Recombinant Human GJC1 Protein: Handling & Usage Protocol

Recombinant Human GJC1 Protein is a research-grade connexin family member that enables scientists to investigate gap junction channel formation, intercellular communication, and associated signaling pathways with high reproducibility.

Protein Name

Recombinant Human GJC1 Protein (Connexin-45, Cx45)

Gene Name

GJC1 (also known as GJA7)

Protein Family

Connexin (gap junction protein) family

Biological Function

Mediates gap junction channel formation and intercellular communication

Recommended Storage

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

SKU

rp368044

Overview of GJC1 and Its Biological Significance

GJC1 (Gap Junction Protein Gamma-1), commonly referred to as Connexin-45 (Cx45), is a member of the connexin multigene family that encodes proteins forming gap junction channels. Gap junctions are specialized intercellular connections that allow the direct exchange of ions, second messengers, and small metabolites between adjacent cells, playing critical roles in tissue homeostasis, cardiac conduction, neuronal synchronization, and developmental signaling.

Recombinant Human GJC1 Protein is produced to support in vitro research applications including protein–protein interaction studies, structural analyses, antibody validation, and functional assays related to connexin biology. Its availability as a recombinant reagent enables researchers to conduct controlled experiments without reliance on variable cell-derived preparations.

Reconstitution and Buffer Preparation

Prior to use, carefully centrifuge the vial at low speed to collect lyophilized material at the bottom of the tube. Reconstitute the protein by adding an appropriate volume of sterile, deionized water or phosphate-buffered saline (PBS, pH 7.2–7.4) to achieve the desired stock concentration. Gently pipette up and down to mix; do not vortex, as mechanical shearing can disrupt protein integrity.

For applications requiring specific buffer conditions (e.g., assays sensitive to salt concentration or detergent), prepare a compatible reconstitution buffer and exchange via dialysis or desalting column if necessary. It is recommended to prepare small-volume aliquots of the reconstituted protein immediately after resuspension to minimize freeze-thaw cycles and prevent activity loss.

Note: Carrier protein (e.g., 0.1% BSA) may be added to the diluent to enhance stability during storage and dilution, particularly at low working concentrations. Consult the product datasheet for product-specific reconstitution volumes and concentrations.

Storage, Stability, and Handling Precautions

Recombinant Human GJC1 Protein should be stored at -80°C upon receipt for long-term stability. Short-term storage (up to one week) may be performed at -20°C or 4°C if repeated access is required; however, prolonged storage at these temperatures is not recommended. Repeated freeze-thaw cycles significantly reduce protein activity and should be avoided by aliquoting the stock upon first use.

Always work on ice when handling the protein to minimize degradation. Use low-protein-binding tubes and pipette tips to reduce adsorptive losses, especially at low concentrations. Avoid exposing the protein to extreme pH, reducing agents incompatible with the formulation, or detergents unless specifically validated for the intended assay.

Monitor protein integrity periodically by SDS-PAGE or Western blot if long-term stability is a concern for your experimental workflow.

Experimental Applications and Working Concentration Guidelines

Recombinant Human GJC1 Protein is suitable for a range of in vitro research applications, including but not limited to:

• ELISA and immunoassays: Use as a standard or coating antigen to develop or validate GJC1-specific detection systems.
• Protein interaction studies: Employ in pull-down, co-immunoprecipitation, or surface plasmon resonance (SPR) assays to identify binding partners of Cx45.
• Western blot positive control: Use as a reference standard to confirm antibody specificity and assay sensitivity.
• Structural and biophysical studies: Apply in circular dichroism (CD), dynamic light scattering (DLS), or other biophysical characterization assays.

Optimal working concentrations will vary by application and must be determined empirically. It is recommended to perform a titration experiment before committing to large-scale assays. Refer to the product datasheet for any application-specific concentration ranges provided by Aladdin Scientific.

Quality Control and Troubleshooting

Each lot of Recombinant Human GJC1 Protein should be verified for purity and integrity prior to use. Common quality control assessments include:

• SDS-PAGE: Confirm expected molecular weight band and assess purity (typically >85% by densitometry, verify on datasheet).
• Western blot: Confirm identity using a validated anti-GJC1 or anti-Cx45 antibody.
• Endotoxin testing: For cell-based assays, ensure endotoxin levels are within acceptable limits (typically <1 EU/µg; verify on datasheet).

Common issues and troubleshooting:

• Protein aggregation: If precipitation is observed, briefly centrifuge the tube and use only the supernatant. Addition of mild non-ionic detergent or optimization of buffer ionic strength may help.
• Loss of activity: Likely due to excessive freeze-thaw cycles or improper storage. Use freshly aliquoted material.
• Unexpected molecular weight on gel: GJC1 may run anomalously on SDS-PAGE due to hydrophobic transmembrane domains; confirm identity by Western blot.

Frequently asked questions

What is the recommended storage condition for Recombinant Human GJC1 Protein?

Recombinant Human GJC1 Protein should be stored at -80°C for long-term stability. To minimize activity loss, aliquot the protein upon first use and avoid repeated freeze-thaw cycles. Short-term storage at 4°C is acceptable for up to one week.

How should Recombinant Human GJC1 Protein be reconstituted?

Reconstitute the lyophilized protein by adding sterile deionized water or PBS (pH 7.2–7.4) to achieve the desired concentration. Mix gently by pipetting; do not vortex. Adding 0.1% BSA to the diluent can help improve stability, especially at low concentrations. Always refer to the product datasheet for lot-specific reconstitution instructions.

What applications is Recombinant Human GJC1 Protein validated for?

Recombinant Human GJC1 Protein is suitable for use as an ELISA standard or antigen, a Western blot positive control, and in protein interaction studies such as pull-down or SPR assays. It can also be used in structural and biophysical characterization. Optimal concentrations should be determined empirically for each application.

Why might GJC1 protein run at an unexpected molecular weight on SDS-PAGE?

GJC1 (Connexin-45) contains multiple hydrophobic transmembrane domains that can cause anomalous migration on SDS-PAGE, resulting in a band that does not correspond precisely to the predicted molecular weight. Western blot confirmation using a validated anti-GJC1 antibody is recommended to verify protein identity.

What is the biological role of GJC1 (Connexin-45) in human physiology?

GJC1 encodes Connexin-45, a gap junction protein that forms intercellular channels allowing the passage of ions and small molecules between cells. It plays important roles in cardiac conduction, neuronal communication, and vascular function. Mutations in connexin genes have been linked to various human diseases, making GJC1 a relevant target for biomedical research.

References

1. Laird DW. Life cycle of connexins in health and disease. Biochem J. 2006;394(Pt 3):527-543. doi:10.1042/BJ20051922
2. Söhl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res. 2004;62(2):228-232. doi:10.1016/j.cardiores.2003.11.013
3. Beyer EC, Bhatt DL, Bhatt J. Connexins: transmembrane proteins of the gap junction. Prog Biophys Mol Biol. 1990;53(2):71-96.

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