Passaging and Protein Structure

Today (January 15th), I went to RPI for my first day of lab work of the new semester! Today was full of really interesting new things. First, JP showed me how to culture tissue, particularly the practice of passaging. Passaging is the process of moving some cells from a previous culture to a new growth medium to allow them to continue to grow. The first thing we had to do was spray everything down with ethanol. Everything must be sterile when working with the tissue, so all materials were sprayed, including our gloves. Next, we got the tissue culture out of the incubator, which maintains the CO₂ level and temperature similar to that of the human body. Before passaging the tissue, I was able to observe the culture under the microscope. The proteins that had been previously cultured were connected in a chicken pattern, and they were attached to the surface of the container with linkages that kept them in place. First, the media was removed from the container with a vacuum line. Then, 10 mL of PBS solution was added to the container to remove dead cells and remove the Fetal Bovine Serum (FBS) that inhibits the enzyme Trypsin from acting on the cells. Trypsin functions to cut the anchor linkages that connect the protein to the container, causing them to float around, which is not wanted while the tissue is being cultured. The PBS was then removed from the container with the vacuum line. Next, 1 mL of Trypsin was added to detach the cells from the container. After the Trypsin was active in the container for a few minutes, I observed the cells in the container again, and they were now floating islands of groups of cells. Next, FBS was added back into the container to re-inhibit the Trypsin. This mixture was then transferred to a conical vile to be spun down in the centrifuge. Once the centrifuge was balanced, it was spun at 1000 rpm for 5 minutes to make a protein pellet separate from the media. While the vile was being spun, we prepared a new container, and labeled it as passage #25 of the cells. 35 mL of new media were then added to the new container. After the vile was done in the centrifuge, the old media was drained from the vile using the vacuum line. We then added 1 mL of media to the vile to re-suspend the protein pellet by using the pipette up and down until the protein pellet broke up and dispersed in the media. 100 microLiters of the suspension were then added to the new container, and it was agitated to spread the cells around the surface of the container. The passage was then complete, and we re-cleaned everything with ethanol and put the new culture in the incubator.

After passaging, JP introduced me to a new computer program for modeling proteins. The program displayed both the amino acid sequence and the three-dimensional structure of the protein. My job was to search through the entire amino acid sequence to highlight certain amino acids that are important to the function of the enzyme. Once I highlighted the amino acids in the sequence, I also highlighted the corresponding structures on the enzyme that those amino acids code for. The purpose of this activity was to show the position of the binding site of the protein structure. After locating the binding site and seeing its structure, we looked at how the signal molecule (which is a sugar) binds to the binding site. This program was really interesting because I am learning about protein synthesis and structure in AP Biology right now!!!!

I can't wait to return next week and find out what's next!