biogenesis, chromatin remodeling, and DNA replication licensing.

GRWD1 interacts with the CRL4-CDT2 E3 ubiquitin ligase complex and has been shown to play a role in regulating histone H3 occupancy at replication origins, thereby influencing replication initiation. Additionally, GRWD1 participates in pre-ribosomal RNA processing and the maturation of the 40S ribosomal subunit, making it a multifunctional regulator at the intersection of genome integrity and translational capacity.

Research into GRWD1 is relevant to cancer biology, as dysregulation of replication licensing and ribosome biogenesis are hallmarks of proliferating tumor cells. Recombinant Human GRWD1 Protein (rp368073) provides a tool for biochemical assays, protein–protein interaction studies, and structural investigations.

Materials and Equipment Required

Before beginning the reconstitution and application protocol, ensure the following materials and equipment are available:

• Recombinant Human GRWD1 Protein, SKU rp368073 (Aladdin Scientific)
• Sterile, ultrapure water or appropriate reconstitution buffer (e.g., PBS, pH 7.4, with carrier protein such as 0.1% BSA if recommended by the datasheet)
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Benchtop microcentrifuge
• Ice bucket or cooling block
• -20°C and/or -80°C freezer for storage
• Relevant downstream assay reagents (e.g., binding buffers for pull-down, SDS-PAGE reagents for purity verification)

Note: Always consult the product-specific datasheet supplied with SKU rp368073 for confirmed specifications including molecular weight, tag information, purity grade, and recommended reconstitution buffer, as these details supersede general guidance provided here.

Step-by-Step Reconstitution and Handling Protocol

Follow these steps carefully to ensure protein integrity and optimal performance in downstream assays:

1. Equilibration: Remove the vial of Recombinant Human GRWD1 Protein (rp368073) from the freezer and allow it to equilibrate at room temperature for 5–10 minutes before opening. Do not heat.
2. Brief centrifugation: Gently centrifuge the vial at low speed (e.g., 2,000 × g for 30 seconds) to collect any lyophilized material from the sides and cap of the tube.
3. Reconstitution: Add the volume of sterile reconstitution buffer specified on the product datasheet along the inner wall of the vial. Gently swirl; do not vortex. Allow the protein to dissolve for 5–10 minutes on ice.
4. Mixing: After dissolution, gently pipette up and down several times to ensure homogeneity. Avoid generating foam or bubbles.
5. Purity check (optional but recommended): Run a small aliquot on SDS-PAGE to confirm the expected molecular weight band and assess purity.
6. Aliquoting: Divide the reconstituted protein into single-use aliquots using low-binding microcentrifuge tubes to minimize repeated freeze-thaw cycles.
7. Storage: Store aliquots at -20°C for short-term use (up to 1 month) or at -80°C for long-term storage. Avoid repeated freeze-thaw cycles, which can reduce activity and cause aggregation.

Important: Adding a carrier protein such as 0.1% BSA may enhance stability during storage. Confirm compatibility with your downstream assay before adding any stabilizer.

Recommended Applications and Assay Guidance

Recombinant Human GRWD1 Protein (rp368073) is suitable for a range of in vitro research applications, including but not limited to:

• Protein–protein interaction studies: Use GRWD1 as bait or prey in co-immunoprecipitation (Co-IP) or GST pull-down assays to identify and validate interaction partners within the CRL4-CDT2 complex or ribosome assembly pathways.
• Biochemical activity assays: Investigate GRWD1's influence on histone occupancy at replication origins using chromatin assembly or nucleosome displacement assays in cell-free systems.
• Western blot standard: Use as a positive control or molecular weight standard when probing for endogenous GRWD1 expression in cell lysates.
• Structural and biophysical studies: Suitable for use in techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or cryo-electron microscopy sample preparation, subject to purity and formulation requirements.
• Antibody production and validation: Can serve as an immunogen or standard for antibody specificity testing.

Optimal working concentrations will vary by application and experimental system. Users are advised to perform titration experiments to determine conditions appropriate for their specific assays.

Quality Control, Safety, and Troubleshooting

Quality Control: Recombinant Human GRWD1 Protein (rp368073) is manufactured under controlled conditions. Each lot is typically assessed for purity by SDS-PAGE and/or HPLC, and biological activity may be confirmed by functional assay. Refer to the Certificate of Analysis (CoA) provided with each shipment for lot-specific data.

Safety: This product is intended for research use only and is not suitable for therapeutic, diagnostic, or food applications. Handle according to standard laboratory biosafety practices. Consult the Safety Data Sheet (SDS) for detailed hazard information.

Troubleshooting common issues:

• Protein precipitation or aggregation: Ensure reconstitution buffer is appropriate (check pH and ionic strength); avoid vortexing; centrifuge briefly at 10,000 × g to pellet aggregates before use.
• Low activity in functional assays: Confirm the protein has not undergone multiple freeze-thaw cycles; verify storage temperature; optimize buffer conditions for the specific assay.
• Unexpected band size on SDS-PAGE: Confirm whether the construct carries an affinity tag (e.g., His-tag, GST-tag) that may shift apparent molecular weight; consult the datasheet.
• Inconsistent results between experiments: Use single-use aliquots; standardize protein concentration measurement using BCA or Bradford assay prior to each experiment.

Frequently asked questions

What is GRWD1 and why is it important for research?

GRWD1 (Glutamate-Rich WD Repeat-Containing Protein 1) is a multifunctional scaffold protein involved in ribosome biogenesis, chromatin remodeling, and DNA replication licensing through interactions with the CRL4-CDT2 E3 ubiquitin ligase complex. Its roles in regulating replication origin activity and pre-rRNA processing make it relevant to cancer biology and cell proliferation research.

How should Recombinant Human GRWD1 Protein (rp368073) be reconstituted and stored?

Allow the vial to equilibrate at room temperature for 5–10 minutes, briefly centrifuge, then add the reconstitution buffer specified on the product datasheet along the vial wall and gently swirl on ice. After reconstitution, aliquot into single-use volumes and store at -20°C (short-term, up to 1 month) or -80°C (long-term). Avoid repeated freeze-thaw cycles to preserve protein activity.

What downstream assays is Recombinant Human GRWD1 Protein suitable for?

Recombinant Human GRWD1 Protein (rp368073) is suitable for protein–protein interaction studies (Co-IP, pull-down assays), biochemical activity assays examining chromatin and replication, Western blot as a positive control or size standard, structural and biophysical studies (SPR, ITC), and antibody production or validation. Optimal working concentrations should be determined empirically for each application.

How can I verify the purity and integrity of the protein after reconstitution?

Run a small aliquot of the reconstituted protein on SDS-PAGE under reducing conditions and compare the observed band to the expected molecular weight indicated on the product datasheet. A single dominant band at the correct molecular weight indicates acceptable purity. The Certificate of Analysis (CoA) provided with each lot contains lot-specific purity data for reference.

What should I do if the protein shows aggregation or reduced activity?

If aggregation is observed, centrifuge the sample at 10,000 × g for 5 minutes to remove aggregates and use the clarified supernatant. Ensure the reconstitution buffer has appropriate pH and ionic strength as specified in the datasheet. Reduced activity often results from multiple freeze-thaw cycles or improper storage; always use freshly thawed, single-use aliquots and verify storage conditions.

References

1. Sugimoto N, et al. A novel protein, GRWD1, is a regulatory subunit of the CRL4-CDT2 E3 ubiquitin ligase complex involved in replication origin regulation. (Specific journal, volume and page details require verification.)
2. Tafforeau L, et al. The complexity of human ribosome biogenesis revealed by systematic nucleolar protein analysis. Molecular Cell. 2013;51(4):539–551. (Relevance of WD-repeat proteins in ribosome biogenesis context; applicability to GRWD1 requires verification.) doi:10.1016/j.molcel.2013.08.011
3. Aladdin Scientific. Recombinant Human GRWD1 Protein, SKU rp368073. Product Datasheet. (Consult current datasheet for confirmed specifications.)

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