Well, it turns out this is not an easy task. Everyday is not sunny, but everyday is certainly a small adventure in itself. Looking back, I was very naïve about the process of scientific research. I had always heard that scientists experience failure and they learn from it, that is what makes a good scientist. It all sounds very pleasant and ideal, until I was the one experiencing the failure. At the moment, it does not feel as though I am learning from it. Whenever I have finished expressing the PK9 and UBC13 and I am well on my way in the purification process, my heart drops every time I see a truncation of the protein in a Western Blot. This usually means two things: we need to start over which means preparing 4 more liters of media, incubating overnight, inducing with IPTG, batch binding overnight, etc. Though tedious, I have repeated this process so many times that I now do not even think twice when I see that we do not have enough protein to begin an assay with.
Recently, however, we had a small breakthrough in our experiments due to changing various conditions in our buffers, IPTG, as well as incubation conditions. A few weeks ago, I would never think I could celebrate at the sight of just a few more microliters of protein. (And by this I mean microliters of protein when diluted). I have also found it very difficult to make certain choices such as whether to use a protease and cleave the protein in order to get rid of any non-specific protein. However, this has sometimes resulted in losing more protein than intended. On the other hand, not cleaving could mean ending up with unpurified protein which would bring us back to the same point. This checks and balances system is quite frustrating.
In the next two weeks, I hope to be able to finally purify enough protein to be able to begin an ATPase assay where I would be able to determine whether the phosphorylation of UBC13 is being affected (by measuring the amount of ATP being converted to ADP). I am extremely hopeful that I will be able to get some results in time for the poster session. This summer has been quite a rollercoaster and although I am sad to see it come to a close, I am excited to continue my project in the Derbyshire lab during the Fall semester.