Eddie Polanco Blog 3

Midway through the program it has been a really good experience so far. I have learned a lot about e. coli cell culture and how to use them to grow up phage for use in experiments. I have also learned a lot about cancer biology, which has been a great experience because in my lab at UCLA I have been studying how the mechanical properties of cancer cells make them more or less invasive, but here we have been focusing on the surface proteins expressed by T-cells and what allows them to attack cancer cells and what cancer cells can do to hide from them. This has been a great learning experience for me to see how different labs attack similar big picture problems and questions from different directions, which is an important aspect of research. I have also had the opportunity to learn new lab techniques that I have a vague idea of how they work but now I have not only a practical understanding of how to use them and what they are used for in addition to understanding the scientific basis for each of the steps which further amplifies my understanding of the techniques. One weird thing I have come to learn about research being in New York is how differently different universities approach it (and how different state laws are). For example pipette tips are sharps in California but not in New York. This seems like a minor detail but it turns out that this leads to a whole different system of waste management which I though was interesting.

Picture 3-1: Using a sonicator to break up the cell membrane of e. coli cells suspended in 6M urea. We use a sonicator because when overexpressing our protein of interest in e. coli it misfolds into occlusion bodies and the sonicator is needed to break up the occlusion bodies. The urea is used to denature the proteins to prevent occlusion bodies from reforming.

Picture 3-2: Purifying our protein of interest using affinity chromatography. Our protein contains a His-tag allowing us to separate the protein using resin beads coated in Ni to coordinate the imidazole group on the Histidine residues. The protein is then eluted off the column using a buffer with increasing concentrations of imidazole.

Picture 3-3: An SDS-PAGE gel showing the purity of our protein after purifying with the affinity column.

Picture 3-4: Forcing the protein to refold by diluting out the urea in the buffer. The buffer containing the purified protein is dripped from a syringe one drop at a time into a buffer with a high Arginine concentration to help keep the protein folded in solution.