Also, I've taken the lack of comments from my blog group on my last post to mean that they don't mind if my blog posts get to be a bit longer, or if I decide to start posting more often. Thanks, guys!
As for new experiences this week (since missing the bus isn't exactly new):
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| My cousin and I in bubble balls. |
Earlier, I was able to watch tests on oocytes (frog eggs). Since I'm not a student at BYU, I can't really work with living cells, but I'm still able to observe (and, of course, ask questions). My trainer, Kelly, injected oocytes with the full length M2 protein, then waited a few days for the cells to express the channels. His results will be a bit more accepted than mine, mostly because oocyte tests are common, while make-your-own-liposome tests are few and far in between. Anyways, moving on. When he was ready to test his oocytes, I followed him into the lab next door and saw this.
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| How come their death ray machine is better than our death ray machine? We have to step up our game. |
I'm glad that I was able to see this experiment, though. Apparently Kelly has been working on this setup for months, and he's only started getting decent results recently. (And, as a side note, I think that we're eventually getting one of these in our lab. Yay for two death ray machines.)
After much more discussion, we've decided to change the M2 protein and lipid concentrations - again. I hope this is the last time, but I wouldn't be surprised if it was changed again while I'm gone for two weeks. (I'm going back to Arizona for some prior commitments, but I'll come back to the lab. And yes, in case you were actually worried, I will still be making blog posts) Naturally, we didn't decide on this until after I made my liposomes, so I'll make liposomes with the correct concentrations when I return.
Everything mostly ran smoothly this week, so I can't really complain. I learned, however, that chemicals and equipment tend to run out rather quickly, especially when you're not in the lab. So I spent some time on Thursday making new CCCP and valinomycin solutions, and I'm willing to bet that those will be gone upon my return. Such is life in the lab. I also learned that some substances just don't like to be dissolved. It turns out that most of the drugs I'm testing have hydrophobic groups.
This week, I think I've almost perfect my vial drying method. Since any water droplet in a test tube or vial could dilute our solutions, we have to shake them out and let them air dry until all visible water droplets are gone. The shaking includes vigorous amounts of arm shaking, along with a bit of wrist flicking. And the best part is, I haven't accidentally let go of the vial while drying it. (Yet.) (It's amazing how much our technology our methods use. It's almost as if I'm back at school.)
Because seven out of my eight minutes of actually testing liposomes is filled by me staring at the clock, waiting for the right time, I've come up with multiple ways to keep myself entertained. Sometimes, I'll try to clean up as much as I possibly can (or add pipette tips to the box) before my minute is up and I have to add something to the liposomes. Other times, it's as if I'm playing Simon Says, except it's Computer Says. I have to make sure I follow what's on the computer screen. To make things more difficult, the computer beeps at different intervals (it was used for similar testing, but the process has changed, and no one bothered to make the beeps on time). I think I'm winning this game of Computer Says so far.
Four out of six of my liposome samples this week were great. As for the other two.... The results from the S31N without drug and S31N with drug look exceedingly similar. Which isn't really that good. The drug is either horrible at blocking this mutant M2 channel, or there's something about the higher concentration of M2 proteins and lipids that's not ideal. Either way, I'm sure I'm going to be testing this same drug when I get back, just to see what the problem is.
I'm sure I'll have plenty of time to explain what "good" and "bad" results are later, but for now, I'll explain about pH meter "noise" (I'm sure I could explain both, but this post is getting to be longer than expected. Hopefully no one minds).
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| With "noise." |
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| Without "noise." |
Pictures, I think, speak louder than words. And maybe you can now appreciate the small scale that we're working on. Also, the numbers on the left are not the actual pH; they're a voltage of some sorts. The pH is somewhere in the 6 to 7 range, but the change in the voltage and the change in the pH are the same.
And happy early pi day! I wish I had been born tomorrow at 9:26 am.
Until next time!
- Lauren
P. S. I think I'm going to claim next week as my "spring break" since I'll be playing soccer all of next weekend, so if I don't post, that's why (I know, I know, I can sense your disappointment).
Hey cool stuff, sorry I haven't been commenting as much as you'd like, I'll be better :)
ReplyDeleteIt seems like you're finally getting some cool results to show us! However you did say "The drug is either horrible at blocking this mutant M2 channel, or there's something about the higher concentration of M2 proteins and lipids that's not ideal"
Which problem do you think it is? If it's part of the second problem, what do you think it is that's not ideal?
It's hard to say. I'm not that experienced with the structures, so I don't have much information to make a guess on. I want to say there was something wrong with the S31N liposomes, but, like I said, I'll have to test this more. I've been told that higher concentrations of M2 proteins and lipids might make it harder for the M2 channels to form correctly in the liposomes. The channels might become warped if there's too many of them. But then again, maybe something went wrong in my process of making the liposomes as well. Or, the S31N proteins that I used might not be good anymore. There are so many different ways to fail in making these, unfortunately.
DeleteFirst off, I seriously want a bubble ball now.
ReplyDeleteA few random questions: what are the axis units on your graphs? You said 'chemicals and equipment tend to run out quickly', what kind of equipment do you use that runs out? Is it like one-time-use stuff? Also on your 'death ray', what are those two needle-like devices on both sides of the binoculars?
Looking forward to seeing where this project takes you!
The bubble balls were pretty fun, especially for soccer, haha. But the x-axis is time (in seconds), and I'm not entirely sure what the y-axis is. I think it's some measure of voltage, since that's what the pH meters are reading. And some of the equipment that runs out are the filters and filter supports that I use for extruding, along with the small glass vials, pipette tips, and eppendorf tubes. All of these are one-time-use since they're so small and cheap; it's better to just get new, (supposedly) sanitized materials rather than try to clean all of the small things.
DeleteLastly, the "needles" are actually clamps. They hold the oocyte in place while fluid (such as drug solutions) moves into the chamber. One of them is a voltage clamp, so it keeps the voltage in the cell constant, since most cells have a negative electric potential. It uses a current to do this, and that current is measured. The oocyte experiment therefore depicts a graph showing the change in current in time, while my liposome experiment depicts a graph showing the change in voltage (or pH) in time.
Even though the graph without noise is more precise, both have a defined general shape to them. How important is it to have less noise?
ReplyDeleteIt's very important. In the graph with noise, the first valinomycin signal (which happens around 70 seconds) can barely be seen. We need a very distinct and clear signal; if there's noise, we might get false signals, or we might miss the signal. That first signal is what tells us how many protons are entering the liposomes per M2 channel per second, so we need pretty exact measurements.
DeleteHey Lauren, first of all sorry for lack of commenting last week (#SpringBreak). I love the narrative style of these posts! It sounds like you're really getting the full lab experience...from the boring wait periods to the exciting "death ray" machines!
ReplyDeleteWith regards to the drug for S3N1, do you think the problem is with a small chemical domain or the whole molecule itself (I.e. can you just use another molecule in the same class, or would you need to try a whole new class of drug to target S3N1?)
That's an interesting question. In theory, all forms of the drugs I'm using have the potential to block the M2 channels, the mutant S31N channel included. I'm using amantadine (which worked in non-mutated M2 channels) derivatives. Since we don't know the exact 3D structure of any of the M2 channels yet (and how they interact with water and other molecules), it's all a guessing game as to which drugs could block the M2 channels.
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