We were unlucky today. Filtering out a relatively lightweight protein with various buffer solutions is already a tedious, time-consuming process, but knowing that you’ll likely have to do it all over again is just heartbreaking. We began the day by running mixtures of acid-base buffers (a solution known as HEPES) along with solutions of tiny carbon-nitrogen rings (imidazole) to extract as much of our precious Nb6B9 as possible. I got in a lot of good practice using micro pipettes, especially when we needed to place little volumes of protein indicators in our filtered solutions. The initial results we got from the indicators weren’t pretty – the image below shows a gradient of color from light brown to blue and back to brown. This seemingly harmless, variegated collection told Dr. Masoudi and I that only a few samples of our solution had a high concentration of protein, meaning we had lost quite a bit of Nb6B9 along the way. Our electrophoresis analysis of the results was even more disappointing. This analysis works by introducing a specific binding agent, called SDS, to each of the proteins. Proteins composed of longer amino acid chains will invariably bind with more of the agent, making the larger structures more net-negative and the smaller structures less net-negative. When a current is applied to the sieve, the protein samples travel different distances based on their relative negativity; the larger proteins have a more difficult time reaching the bottom of the cylinder compared to the smaller proteins. Through this process, we were able to identify that we had some Nb6B9 but not nearly enough. We’ll probably have to go through the entire filtration process again to increase our yield… 🙁
Regardless, there’s still hope for tomorrow! Stay tuned!
