User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/10/28/Lipid comparisons

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Contents

Lipids

So I am going to consolidate all my knowledge so far on lipids as passivators here. The lipids I have are:

  • PC
  • PE-PEG
  • PE
  • TAP (positive lipid head group)
  • PG (negative lipid head group)

I'm going to look at all of them using the same procedure to make a slide and we shall see what happens.

Reconstitution

So I reconstituted the PG, TAP, and PE in a 1:1 solution of Methanol:Ethanol. Everything seems to be going into solution so this is great!

I should also note that I have made the solutions 10 mg/mL. I got 25mg and put 2.5mL of 1:1 MeOH:EtOH.

  • Also, it seems that the PG is not completely going into solution. I'm a bit concerned but I don't think I will ever make a slide is totally PG.
  • I also made a nice little stand for the lipids out of Styrofoam.

Flow cell preparation

Just like every other time I have done this, I will start off with using the hydrophobic slips.

  • I first need to cut them down to size so that they fit in the centrifuge.
  • I will then spin coat the lipids at 10 mg/mL on them using 100 ┬ÁL of solution.
  • I will construct a flow cell using the kinesin flow cell protocol with a modification for using 2 hydrophobic slips instead of a slip and a slide.

Motility check

So I went ahead and made sure that my motility worked before trying to look at lipid passivation. Well, it works and not only does it work, but it is still trucking along after sitting for an hour! I'm super excited!

  • Motility check

  • Motility check after 1 hour of sitting on the microscope (went to lunch). It still works!

  • Oh yeah! 3 hour check and it still works! And yes, it took me 2 hours to prepare the samples below that ended up not working. But, who cares? I'm jazzed that this worked so spectacularly.

SJK 02:05, 29 October 2009 (EDT)
02:05, 29 October 2009 (EDT)Hot Diggity?  Do they maybe get more wiggly and jittery (ends detaching) over time?
02:05, 29 October 2009 (EDT)
Hot Diggity? Do they maybe get more wiggly and jittery (ends detaching) over time?

New lipids

I want to point out that I am rather concerned about the new lipids I got in powder form not going into solution. This is the first time I have ever tried using lipids in powder form and I don't think I was able to get them completely in solution as is apparent from the splotchy nature of the slides I prepared. Oh well. I guess I will just have to run with it for now. I'm going to send out an email to Avanti to see what they suggest.

Andy Maloney 18:18, 28 October 2009 (EDT): Update

So after preparing the slides I went ahead and tried to add the PEM to the flow cell. Well, I'm using hydrophobic slips and guess what, no PEM would go into the flow cell. This means that I do not have a coating of lipid molecules in the flow cell.

So what could this mean exactly? I'm not sure but I think it is because of the melting temperature of PE being so high. I'm going to try and heat up the slide on the hot plate to see if I can get the lipids to melt into a coating. We shall see. It also could be that a 1:1 MeOH:EtOH is not going to work for reconstituting lipids from the powder form. This might mean that I have to buy the chloroform stuff always to ensure that I have lipids in solution. I should point out that the only reason why I didn't get the chloroform stuff is because the hydrophobic slides I am using are made of plastic. Chloroform eats this stuff but the MeOH:EtOH sauce doesn't.

Hot plate

Well I did try heating the slide and yep, the bead of PEM I had on the flow cell immediately went in the cell after heating it up. I have no idea if this is because I melted the slide or if I was able to get the lipids above their melting temperature. Suffice it to say, I cannot go the route of using plastic slides any more. I can't wait to learn the silane chemistry tomorrow!

Cells that worked

So I made a cell with PC on it and I was able to get PEM to go in it. I also made one with 10% PG and 90% PE and I got a solution to go in that.

Hmm...I don't know what is going on then.

Andy Maloney 18:56, 28 October 2009 (EDT):

When trying to get kinesin in the flow cell for the PG stuff, I couldn't get it to go in. So, I really don't know what is going on.

Andy Maloney 19:46, 28 October 2009 (EDT): Update

Even though nothing worked, I was still able to get the PC slide to work. I allowed more light to come through the sample stage and you can see, catastrophe occurs rather rapidly when I do.

SJK 02:10, 29 October 2009 (EDT)
02:10, 29 October 2009 (EDT)Wow, that is one long-ass mofo that disintegrates in that last video!  I'm sorry this didn't work, but very glad you got such great videos.  I'm not saying we should spend much time thinking about it, but there is probably something to be learned from the mysteries in these videos.  What is it that is causing them to be more light sensitive?  I've seen this light-sensitivity problem in my Sandia work, and I think it's well-known.  (Haiqing sent us that paper on ascorbic acid supposedly reducing the problem, in addition to photobleaching.)  But what is it about PC lipids that render antifade innefective?  From the way it looks, it would seem like something extra (a small molecule) is in solution?  I really have no idea.  Looks to me like you had a great day of research, despite the negative results.
02:10, 29 October 2009 (EDT)
Wow, that is one long-ass mofo that disintegrates in that last video! I'm sorry this didn't work, but very glad you got such great videos. I'm not saying we should spend much time thinking about it, but there is probably something to be learned from the mysteries in these videos. What is it that is causing them to be more light sensitive? I've seen this light-sensitivity problem in my Sandia work, and I think it's well-known. (Haiqing sent us that paper on ascorbic acid supposedly reducing the problem, in addition to photobleaching.) But what is it about PC lipids that render antifade innefective? From the way it looks, it would seem like something extra (a small molecule) is in solution? I really have no idea. Looks to me like you had a great day of research, despite the negative results.
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