This week, I had to relabel PKA-R W260A mutation with cy5 again. The concentration that I had started with ended up being too low around absorbance of .6 that when I tried to remove the cAMP by running it in the centrifuge, some of the protein got lost as well. Losing protein in the process is common, but for this reason, it is a good practice to have a protein absorbance of around 2 in the beginning, and as shown, my initial protein absorbance was too low. This resulted in the protein concentration being too low to be detected by the nanodrop after running it through the NGC to remove excess dye after labeling.
Then this week, I was able to successfully remove the cAMP in PKA-R as the ratio of A260/A280 dropped from .8 to .5. I started by concentrating my protein to have an absorbance of 1.9 initially. After labeling it with dye and then running it through NGC, the results were successful with a satisfying labeling efficiency yield and protein absorbance reading. I found out the importance of troubleshooting when there is an issue, to not just continue on without knowing what the issue is, but trying to find the issue at its core so that the next trials would be able to fix the issue.
Hi! My name is Erica Hahn, and I am a rising sophomore at Georgetown University, majoring in Biochemistry.
This summer, I will be working with Dr. Rodrigo Maillard from the Chemistry Department. The Maillard lab focuses on the mechanisms of regulation and signal transduction of Protein Kinase A (PKA) which is a family of proteins that works to phosphorylate other proteins in complex signaling pathways in cells. Because PKA is involved in many critical signaling pathways, it has been associated with many diseases including Carney Complex and Acrodysostosis, making it an important therapeutic drug target. My project over this summer would be to observe and detect the behavioral changes of PKA using FRET (fluorescence resonance energy transfer) sensor that our lab developed. Specifically, I will be observing the interaction between the catalytic subunit of PKA and smoothened protein in hedgehog signaling which has shown to regulate PKA activity, different from the traditional PKA regulator cAMP and regulatory subunit. This research will be conducted in collaboration with Dr. Ben Myers from the University of Utah.
I am very excited to pursue this research opportunities and to connect with various scholars. Please do not hesitate to reach out!
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