AS_Comments

[adamsychla]

AS_Liposome_Protocol.docx Materials: have concentrations written for POPC and Lyss-Rhod-PE

Step 2.2: a missing "1" after the word "step"

Step 2.7: a more thorough description of what is visually expected would help. The most ambiguity arises here because I got pellet at the bottom, the aqueous layer, some sort of floating aggregate, and the oil layer. It is not clear whether the aggregate should be removed or not.

I ran the protocol twice. The first time I did 4 preps and did get approximately 1 vesicle/10uL sample. In the second run I did 3 prep. The first worked very well with many vesicles. The other two also had vesicles but the emulsions solidified for those two leading to difficulty in transferring to the centrifuge buffer so the yields were much lower.

Overall the protocol is pretty accessible. I could follow it the first time and think the second time worked better largely due to being more familiar with the steps.

[zjuradoq]

Hi Adam,

Thank you for your comments. As for your second point, the aggregate I believe you are referring to is the a lipid raft that forms between the oil and aqueous layer. This is "normal" and should not be removed when removing the vesicles.

Also, when you say solidified, do you mean like clumped up? I saw this happen once before to me, and also resulted in a lower yield. I have images of the solution for reference, but it would be curious to see if this happens to others and why. @OneScientista @PaolaTorre have you ever seen this?

Between your first and second trials can you think of anything you may have done differently, even if it is something small (like blowing on the tubes like one would do in a game of craps).

-Zoila

[OneScientista]

@adamsychla @zjuradoq Hi Adam, it's great that you've run through the protocol. Can you attach your protocol here? You can just edit your previous post with the attachment.

Step 2.7: a more thorough description of what is visually expected would help. The most ambiguity arises here because I got pellet at the bottom, the aqueous layer, some sort of floating aggregate, and the oil layer. It is not clear whether the aggregate should be removed or not.

Your observations are in line with literature and my own observations. You should remove and discard the inter-layer while removing the oil after the first centrifugation step. You only want to collect the pellet at the bottom of the tube.

For a better visual reference: This image above is from: Liposome display for in vitro selection and evolution of membrane proteins Satoshi Fujii1, Tomoaki Matsuura1,2, Takeshi Sunami1,3, Takehiro Nishikawa1, Yasuaki Kazuta1 & Tetsuya Yomo1,3,4 Here are some of my own pictures and a protocol: Build-A-Cell_10_05_18_HPTS_vesicles.pdf

The layer between aqueous and oil phase, to the best of mine and others' understanding is a complex mixture of aqueous/lipid/oil phases. I've examined this mixture with a microscope and it looks like this:

I ran the protocol twice. The first time I did 4 preps and did get approximately 1 vesicle/10uL sample. In the second run I did 3 prep. The first worked very well with many vesicles. The other two also had vesicles but the emulsions solidified for those two leading to difficulty in transferring to the centrifuge buffer so the yields were much lower.

This seems like very low yield. How solid did the emulsion become? What lipids did you use?

[adamsychla]

My yield in the run that worked was in in thousands.

The two that solidified ended with approximately the consistency of lard. I used POPC, Cholesterol, and Lyss-Rhod-PE as described in the protocol.

The key difference is that is used a different stock of Lyss-Rhod-PE. My first run had flat bottom 20mL vials for Step 0 while my second one glass test tubes. The latter was much easier to work with and I think the oil preparation was better. Besides that, I think that it was helpful to run a second time simply because moving between steps was more fluid.