A Day in the Derbyshire Lab

My typical day begins at 8:15am on Mondays and Fridays with a debriefing session with my mentor. We usually go over the order of business for the day which includes the tasks that should be accomplished that day. Most of the time, we have had overnight cultures growing in one of the Shaker rooms and I begin by completing the prepping for the cultures so we can begin our experiments. As described by the purpose of my project, we are trying to determine whether the compounds that were screened by a researcher in our lab are directly or specifically targeting PfPK9. We can determine this by matching the IC50 value of the cell-based assays with our assays in vitro. My day occurs in the following steps:

  1. Expressing PfPK9 and its substrate PfUBC13. Essentially, I want to find the optimal conditions so that I can get the most amount of pure protein.
  • In expression, I have a plasmid with a GST site linked to our PfPK9 site and an ampicillin resistant site. So I use high expression cells BL21 in order to ensure I have an adequate amount of PK9 to purify.
  • After growing the cultures overnight with Lbamp, I induce the lac operon with IPTG which mimics the allo-lactose sugar that turns on the DNA to allow for expression and I begin the purification process.
  1. I then use three main methods of purification: a GST column, gel filtration, and an anion exchange column
  • In the GST (glutathione s-transferase), I use an affinity purification method where I bind the GST-PfPK9 protein to the resin which has glutathione. I then wash to remove non-specific binders and then compete off with an excess of glutathione so that the protein diffuses off the resin more pure. I then further purify the elute
  • Another method I use for purification purposes is gel filtration which is a form of separation based solely on size. I still use resin however, these beads have pores through which the large proteins cannot diffuse through therefore the larger contaminants will flow through the fastest and the smallest proteins come off last. Since I know the size of PfPK9, I can then speculate at which point it will flow through the resin, but I also collect fractions from the whole column elution and test it for PfPK9.
  • In the event that we have not achieved optimal purification, I then use an anion exchange column in which I know that the resin is positively charged while our protein is negatively charged. Therefore, we can load at low concentrations and then gradually increase the salt concentration which allows the negative chloride ions to slowly build up and compete off proteins.
  1. In order to check whether we have purified enough protein to begin an assay, I perform a gel electrophoresis use coomassie and SDS/PAGE buffer. I then perform a western blot by using various imaging buffers in order to ensure that I can clearly visualize the bands. Darker bands at the right size indicate that we have purified enough protein to begin an assay. To confirm, we run MALDI or check our values using a nano-drop machine which is similar to a Bradford assay.
  2. Although I have not yet reached this step, after obtaining enough pure protein, the next step would be to perform an ATPase activity “hydrolysis” assay. This in vitro assay will allow me to determine what the level of inhibition is. I expect that there should be more ATP hydrolysis in the presence of UBC13 because it should be phosphorylated by PK9.

After a long day of purifying protein (successfully or not), there is a new to-do list on the board for the next protein purification steps for the next day. As a side note, we do not simultaneously purify UBC13 with PK9. Once we are at the purification stage for PK9, we begin expressing UBC13.

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