Purification Protocol for Ranavirus GST-Major Capsid
Presentation Type
Poster
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
26-4-2024 2:15 PM
End Date
26-4-2024 3:15 PM
Description
Protein purification is a crucial preliminary step in the systematic evolution of ligands by exponential enrichment (SELEX) process for aptamer discovery. It enables the isolation of the target protein from complex expressed protein pellets allowing the evaluation of aptamer binding affinity and thus contributing to the development of rapid field tests for detecting and monitoring Ranavirus infections. This study presents a detailed procedure for purifying a specific protein of Ranavirus, the major capsid protein bound to the glutathione S-transferase tag (GST-MCP). The cell suspension, prepared with 1X phosphate-buffered saline (PBS) buffer containing 1 mM dithiothreitol (DTT) and protease inhibitors, is subjected to sonication and centrifugation to obtain the soluble protein fraction. The resin is equilibrated with a cold 1X PBS buffer before loading the lysate, followed by column washing to remove nonspecifically bound proteins. Elution uses a buffer containing 50 mM Tris-HCl and 20 mM reduced glutathione at pH 8.0. The eluted fractions are pooled, concentrated, and exchanged into 1X PBS buffers. Careful attention is paid to maintaining cold temperatures and proper pH conditions throughout the process. The final purified protein is obtained in microliter volumes with concentrations ranging from 1.47 to 18.3 μg/μL and is ready for further analysis using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results of SDS-PAGE confirms the purity of the protein, showing a band corresponding to a molecular weight of 66 kDa. By assessing the binding affinity of the generated aptamers to the purified protein, we can evaluate their effectiveness in specifically targeting the viral protein. This innovative approach holds promise for developing rapid field tests that provide real-time results, addressing the challenges associated with detecting and monitoring Ranavirus infections.
Purification Protocol for Ranavirus GST-Major Capsid
Protein purification is a crucial preliminary step in the systematic evolution of ligands by exponential enrichment (SELEX) process for aptamer discovery. It enables the isolation of the target protein from complex expressed protein pellets allowing the evaluation of aptamer binding affinity and thus contributing to the development of rapid field tests for detecting and monitoring Ranavirus infections. This study presents a detailed procedure for purifying a specific protein of Ranavirus, the major capsid protein bound to the glutathione S-transferase tag (GST-MCP). The cell suspension, prepared with 1X phosphate-buffered saline (PBS) buffer containing 1 mM dithiothreitol (DTT) and protease inhibitors, is subjected to sonication and centrifugation to obtain the soluble protein fraction. The resin is equilibrated with a cold 1X PBS buffer before loading the lysate, followed by column washing to remove nonspecifically bound proteins. Elution uses a buffer containing 50 mM Tris-HCl and 20 mM reduced glutathione at pH 8.0. The eluted fractions are pooled, concentrated, and exchanged into 1X PBS buffers. Careful attention is paid to maintaining cold temperatures and proper pH conditions throughout the process. The final purified protein is obtained in microliter volumes with concentrations ranging from 1.47 to 18.3 μg/μL and is ready for further analysis using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results of SDS-PAGE confirms the purity of the protein, showing a band corresponding to a molecular weight of 66 kDa. By assessing the binding affinity of the generated aptamers to the purified protein, we can evaluate their effectiveness in specifically targeting the viral protein. This innovative approach holds promise for developing rapid field tests that provide real-time results, addressing the challenges associated with detecting and monitoring Ranavirus infections.
Comments
Additional Author: Ulrich Gubler