Recombinant Human GOLGA8N Protein: Handling & Usage Protocol

Recombinant Human GOLGA8N Protein is a purified research-grade reagent designed to support investigations into Golgi apparatus structure, membrane trafficking, and golgin-family protein function in human cellular systems.

Product Name

Recombinant Human GOLGA8N Protein

SKU

rp377479

Protein Family

Golgin family (GOLGA subfamily), Golgi-associated coiled-coil protein

Species Origin

Human (Homo sapiens)

Application

In vitro studies, protein interaction assays, antibody validation, functional studies of Golgi trafficking

Storage Recommendation

Avoid repeated freeze-thaw cycles; aliquot upon reconstitution and store at -20°C or -80°C for long-term stability

Background: GOLGA8N and the Golgin Protein Family

GOLGA8N (Golgin A8 Family Member N) is a member of the golgin family of proteins, which are characteristically large coiled-coil proteins associated with the cytoplasmic face of the Golgi apparatus. Golgins play critical structural and functional roles in maintaining Golgi morphology, mediating vesicular tethering, and coordinating intracellular membrane trafficking pathways.

GOLGA8N belongs to the GOLGA8 subfamily, a group of primate-specific or human-enriched golgins encoded by genes clustered in segmentally duplicated genomic regions. Members of this subfamily have been implicated in Golgi ribbon formation and may participate in the regulation of secretory pathway fidelity. Research into GOLGA8N and related family members contributes to our understanding of organelle biogenesis and the molecular basis of diseases associated with Golgi dysfunction.

Recombinant Human GOLGA8N Protein provides a defined, reproducible reagent for dissecting the biochemical and cellular functions of this protein outside of endogenous expression contexts.

Materials Required

Before beginning, ensure the following materials are available:

• Recombinant Human GOLGA8N Protein (SKU: rp377479, Aladdin Scientific)
• Sterile, nuclease-free deionized water or reconstitution buffer as specified on the product datasheet
• Microcentrifuge tubes (low-binding, 1.5 mL recommended)
• Calibrated micropipettes and sterile tips
• Refrigerated microcentrifuge
• Dry ice or liquid nitrogen for snap-freezing aliquots
• Appropriate assay buffers compatible with downstream applications (e.g., PBS pH 7.4 with carrier protein if required)
• Protein quantification reagents (e.g., BCA or Bradford assay kit)

Note: Always consult the specific product datasheet supplied with SKU rp377479 for the most up-to-date reconstitution buffer, concentration, and purity specifications, as these may be updated by the manufacturer.

Step-by-Step Reconstitution and Handling Protocol

Step 1 – Thawing: Remove the vial from storage and allow it to thaw slowly on ice or at 4°C. Do not thaw at room temperature rapidly or use a water bath, as this may compromise protein integrity.

Step 2 – Centrifugation: Briefly centrifuge the vial (e.g., 5–10 seconds at low speed, ~300–500 × g) to collect any residual material from the walls and cap of the tube before opening.

Step 3 – Reconstitution: Add the reconstitution buffer or sterile deionized water as directed on the product datasheet to achieve the recommended working concentration. Add the buffer gently along the side of the tube. Do not add directly onto the lyophilized cake if supplied in lyophilized form.

Step 4 – Gentle mixing: Gently swirl or pipette slowly to dissolve. Avoid vortexing, which can cause protein denaturation and aggregation. Allow 10–15 minutes at room temperature or on ice to ensure complete dissolution.

Step 5 – Clarification: Centrifuge the reconstituted solution at 10,000 × g for 1–2 minutes at 4°C to pellet any insoluble aggregates. Transfer the supernatant to a fresh low-binding tube.

Step 6 – Quantification: Perform a protein concentration measurement (BCA or Bradford assay) to confirm the actual concentration before use in downstream assays.

Step 7 – Aliquoting: Prepare single-use aliquots appropriate for your experimental needs to minimize repeated freeze-thaw cycles. Label each aliquot with date, concentration, and passage number of freeze-thaw.

Step 8 – Storage: Store working aliquots at -20°C for short-term use (up to 1 month) or at -80°C for long-term storage. Refer to the product datasheet for the manufacturer's specified shelf life under recommended storage conditions.

Quality Control and Troubleshooting

Verifying Protein Integrity: SDS-PAGE followed by Coomassie staining or Western blotting can be used to confirm the molecular weight and purity of the reconstituted protein. Compare observed band size against the theoretical molecular weight of GOLGA8N as stated on the product datasheet.

Activity Verification: If the protein is intended for functional assays (e.g., protein–protein interaction studies, co-immunoprecipitation), include a positive control using a validated binding partner or standardized assay format to confirm biological activity.

Troubleshooting Common Issues:

• Protein aggregation or turbidity: Ensure reconstitution buffer conditions (pH, ionic strength) are optimal. Consider adding a carrier protein (e.g., 0.1% BSA) to stabilize the protein in dilute solutions.
• Low recovery or activity: Check that the freeze-thaw history of the vial has not exceeded manufacturer recommendations. Verify storage temperature was consistently maintained.
• Unexpected band sizes on gel: Some recombinant proteins may migrate anomalously due to post-translational modification tags, high coiled-coil content, or glycosylation; consult the datasheet for expected band size and tag information.

Experimental Applications and Recommended Assay Conditions

Recombinant Human GOLGA8N Protein is suitable for a range of in vitro research applications. Typical uses include:

• Protein–Protein Interaction Studies: Use as bait or prey in pull-down assays or surface plasmon resonance (SPR) experiments to identify binding partners within the Golgi tethering machinery.
• Antibody Validation: Use as a positive control antigen to validate the specificity and sensitivity of anti-GOLGA8N antibodies in ELISA, Western blot, or immunoprecipitation formats.
• Structural Studies: As a defined recombinant source, GOLGA8N protein may be employed in biophysical characterization experiments such as circular dichroism (CD) spectroscopy to assess secondary structure content.
• Cell-Free Reconstitution Assays: Investigate the role of GOLGA8N in Golgi membrane tethering or vesicle fusion events using reconstituted in vitro systems.

General Assay Tips: Titrate the protein concentration in each assay system, as optimal concentrations will vary by application. Include appropriate negative controls (e.g., buffer only, irrelevant recombinant protein of similar molecular weight) to account for non-specific effects. Ensure all downstream assay components (buffers, co-factors, binding partners) are compatible with the reconstitution buffer used for GOLGA8N.

Frequently asked questions

How should Recombinant Human GOLGA8N Protein (rp377479) be reconstituted?

Reconstitute the protein using the buffer type and volume specified on the product datasheet provided with SKU rp377479. Generally, sterile deionized water or a physiological buffer (e.g., PBS pH 7.4) is used. Add buffer gently along the tube wall, swirl gently, and allow 10–15 minutes for complete dissolution. Avoid vortexing to prevent denaturation.

How many freeze-thaw cycles can Recombinant Human GOLGA8N Protein tolerate?

As with most recombinant proteins, repeated freeze-thaw cycles should be minimized to preserve protein integrity and activity. It is strongly recommended to aliquot the reconstituted protein into single-use volumes before freezing. Consult the specific product datasheet for rp377479 for the manufacturer's stated tolerance, and flag any results obtained from samples subjected to more than three freeze-thaw cycles.

What is the biological function of GOLGA8N, and why is it relevant to my research?

GOLGA8N is a member of the golgin family of Golgi-associated coiled-coil proteins involved in maintaining Golgi structure and facilitating intracellular membrane trafficking. Research on GOLGA8N is relevant to studies of organelle biogenesis, secretory pathway regulation, and potentially to understanding diseases linked to Golgi dysfunction. Its human-specific genomic context also makes it of interest in comparative and evolutionary cell biology.

Can Recombinant Human GOLGA8N Protein be used for in vivo studies?

Recombinant Human GOLGA8N Protein (rp377479) is supplied for research use only and is intended for in vitro laboratory applications. It is not validated or approved for clinical, therapeutic, or diagnostic use in humans or animals. Researchers considering in vivo model studies should consult their institutional biosafety and ethics guidelines and contact Aladdin Scientific for further technical guidance.

How do I verify the quality of reconstituted Recombinant Human GOLGA8N Protein?

Quality can be assessed by running an SDS-PAGE gel and comparing the band size to the expected molecular weight stated on the product datasheet. Western blotting with a validated anti-GOLGA8N antibody provides additional confirmation of identity. For functional applications, include a positive control (e.g., a known binding partner or standardized assay) to verify biological activity.

References

1. Barr FA. Review series: Rab GTPases and membrane identity: causal or inconsequential? J Cell Biol. 2013;202(2):191–199. [General reference on Golgi-associated proteins and membrane trafficking; specific GOLGA8N data not confirmed from source material]
2. Munro S. The golgin coiled-coil proteins of the Golgi apparatus. Cold Spring Harb Perspect Biol. 2011;3(6):a005256. [Foundational review of golgin family proteins; specific GOLGA8N functional data requires verification]
3. Aladdin Scientific. Product Datasheet: Recombinant Human GOLGA8N Protein, SKU rp377479. [Primary source for product-specific specifications; consult current datasheet for authoritative values]

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