User:Emmalee Jones/Notebook/Lab Notebook/2010/04/23

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Flow Cells

  • Made up a flow cell with MTs and lipids
  • Had to make more Antifade (remember 12, 12, 6 are the proportions, and BME is in centrifuge tube in the fridge)
  • Results from first cell inconclusive. Thoughts are that lysine coverage may not be uniform. That means there might be bare glass which vesicles would attach to and burst. (Partly because vesicles don't seem big enough)
  • Maybe need to add something to coat bare glass, like casein or BSA. First tried adding whole casein as part of the wash
  • Casein with pem is in nalgene 2 mL vials
  • So new procedure would be:

1) Wash flow cell with casein/pem
2) Put in MTs, let sit for 10 minutes
3) Wash with casein/pem/taxol
4) Put in lipids, let sit for 10 minutes
5) Wash with casein/pem/taxol, seal

  • Results were weird, weird, weird. It looks like the vesicles show up better (they are bigger) and are definitely intact. And they are flowing more than they were. But the MTs have disappeared. There are also all sorts of weird shapes that look like hairs or something, and then weird fluorescent and black clumps. Maybe the casein also stuck to the lysine, and did so in weird clumps, and then the MTs had nothing to stick to?
  • Notes and things to research:
     Interaction of lysine with TR, casein, BSA
     My vials of casein/pem have a green line through the label
     Order more lysine - DONE - delivering to CBME
     Could also look at BSA instead of casein
  • So just a preliminary search reveals that lysine-coated slides have been used to stick casein micelles down to examine them. (Supramolecular Structure of the Casein Micelle, J. Dairy Sci. 91:1709–1721) That probably means that they stick to each other.
  • Quoting from the Dairy Science paper: "Proteins that contain negatively charged groups, such as carboxylate or phosphoserine groups, will bind to the poly-L-lysine, and the complete surface of the grid becomes covered with immobilized protein."
  • This paper talks about interaction of Egg PC (70% of the lipids in the vesicles) and casein: Fang, Dalgleish - Studies on Interactions between Phosphatidylcholine and Casein - Langmuir - 1995. Casein does not seem to interact with vesicles once they are made, but it can be incorporated into vesicles.
  • This article talks more about lipid and casein interaction: Membrane interactions and lipid binding of casein oligomers and early aggregates, Sokolovski, Biochimica et Biophysica Acta, 2008. Basically, lipids bind to casein more when they have a charge.