As the summer continues to progress, so does my project. After the last round of purification, detailed in my previous blog post, I am ready for another round. During this purification, I will be working with SH3G3 and PACN1. PACN1 is responsible for cytoskeletal remodeling through regulation of several other proteins, as well as neuronal vesicle endocytosis, among other things. It is thought that SH3G3 is more specialized in aiding endocytosis. Luckily, the purification of both proteins was successful, although that’s not to say everything went to plan.
The tags on these proteins allowed for multiple possible binding possibilities, meaning we had different avenues we could try for the purification. In the first attempt, we used SBP resin. In theory, the SBP would bind our protein of interest, allowing us to purify it, but in practice, the binding affinity was disappointingly low. In an attempt to salvage the purification, we then ran through a nickel purification, reverse nickel purification and size exclusion chromatography. Because of the design of the tag, our protein binds Nickel, but some proteins that occur naturally will also bind Nickel, so after collecting all of the nickel binding proteins, we can cut off the tag on our protein, using TEV, leaving it unable to bind Nickel. This means that is we mix nickel resin with the proteins; everything should bind the Nickel except our protein. After this, we ran it through a column with gel beads that contain tiny pores. Small proteins get trapped in the pores while larger proteins glide through, this gives us an additional way to purify our protein. For complete methods and data for SH3G3 and PACN1 purifications, look to my posts on Zenodo.