To improve the purification of viral proteins, especially when facing challenges with the Sephadex column after using the His-Trap column, consider the following suggestions based on the information from the provided sources:

1. Optimize Purification Methods:

• Avoid Multiple Purification Steps: Reduce the number of purification steps to minimize protein loss.
• Consider Alternative Methods: Explore different purification techniques like size-exclusion chromatography (SEC) or ion-exchange chromatography (IEC) mentioned in the sources.

2. Enhance Protein Stability:

• Maintain Optimal Conditions: Ensure proper buffer conditions, pH, and temperature throughout the purification process to prevent protein denaturation.
• Use Suitable Buffers: Employ buffers that are compatible with the protein of interest to maintain stability.

3. Troubleshooting Protein Loss:

• Check Protein Concentration: Monitor protein concentration at each purification step to identify where losses occur.
• Evaluate Column Performance: Assess the efficiency and suitability of the Sephadex column for your specific protein purification needs.

4. Consider Specific Protein Characteristics:

• Protein Size and Charge: Understand the size and charge properties of the viral protein to select the most appropriate purification method.
• Protein Interactions: Consider any specific interactions or characteristics of the NS5 protein of JEV that might affect its purification.

Refer these articles:

1. Article Purification Methods and the Presence of RNA in Virus Partic...

2. 10.1016/S0166-0934(02)00069-1

3. https://www.sartorius.com/en/knowledge/science-snippets/chromatography-applications-for-new-modalities-viruses-1152796