Recombinant Human GOLGA8J Protein: Handling & Usage Protocol

Recombinant Human GOLGA8J Protein is a research-grade recombinant protein derived from the Golgin subfamily A member 8J gene, used to investigate Golgi apparatus structure, membrane trafficking, and associated cellular processes in vitro.

Product Name

Recombinant Human GOLGA8J Protein

SKU

rp377476

Protein Family

Golgin subfamily A (GOLGA), Golgi autoantigen family

Source Organism

Human (Homo sapiens)

Application

In vitro research; cell biology, proteomics, and biochemical assays

Storage Recommendation

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

Supplier Reference

absea#PP-11639

Background: GOLGA8J and the Golgin Protein Family

GOLGA8J (Golgin Subfamily A Member 8J) belongs to the Golgin family of coiled-coil proteins that are integral to the structural organization and functional maintenance of the Golgi apparatus. Golgins act as tethering factors facilitating vesicle docking and membrane trafficking between Golgi cisternae and other cellular compartments.

As a member of the GOLGA8 paralog group, GOLGA8J is encoded within a region of chromosome 15 that contains multiple duplicated golgin-like sequences. Research into GOLGA8J contributes to the broader understanding of Golgi biogenesis, intracellular transport fidelity, and the molecular basis of diseases linked to Golgi dysfunction. Recombinant expression of GOLGA8J enables in vitro biochemical characterization, interaction studies, and antibody validation without reliance on endogenous cellular sources.

Materials and Equipment Required

• Recombinant Human GOLGA8J Protein (rp377476, Aladdin Scientific)
• Sterile, nuclease-free deionized water or recommended reconstitution buffer (e.g., PBS, pH 7.4)
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Calibrated micropipettes and sterile tips
• Benchtop microcentrifuge
• Ice bucket or cooling block
• -20°C and/or -80°C freezer for storage
• Protein quantification reagents (e.g., BCA or Bradford assay kit)
• SDS-PAGE gel system and relevant molecular weight markers (for QC verification)
• Assay-specific buffers and reagents appropriate to downstream application

Step-by-Step Protocol: Reconstitution and Aliquoting

1. Thaw and equilibrate: Remove the vial of Recombinant Human GOLGA8J Protein from -20°C or -80°C storage and allow it to equilibrate to room temperature for 10–15 minutes. Do not use heat to accelerate thawing.
2. Brief centrifugation: Briefly centrifuge the vial (1,000–2,000 × g, 30 seconds) to collect any lyophilized material from the cap and sides.
3. Reconstitution: Reconstitute the protein by adding the volume of sterile reconstitution buffer specified in the product datasheet. Gently pipette up and down to mix; do not vortex, as this may denature the protein.
4. Incubation: Allow the reconstituted solution to stand on ice for 5–10 minutes to ensure complete dissolution.
5. Protein quantification: Determine protein concentration using a BCA or Bradford assay according to the manufacturer's instructions. Compare to the expected concentration provided on the Certificate of Analysis (CoA).
6. Quality control: Run a small aliquot on SDS-PAGE under reducing conditions and stain with Coomassie Brilliant Blue to verify molecular weight and purity.
7. Aliquoting: Divide the reconstituted protein into single-use aliquots in low-binding microcentrifuge tubes to minimize freeze-thaw cycles. Label each tube with date, concentration, and lot number.
8. Storage: Snap-freeze aliquots in liquid nitrogen or on dry ice and store at -80°C for long-term use, or at -20°C for short-term use (up to 1 month). Avoid repeated freeze-thaw cycles.

Note: Always consult the product-specific Certificate of Analysis (CoA) and datasheet supplied with SKU rp377476 for exact reconstitution volumes, recommended buffers, and confirmed purity data.

Downstream Application Guidelines

Recombinant Human GOLGA8J Protein is suitable for a range of in vitro research applications. Typical uses include:

• Protein–protein interaction studies: Use the recombinant protein as bait or prey in pull-down or co-immunoprecipitation assays to identify GOLGA8J binding partners involved in Golgi trafficking.
• Antibody validation: Employ purified GOLGA8J protein as a positive control antigen to confirm antibody specificity in Western blot, ELISA, or immunofluorescence experiments.
• Biochemical assays: Use as a substrate or reference standard in enzymatic or binding assays relevant to Golgi function research.
• Structural studies: Suitable for biophysical characterization techniques such as circular dichroism (CD) spectroscopy or size-exclusion chromatography (SEC) when working concentration and buffer conditions are optimized.

Optimize working concentrations empirically for each application. Pilot experiments with a small volume of the aliquot are recommended before committing the full stock to any single assay.

Troubleshooting and Best Practices

Problem	Possible Cause	Recommended Action
Protein precipitation after reconstitution	Buffer incompatibility or protein aggregation	Try a different reconstitution buffer (e.g., add mild detergent such as 0.1% Tween-20 or increase salt concentration); centrifuge at low speed and use supernatant
Lower than expected protein concentration	Incomplete reconstitution or pipetting error	Ensure complete dissolution; repeat quantification; consult CoA for lot-specific yield
Degradation bands on SDS-PAGE	Protease contamination or repeated freeze-thaw	Add protease inhibitor cocktail; ensure single-use aliquots; verify cold-chain maintenance
Loss of biological activity	Denaturation due to vortexing or temperature excursion	Handle gently; maintain cold chain; avoid temperatures above 4°C for extended periods after reconstitution

Always record lot numbers and storage conditions in your laboratory notebook to facilitate reproducibility and troubleshooting.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GOLGA8J Protein (rp377476)?

The reconstitution buffer and volume are specified on the product-specific Certificate of Analysis (CoA) and datasheet supplied with each lot of rp377476. Sterile PBS (pH 7.4) is a commonly used general reconstitution buffer for recombinant proteins; however, always consult the CoA for lot-specific guidance before proceeding.

How should Recombinant Human GOLGA8J Protein be stored to maintain stability?

Lyophilized protein should be stored at -20°C or -80°C in a frost-free freezer. After reconstitution, aliquot the protein into single-use volumes and store at -80°C for long-term use. Avoid repeated freeze-thaw cycles, as these can cause protein aggregation and loss of activity.

What applications is Recombinant Human GOLGA8J Protein suitable for?

Recombinant Human GOLGA8J Protein is suitable for in vitro research applications including protein–protein interaction studies, antibody specificity validation (e.g., Western blot, ELISA, immunofluorescence), biochemical assays related to Golgi function, and biophysical characterization. Confirm suitability for each specific application by consulting the product datasheet and performing pilot experiments.

How do I verify the quality of the reconstituted GOLGA8J protein?

Run a small aliquot on SDS-PAGE under reducing conditions and stain with Coomassie Brilliant Blue to confirm the expected molecular weight and purity. Compare results to the Certificate of Analysis provided with your lot. Protein concentration should also be measured using a BCA or Bradford assay and compared against the CoA specification.

Can Recombinant Human GOLGA8J Protein be used for in vivo studies?

Recombinant Human GOLGA8J Protein (rp377476) is intended for in vitro research use only and is not approved for clinical, therapeutic, or in vivo diagnostic applications. Researchers should adhere to all institutional biosafety and ethical guidelines when designing experiments.

References

1. Gillingham AK, Munro S. The small G proteins of the Arf family and their regulators. Annual Review of Cell and Developmental Biology. 2007;23:579-611. [General reference for Golgin/Golgi tethering factor biology; specific GOLGA8J data not cited in source material]
2. Barr FA. Review series: Rab GTPases and membrane identity: causal or inconsequential? Journal of Cell Biology. 2013;202(2):191-199. [Background reference for Golgi membrane trafficking; specific GOLGA8J data not cited in source material]

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