What step can be taken if GFP appears to be aggregated after purification?

When GFP appears to be aggregated after purification, implementing a refolding step or adjusting buffer conditions is the most appropriate and effective approach. Aggregation can occur when proteins are improperly folded, which can be influenced by the conditions during purification, such as the concentration of salt, pH, and temperature.

Refolding involves creating an environment that allows the protein to regain its proper conformation. This can be achieved through the careful adjustment of buffer conditions—such as pH optimization, ionic strength, or the addition of specific additives that promote solubility and proper folding (like glycerol or arginine). Furthermore, refolding protocols often include dilution, which helps to reduce the concentration of aggregated proteins, allowing the remaining proteins to refold properly.

This step is essential because protein aggregation can lead to loss of function and decreased yield, meaning that the GFP may not perform as expected if it remains in an aggregated state. Implementing effective refolding strategies is crucial in the purification process to ensure that the protein is functional and retains its fluorescent properties.