I was ready for a new approach with my third attempt to purify histone H4. Instead of blindly running a race as before, I decided to grow up many colonies and run their proteins on a gel in order to check which, if any, were expressing the protein well. Kiyoe and I had been discussing the use of a chemical that induces expression of proteins in cells (i.e. kicks it up a notch). Kiyoe thought that adding the chemical would result in over expression and would poison the cells since our protein binds to DNA. I didn’t think we were in any danger of expressing too much of this particular protein and, at this point, was willing to try anything. We reached a compromise- I would split the cultures of each colony in half and would induce half and leave the rest alone. We could then compare the methods side-by-side. On this morning, there were only 10 colonies on my plates from the previous evening- that meant that I would end up running 20 samples on a gel, not a bad number at all.
I messed around with the size-exclusion column some, trying to get the top of it level and at just the right height, but the time soon came to finally load my sample on it. It was running very slowly since it was taller and skinnier than my previous columns, I had the pump at one-third the speed I normally did. I checked on it periodically as it collected fractions but, unlike previously, it did not run into any problems.
For lunch I found a new hot dish in the co-op, Chinese beef and vegetables in XO sauce, a “spicy” sauce that is made of garlic mixed with dried shrimp and scallops. It wasn’t spicy, but the curry wonton soup that I had did have a little kick to it. The column finished running after lunch. To read the protein levels this time, I transferred a small amount of each fraction to a plastic plate with small wells in it to which one adds a substrate that then changes color in the presence of protein. I had not been able to use this method previously since some of the chemicals in my initial purification steps interfered with the colorimetric substrate, hence the use of the Nanodrop instead. I didn’t add the substrate at this time, though, since it was time to go to Kanji Table.
The class started with a speech by a student again, an Italian computer scientist who gave a PowerPoint presentation about his hometown, near Venice. Then, as was also the custom, the group leader picked a few people to stand up and introduce themselves to the rest of the class. I was chosen on this particular day, so I told everyone my name, where I was from, my field, and that I was working at the Medical School. I spent the next hour doing the usual reading and writing in Japanese.
After I returned to lab, I added the color substrate to my fractions and put the plate with the colored fractions into a machine that would read how dark the color change was. I could already tell without the machine that I had a good amount of protein complex in some of the fractions- apparently the purification of the H2A-H2B complex had worked! Formation of the second complex, however, required that I have purified histone H4, I therefore checked on my H4-expressing cells. Kiyoe was right, the treatment with the chemical had killed off many of the cells but a few were still hanging in there. I reasoned that, if they were expressing enough of the protein, it would make up for the fact that there were fewer of them. . I prepped the cells that might be expressing this protein for running their contents on a gel, but decided that actually running it could wait, since it was about 5:30 and I wanted to leave for church in time to have a real meal again. I put the samples and the gel into the cold room and left by 6.
I went to Cavs Coffeehouse nearby the place I had gotten tako yaki a few weeks prior. I ordered a hamburger patty over soba noodles and fried rice. When I was done eating, I still had time to kill, so I walked to the huge electronics store nearby the church building and looked for a cell phone, knowing that Trudy would want to have a phone when she arrived. Up to this point, I hadn’t been in a hurry to get one- since I didn’t really have anyone to call during the 12 hours I spent at the house each day. It took me a while to locate to one that I wanted, and by then it was too late to go through the steps of getting it activated- so I decided to come back before church on Sunday and pick it up.
Church was good- we watched a video of the minister from the church in Tokyo give a lesson about the Holy Spirit, which Fiona translated for me. She only hesitated some when it came to the Japanese word for blaspheme, but eventually figured out what it meant. I assured her that it wasn’t a word we used a lot in casual conversation in America, either. I got home at 11 pm and went straight to bed.