I am trying to extract lipid from Y. lipolytica. I had done sonication of the cells suspended chloroform-methanol （2:1). But after addition of 1% NaCl, some of the samples form bilayer spontaneously, but not for the others. Can i know the possible reasons for this inconsistency?
P/s:
1. Can I also know what is the white layer in between the bilayer? Are them proteins?
2.Does it matter if the white stuff is suspended in either of the top or bottom layer once adding NaCl solution? (Some of the samples have the stuff suspended at the top but some of them is suspended at the bottom layer.)
Thanks in advance!
Based on a quick scan through literature it seems that the main part of the lipids extracted are non-bilayer forming lipids.
A good check on the best extraction method might be worthwhile too.
See for example:
Article Bio-Based Solvents for Green Extraction of Lipids from Oleag...
The method you used can indeed extract some (membrane) proteins too which might (in part) explain your observations.
See also the care you have to take regarding things like evaporate all solvent carefully. See for discussions on this for example:
https://www.researchgate.net/post/Are_there_alternatives_to_Nitrogen_drying_for_evaporating_chloroform_from_lipids
https://www.researchgate.net/post/How_much_time_do_we_need_to_evaporate_organic_solvents_in_a_2-mL_solution_frozen_under-80_degrees_using_a_rotary_vacuum_Scanvac
https://www.researchgate.net/post/What_is_the_best_method_of_removing_solvents_like_chloroform_THF_dichloromethane_without_using_a_rotary_evaporator
Hope this is of any help to you.
Rob, many thanks for the suggestions and information given!
However, I have a thinking that whether the separation process is interfered by biosurfactant produced by Y. lipolytica (if it is present). Because from the article given (http://ijirts.org/volume4issue3/IJIRTSV4I3009.pdf), I learnt that Y. lipolytica can actually secrete biosurfactant.
So, how do you think?
Thanks in advance!
Dear Lee Yee Seng,
I am no expert in the field of Y. lipolytica , but I notice that almost every paper (including the one you mention) mentioning Biosurfactant tried to characterize its emulsifier properties but none really tried to identify what component (or components) cause this.
I found one example:
https://s3.amazonaws.com/academia.edu.documents/38170274/proc_biochem_41_2006_1894.pdf?AWSAccessKeyId=AKIAIWOWYYGZ2Y53UL3A&Expires=1549839446&Signature=86wR9jgKLEeQzmxBFOyir4VvjI8%3D&response-content-disposition=inline%3B%20filename%3DShort_communication_Production_and_chara.pdf
This paper mentioned a carbohydrate-protein-lipid complex. Anyway most papers mention methods to optimize the amount of Biosurfactant (so to me it is unclear whether under ‘normal’ circumstances like yours this amount is significant.
I have to repeat my assumption that (based on the overall lipid(like) composition) the extract is presumably not able to form (stable) bilayers. I am not sure why you want this (studying protein-lipid interactions etc.), but if so I would recommend:
-Select the best extraction method based on what can be found in the literature.
-Include a purification step in order to get rid of possible protein content and/or to get a pure lipid fraction (phospholipids and glycolipids). What purification method to choose depends very much on the quantities you need (and the possibilities in the lab you work in).
Good luck with your work!
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