Recombinant Human GRASP65 Protein: Protocol & Usage Guide

Recombinant Human GRASP65 Protein is a eukaryotic secretory pathway regulator critically involved in Golgi ribbon formation, stacking, and membrane trafficking, making it an essential tool for researchers investigating organelle biogenesis and cell division.

Product Name

Recombinant Human GRASP65 Protein

SKU

rp375938

Gene Name

GORASP1 (encodes GRASP65)

Protein Function

Golgi reassembly and stacking; regulation of Golgi ribbon formation and membrane trafficking

Host Expression System

Recombinant (expression system to be confirmed with datasheet)

Recommended Storage

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

Primary Applications

Western blot, ELISA, functional assays, Golgi structure studies

Background: GRASP65 and Its Biological Role

GRASP65 (Golgi Reassembly and Stacking Protein of 65 kDa), encoded by the GORASP1 gene, is a peripheral membrane protein anchored to the cis-Golgi network. It plays a pivotal role in Golgi ribbon formation, cisternal stacking, and the regulation of anterograde protein secretion. During mitosis, GRASP65 is phosphorylated, leading to Golgi fragmentation, a process required for accurate partitioning of the organelle between daughter cells. Beyond its structural role, GRASP65 has been implicated in unconventional protein secretion pathways and has emerged as a potential biomarker in cancer biology and neurological disease research.

Recombinant Human GRASP65 Protein (SKU: rp375938) supplied by Aladdin Scientific enables researchers to investigate these processes in cell-free and cell-based systems, providing a biochemically defined tool for mechanistic studies.

Reconstitution and Storage Protocol

Materials Required:

• Sterile deionized water or the recommended reconstitution buffer (refer to product datasheet for exact buffer composition)
• Microcentrifuge tubes (low-binding recommended)
• Ice or cooling block

Reconstitution Steps:

1. Briefly centrifuge the vial (1,000–2,000 × g, 30 seconds) to collect lyophilized material at the bottom before opening.
2. Add the volume of sterile reconstitution buffer specified on the product datasheet to achieve the desired working concentration. Gentle pipetting is recommended; avoid vortexing.
3. Allow the solution to stand on ice for 5–10 minutes to ensure complete dissolution.
4. Aliquot into single-use volumes to minimize freeze-thaw cycles and label with date and concentration.

Storage Conditions:

• Store lyophilized protein at -20°C or -80°C, protected from light and moisture.
• Reconstituted protein should be stored at -80°C for long-term use or at 4°C for short-term use (within 1 week). Consult the product datasheet for stability data specific to this lot.
• Avoid repeated freeze-thaw cycles, as this may reduce protein activity and integrity.

Experimental Applications

Recombinant Human GRASP65 Protein is suitable for a range of in vitro and biochemical research applications:

• Western Blot (WB): Use as a positive control to validate anti-GRASP65 antibody performance or to assess endogenous GRASP65 expression levels in cell lysates.
• ELISA: Serve as a standard or capture antigen for quantitative measurement of GRASP65 in experimental samples.
• Protein–Protein Interaction Studies: Investigate GRASP65 binding partners, including GM130 and other Golgi matrix proteins, using pull-down or co-immunoprecipitation assays.
• In Vitro Golgi Reassembly Assays: Reconstitute Golgi stacking and fusion events in cell-free systems to dissect the biochemical requirements for cisternal reformation.
• Phosphorylation and Kinase Assays: Study mitotic phosphorylation of GRASP65 by CDK1/cyclin B or Polo-like kinases to understand Golgi fragmentation during cell division.
• Structural Biology: Suitable as a substrate for crystallography or cryo-EM studies aimed at resolving GRASP65 domain architecture.

Always titrate the protein empirically for each application. Refer to published literature and the Aladdin Scientific product datasheet for recommended starting concentrations.

Quality Control and Handling Tips

Quality Control: Recombinant Human GRASP65 Protein from Aladdin Scientific (SKU: rp375938) is manufactured under controlled conditions. Each lot should be accompanied by a Certificate of Analysis (CoA) specifying purity (typically assessed by SDS-PAGE and/or HPLC), endotoxin levels, biological activity where applicable, and protein concentration. Always request and review the CoA before use.

Handling Tips:

• Work on ice at all times after reconstitution to preserve protein integrity.
• Use low-binding microcentrifuge tubes and pipette tips to minimize protein adsorption losses.
• Prepare single-use aliquots immediately after reconstitution to avoid repeated freeze-thaw cycles.
• Include appropriate positive and negative controls in every experiment.
• If turbidity or precipitation is observed after reconstitution, centrifuge briefly at 10,000 × g and use the clear supernatant.
• Do not use protein that shows visible aggregation, abnormal color, or has exceeded its expiration date.

Troubleshooting

The following guidance addresses common issues encountered when working with Recombinant Human GRASP65 Protein:

Issue	Possible Cause	Suggested Action
No signal in Western Blot	Protein degradation; incorrect loading amount; antibody incompatibility	Verify protein integrity by SDS-PAGE; optimize loading; confirm antibody validated for recombinant GRASP65
Protein precipitation after reconstitution	Incorrect buffer; rapid temperature change; protein aggregation	Use recommended buffer from datasheet; reconstitute slowly on ice; centrifuge and use supernatant
Low biological activity	Repeated freeze-thaw; improper storage	Use freshly aliquoted protein; store at -80°C; avoid temperature fluctuations
High background in ELISA	Endotoxin contamination; non-specific binding	Check endotoxin level on CoA; optimize blocking conditions; use endotoxin-free buffers

Frequently asked questions

What is GRASP65 and why is it important in cell biology research?

GRASP65 (Golgi Reassembly and Stacking Protein of 65 kDa) is a peripheral Golgi membrane protein encoded by GORASP1. It is essential for Golgi ribbon formation, cisternal stacking, and regulated protein secretion. During mitosis, its phosphorylation drives Golgi fragmentation, making it a key regulator of organelle inheritance and cell cycle progression.

How should Recombinant Human GRASP65 Protein be reconstituted and stored?

Reconstitute the lyophilized protein using the sterile buffer specified in the product datasheet, gently pipetting on ice until fully dissolved. Aliquot immediately into single-use volumes and store at -80°C for long-term stability. Avoid repeated freeze-thaw cycles, which can compromise protein activity and structural integrity.

What experimental applications is Recombinant Human GRASP65 Protein suitable for?

Recombinant Human GRASP65 Protein is suitable for Western blot (as a positive control), ELISA (as a standard or capture antigen), protein-protein interaction studies, in vitro Golgi reassembly assays, kinase/phosphorylation assays, and structural biology applications including crystallography and cryo-EM. Empirical titration is recommended for each application.

How can I verify the quality of the GRASP65 protein before use?

Request the Certificate of Analysis (CoA) from Aladdin Scientific for the specific lot number. The CoA should confirm protein purity by SDS-PAGE or HPLC, provide endotoxin levels, state the protein concentration, and include activity data where applicable. Running an SDS-PAGE or Western blot with the reconstituted protein before your main experiment is also recommended.

What precautions should I take to avoid protein aggregation?

Reconstitute the protein slowly on ice using the recommended buffer and avoid vortexing. Use low-binding tubes and tips to minimize surface adsorption. If aggregation occurs after reconstitution, centrifuge at 10,000 × g for 5 minutes and use the clear supernatant. Prepare single-use aliquots and store at -80°C to prevent repeated freeze-thaw-induced aggregation.

References

1. Barr FA, Puype M, Vandekerckhove J, Warren G. GRASP65, a protein involved in the stacking of Golgi cisternae. Cell. 1997;91(2):253-262. doi:10.1016/S0092-8674(00)80408-7
2. Wang Y, Seemann J, Pypaert M, Shorter J, Warren G. A direct role for GRASP65 as a mitotically regulated Golgi stacking factor. EMBO J. 2003;22(13):3279-3290. doi:10.1093/emboj/cdg317
3. Aladdin Scientific. Recombinant Human GRASP65 Protein, SKU rp375938. Product Datasheet. Aladdin Scientific; [current year].

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