Recombinant Human GOLGA4 Protein: Usage Protocol Guide

Recombinant Human GOLGA4 Protein is a research-grade recombinant protein targeting the Golgi apparatus autoantigen GOLGA4, designed to support studies in Golgi structure, membrane trafficking, and related cell biology applications.

Product Name

Recombinant Human GOLGA4 Protein

SKU

rp372302

Target Protein

GOLGA4 (Golgin subfamily A member 4)

Species

Human (Homo sapiens)

Protein Type

Recombinant protein

Primary Application

Cell biology research, Golgi apparatus studies, membrane trafficking assays

Background: GOLGA4 and Its Biological Role

GOLGA4 (Golgin subfamily A member 4), also known as Golgin-245 or p230, is a large coiled-coil protein predominantly localized to the trans-Golgi network (TGN). It belongs to the golgin family of proteins, which are characterized by extended coiled-coil domains that play structural roles in maintaining Golgi apparatus morphology and facilitating vesicular trafficking.

GOLGA4 functions as a tethering factor involved in the docking and fusion of transport vesicles at the TGN. It interacts with small GTPases, including members of the Rab and Arl GTPase families, and participates in the sorting and delivery of cargo proteins to the cell surface and endosomal compartments. Disruption of GOLGA4 function has been linked to altered Golgi morphology and defects in secretory pathways.

In addition, GOLGA4 has been identified as an autoantigen in certain autoimmune conditions, making it relevant for immunological research. Recombinant Human GOLGA4 Protein (rp372302) enables researchers to investigate these functions in vitro and to develop or validate immunoassay tools.

Reconstitution and Storage Protocol

Materials Required:

• Recombinant Human GOLGA4 Protein (rp372302)
• Sterile deionized water or manufacturer-recommended reconstitution buffer
• Low-protein-binding microcentrifuge tubes
• Ice or cold block

Reconstitution Steps:

1. Briefly centrifuge the vial at low speed (e.g., 300–500 × g for 30 seconds) before opening to consolidate the lyophilized material at the bottom of the tube.
2. Reconstitute the lyophilized protein by adding the volume of sterile deionized water or reconstitution buffer specified on the product datasheet to achieve the desired working concentration.
3. Gently pipette up and down or swirl the vial to mix. Do NOT vortex, as this may cause protein denaturation or aggregation.
4. Allow the protein to dissolve completely at room temperature for 5–10 minutes if needed.
5. Briefly centrifuge after reconstitution to collect any undissolved material.

Storage Conditions:

• Prior to reconstitution: Store lyophilized protein at –20 °C or –80 °C as indicated on the datasheet, protected from light and moisture.
• After reconstitution: Aliquot into single-use volumes to avoid repeated freeze-thaw cycles. Store aliquots at –80 °C for long-term storage or at –20 °C for up to one month.
• Avoid more than two freeze-thaw cycles to maintain protein integrity and activity.

Experimental Applications and Working Concentrations

Recombinant Human GOLGA4 Protein (rp372302) is suitable for a range of in vitro research applications. Recommended usage areas include:

• ELISA and Immunoassays: Use as a coating antigen or standard for the detection of GOLGA4-specific antibodies. Typical coating concentrations range from 0.5–2 µg/mL; optimize empirically for your specific assay format.
• Western Blotting (Positive Control): Load 50–200 ng of recombinant protein per lane as a positive control to confirm antibody specificity and molecular weight.
• Antibody Validation: Use as a reference antigen for validating anti-GOLGA4 antibodies in immunoprecipitation or dot-blot assays.
• Protein Interaction Studies: Employ in pull-down or co-immunoprecipitation experiments to identify binding partners of GOLGA4 in the context of Golgi trafficking.
• Autoantibody Detection Research: Useful as a substrate antigen in research aimed at identifying or characterizing GOLGA4 autoantibodies in patient samples.

Note: Optimal working concentrations should be determined by the end user through titration experiments, as results may vary depending on the experimental system, antibody clone, and detection method.

Quality Control and Troubleshooting

Quality Control Checks:

• Confirm protein identity and purity by SDS-PAGE and, where available, mass spectrometry data provided on the product certificate of analysis (CoA).
• Verify protein concentration using absorbance at 280 nm (A280) with the appropriate extinction coefficient, or by a colorimetric assay such as BCA or Bradford.
• Check for aggregation by visual inspection (solution should be clear) and, if required, by dynamic light scattering (DLS).

Common Issues and Solutions:

Issue	Possible Cause	Recommended Action
Protein precipitation after reconstitution	Protein aggregation due to improper reconstitution or freeze-thaw damage	Reconstitute gently; avoid vortexing; add carrier protein (e.g., 0.1% BSA) if appropriate
Low signal in ELISA	Suboptimal coating concentration or buffer	Optimize coating concentration; try carbonate-bicarbonate buffer (pH 9.6) for coating
Multiple bands on Western blot	Protein degradation or non-specific antibody binding	Use fresh aliquots; verify antibody specificity with blocking peptide
No detectable protein activity	Loss of activity due to excessive freeze-thaw or improper storage	Use fresh single-use aliquots stored at –80 °C; add stabilizers if recommended

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GOLGA4 Protein (rp372302)?

The recommended reconstitution buffer is specified on the product datasheet and certificate of analysis (CoA) provided with each lot. In general, sterile deionized water or a physiological buffer such as PBS (pH 7.4) is commonly used. Always refer to the specific product documentation for the most accurate guidance.

How should Recombinant Human GOLGA4 Protein be stored to maintain stability?

Prior to reconstitution, store the lyophilized protein at –20 °C or –80 °C as directed on the datasheet. After reconstitution, aliquot the protein into single-use volumes and store at –80 °C for long-term use. Avoid repeated freeze-thaw cycles (maximum two cycles recommended) as this can significantly reduce protein activity and integrity.

What applications is Recombinant Human GOLGA4 Protein (rp372302) validated for?

Recombinant Human GOLGA4 Protein is suitable for use as an antigen in ELISA, as a positive control in Western blotting, and in antibody validation and protein interaction studies. It is also used in autoantibody detection research. Users should validate the protein for their specific experimental conditions, as optimal concentrations may vary.

What is the biological significance of GOLGA4 in cell biology?

GOLGA4 is a key structural and functional component of the trans-Golgi network (TGN), where it acts as a tethering factor facilitating vesicle docking and fusion. It interacts with Rab and Arl GTPases and plays a role in cargo sorting and secretory pathway regulation. GOLGA4 is also recognized as an autoantigen in certain autoimmune diseases, broadening its research relevance.

References

1. Gleeson PA, Lock JG, Luke MR, Stow JL. Domains of the TGN: coats, tethers and G proteins. Traffic. 2004;5(5):315-326.
2. Luke MR, Kjer-Nielsen L, Brown DL, Stow JL, Gleeson PA. GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network. J Biol Chem. 2003;278(6):4216-4226.
3. Chia PZ, Gleeson PA. Membrane tethering. F1000Biol Rep. 2011;3:24.

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