as i am waiting for my baked salmon topped with feta cheese and parsley to cook in the oven, i just wanted to write how frustrated i am today. so frustrated that i wanted to hit anybody. or anything...
the day started out okay. i woke up early enough to prepare breakfast before coming to work, which is a good sign because it's always a good day when i come to work on a full stomach. although i arrived at work midmorning, which i didn't initially plan on doing, i was still able to finish the experiments i needed to do in the morning. it all went downhill late in the afternoon when i was purifying my already "purified" bitchy little looping protein. i put purified in quotation marks because i thought it was highly pure when i ran it on an affinity column. turns out the brat brought other stuff that i don't like. so this time, i ran the sample on a 120-mL size exclusion column, in the hopes that i can isolate each of the other stuff. for the non-science geeks out there, a size exclusion column is used to isolate the components of a protein mixture, as long as the difference in size is huge, i.e. 39 kDa vs. 29 kDa or even better, 39 kDa vs. 10 kDa. the column has little beads with even little holes on it that act as a molecular sieve. as the mixture passes through the column, the small-sized components pass through the holes and get stuck inside the beads while the large components just pass outside the bead. ideally, the big ones get out of the column first and the little ones elute out last.
here's a quick background info on my bitchy little protein..it's tiny with a huge GST tag attached to it (for ease of purification and to keep it well-behaved). supposedly, it should come out clean after a GST affinity chromatography purification. however, the bugger started breaking up after a few days in the fridge, indicating there was something eating it up. in short, the "pure" fusion protein actually had three components: the fusion protein, the GST tag and the untagged bitch itself. i have proceeded to the next step which was proving whether it binds to my DNA (which it did), but i'm getting so many complexes because both the GST-tagged and the untagged protein bind the DNA and there are three ways these two proteins can combine to bind the DNA. bottom line: the more stuff you see, the more complicated life becomes.
to make the story short, the purification worked alright, except that (1) there were a lot more components than what i expected to see, (2) the separation of these components wasn't clean enough, even if the sample was dripping through the column at 1 mL/minute, and the most frustrating of all, (3) the last component that came out (which could be the stuff that i want) went to the waste container. the instrument that i was using is actually automated so there's no reason that i would not be able to collect that last fraction. unfortunately, the bitchy little protein defied all odds by eluting out of the column way after the void volume unlike the rest of the components. what makes me so upset is i was sitting through the whole experiment, staring at the computer screen and manually controlling the fractionation of the desired components. just when i saw the telltale sign that the sample mixture has passed through the column (hence, the void volume), i started cleaning up my working area in the other lab room on the opposite end of the hallway. i was just gone 10 minutes before i checked on the said instrument (since it was still running the program) before calling it a day and when i saw the signal from that last component, my happiness quotient just went poof. ten minutes!!! how would i know that friggin' bitch would hang on a wee bit longer on the column before deciding to come out?!?! all those three hours of purification down the drain. although i was able to collect the rest of the components, thinking about that one fraction that could most likely be the one that would get me into the next step, or get me to graduate...why didn't i wait for another ten minutes before cleaning up my stuff?!
aaaaaarrrrggggh!
in other news, it's halloween night and i don't know what to wear for a costume party. i have an idea but that would mean i better brace myself for a major embarrassment. darn, i should've bought a mask or something to cover my face so no one would recognize me!
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