Recombinant Human GOLPH3L Protein: Handling & Application Protocol

Recombinant Human GOLPH3L Protein is a research-grade recombinant protein targeting the Golgi phosphoprotein 3-like (GOLPH3L) gene product, used in studies of Golgi membrane dynamics, vesicular trafficking, and related oncogenic signaling pathways.

Product Name

Recombinant Human GOLPH3L Protein

SKU

rp367264

Gene Target

GOLPH3L (Golgi Phosphoprotein 3-Like)

Species

Human (Homo sapiens)

Application

Biochemical assays, binding studies, antibody validation, cell-based assays

Storage Recommendation

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

Background: GOLPH3L Biology and Research Significance

GOLPH3L (Golgi Phosphoprotein 3-Like) is a paralog of GOLPH3, a protein primarily localized to the Golgi apparatus and implicated in the regulation of Golgi membrane morphology and vesicular trafficking. GOLPH3L shares structural homology with GOLPH3 and is thought to interact with phosphatidylinositol 4-phosphate [PI(4)P] on Golgi membranes, thereby modulating membrane curvature and cargo sorting.

Research interest in GOLPH3L has grown alongside studies linking GOLPH3 to oncogenic signaling via the mTOR and MYO18A pathways. Understanding GOLPH3L's distinct and overlapping roles relative to GOLPH3 may provide new insights into Golgi biology and cancer cell physiology. Recombinant Human GOLPH3L Protein (rp367264) enables researchers to investigate protein–protein interactions, enzymatic activities, and structural features of GOLPH3L in a controlled in vitro environment.

Materials and Equipment Required

• Recombinant Human GOLPH3L Protein, rp367264 (Aladdin Scientific)
• Sterile, nuclease-free water or appropriate reconstitution buffer (e.g., PBS, pH 7.4, or manufacturer-recommended buffer)
• Microcentrifuge tubes (1.5 mL, low-protein-binding recommended)
• Calibrated micropipettes and sterile tips
• Tabletop microcentrifuge
• Ultrafreezer (-80 °C) for long-term storage
• Ice or cooling block for bench-top handling
• SDS-PAGE or Western blot apparatus (for quality verification)
• BCA or Bradford protein quantification kit
• BSA protein standards

Note: Always handle recombinant proteins on ice to minimize degradation. Use low-protein-binding tubes to reduce adsorption losses.

Step-by-Step Protocol: Reconstitution, Aliquoting, and Storage

Step 1: Preparation

Remove the vial of Recombinant Human GOLPH3L Protein (rp367264) from -80 °C storage. Allow the vial to equilibrate briefly at 4 °C (do not thaw at room temperature) to minimize protein aggregation.

Step 2: Reconstitution

Briefly centrifuge the vial (1,000 × g, 30 seconds) to collect any lyophilized material from the walls and cap. Reconstitute the protein by adding the recommended volume of sterile reconstitution buffer dropwise to the vial. Gently pipette up and down or swirl carefully — do NOT vortex, as vigorous agitation can cause protein denaturation and aggregation.

Step 3: Protein Quantification

Determine the protein concentration using a BCA or Bradford assay with BSA as the standard. Compare to the stated concentration on the Certificate of Analysis (CoA) as a quality check.

Step 4: Aliquoting

Immediately divide the reconstituted protein into single-use aliquots in low-protein-binding microcentrifuge tubes. This minimizes the number of freeze-thaw cycles, which can significantly impair protein integrity and activity.

Step 5: Storage

Snap-freeze aliquots in liquid nitrogen and store at -80 °C. For short-term use (up to 1 week), aliquots may be stored at 4 °C; however, -80 °C is strongly recommended for prolonged preservation. Do not store at -20 °C, as the freeze-thaw cycle associated with frost-free freezers can damage the protein.

Recommended Applications and Working Concentrations

Recombinant Human GOLPH3L Protein (rp367264) is suitable for a range of in vitro research applications. Researchers should optimize conditions empirically for their specific experimental systems.

• SDS-PAGE / Western Blot (Identity Verification): Load 1–5 µg of protein per lane under denaturing, reducing conditions to confirm apparent molecular weight and purity.
• ELISA / Binding Assays: Use as a coating antigen or binding partner at concentrations typically ranging from 0.1–2 µg/mL; titrate for optimal signal-to-noise ratio.
• Protein–Protein Interaction Studies (e.g., co-IP, pull-down): Use at concentrations determined empirically based on binding affinity; starting concentrations of 1–10 µg per reaction are common.
• Cell-Free Biochemical Assays: Optimize protein concentration based on assay sensitivity and expected activity range.
• Antibody Validation / Immunogen: Use as a positive control antigen to verify antibody specificity in immunoassays.

Important: Always include a negative control (buffer only) and a positive control in every experiment. Specific optimal concentrations may vary; empirical optimization is essential.

Troubleshooting and Quality Control

Problem: No or Low Signal in Functional Assay

Possible causes include protein inactivation due to improper reconstitution, repeated freeze-thaw cycles, or use of inappropriate buffer conditions. Verify protein integrity by SDS-PAGE before use. Ensure the reconstitution buffer is compatible with downstream assays.

Problem: Protein Aggregation

Aggregation may occur if protein is thawed rapidly at room temperature or vortexed. Always thaw on ice, handle gently, and consider adding a carrier protein (e.g., 0.1% BSA) if aggregation persists. Centrifuge at 10,000–14,000 × g for 5 minutes at 4 °C to remove aggregates before use.

Problem: Multiple Bands on SDS-PAGE

Minor degradation bands can appear with improper storage. Check that -80 °C storage conditions have been maintained and that the protein has not been subjected to multiple freeze-thaw cycles. Compare banding pattern to the Certificate of Analysis.

Quality Control Checklist

• Confirm protein concentration by BCA/Bradford assay upon reconstitution.
• Run SDS-PAGE to verify molecular weight and purity.
• Refer to the Certificate of Analysis (CoA) for lot-specific QC data.
• Document all storage conditions and usage dates for traceability.

Frequently asked questions

What buffer should I use to reconstitute Recombinant Human GOLPH3L Protein?

The optimal reconstitution buffer will be specified on the product's Certificate of Analysis (CoA). In general, sterile PBS (pH 7.4) or a buffer compatible with your downstream application is recommended. Avoid buffers containing strong reducing agents or detergents unless specifically required by your assay.

How many freeze-thaw cycles can Recombinant Human GOLPH3L Protein tolerate?

It is strongly recommended to minimize freeze-thaw cycles to preserve protein integrity and activity. Best practice is to aliquot the protein into single-use volumes immediately after reconstitution and store at -80 °C. Repeated freeze-thaw cycles can lead to protein denaturation, aggregation, and loss of functional activity.

What applications is Recombinant Human GOLPH3L Protein validated for?

Recombinant Human GOLPH3L Protein (rp367264) is suitable for use in SDS-PAGE/Western blot identity verification, ELISA, protein–protein interaction studies (pull-down, co-immunoprecipitation), cell-free biochemical assays, and antibody validation. Users should verify suitability for their specific experimental conditions and optimize working concentrations empirically.

How should I verify the quality of the reconstituted protein before use?

Run the reconstituted protein on an SDS-PAGE gel under denaturing, reducing conditions and compare the band pattern and apparent molecular weight to the Certificate of Analysis (CoA). Additionally, quantify protein concentration using a BCA or Bradford assay. Any significant deviation from the expected profile should be investigated before proceeding with experiments.

Can Recombinant Human GOLPH3L Protein be used in cell-based assays?

While Recombinant Human GOLPH3L Protein is primarily intended for in vitro biochemical and binding applications, its use in cell-based assays may be possible depending on delivery method and experimental design. Researchers should validate the protein's activity and biocompatibility for any cell-based application and ensure the formulation is free of components toxic to cells.

References

1. Dippold HC, et al. GOLPH3 bridges phosphatidylinositol-4-phosphate and actomyosin to stretch and shape the Golgi to promote budding. Cell. 2009;139(2):337-351. doi:10.1016/j.cell.2009.07.052
2. Scott KL, et al. GOLPH3 modulates mTOR signaling and rapamycin sensitivity in cancer. Nature. 2009;459(7250):1085-1090. doi:10.1038/nature08109
3. Mayinger P. Phosphoinositides and vesicular membrane traffic. Biochimica et Biophysica Acta. 2012;1821(8):1104-1113. doi:10.1016/j.bbalip.2012.01.001

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