I need help with the membrane fatty acid determination - (Sep/15/2009 )

Hello everyone,
I'm going to determine the fatty acid profile of my E. coli strains but the available method we can use is just for total fatty acid analysis. The questions are: For membrane fatty acid analysis, I can use the membrane fraction by ultracentrifugation or sucrose gradient method? How can the fatty acids be separated from the phospholipids they attached to in the membrane? and Using the ultracentrifugation, would the membrane fatty acids be contaminated with other cellular free fatty acids?
I really need the experience of someone who got familiar with that.
Thank you in advance.

I am looking at free fatty acid concentration in media. It has given me gray hairs trying to sort out a proper protocol. I eventually had to splice a few protocols together to make something appropriate. What analytical technique are you using? Gas Chromatography?
If you find any useful resources please paste because I am feeling just as lost!

Stephan on Sep 16 2009, 03:00 PM said:

what i have in hand is the Whole cell fatty acid analysis (MIDI) using the GC. There are two methods (I heard from the technician): ethyl esters and methyl esters, but I can use only the latter (Fatty acid methyl esters - FAMEs). The methylated fatty acids will be detected by gas-liquid chromatography on an HP 6890A gas chromatograph equipped with a flame inonzation detector. Conditions: injector temperature 250 degree, detector temperature 300 degree, initial column temperature 170 degree, increasing by 5 degree/minute to 270 deg. in 20 min, carrier gas flow rate 50 degree/min, sample volume 1 ul.
I think your critical point is extracting the fatty acids from the medium, right? In my case, it's isolating the membrane fraction , I don't know which method is appropriate.

Well that sounds pretty much the same as what I'm doing except for difference in fatty acid source. My sample sizes are only 200ul which made most protocols unuseable unless I adapted them. Also most require some sort of solid sample to extract from which I also didnt have.

I'm not sure how far ur reading has taken you but look for the Folch method of lipid extraction and the Bligh and Dyer (1959) method. These two are the most popular.

I found www.cyberlipid.org a very good website for background reading.

I'll give you a brief on the method
1) extract lipids with chloroform:methanol
2) Purify Lipids
3) Derivitize (be that methyl or ethyl)
4) Run on GC

Obviously that is a nutshell in a nutshell but I'm not sure how far you are?

If you have a step by step protocol I would love to give it a read so I could compare.
I have a protocol which is very easy to understand if you are interested?

Stephan on Sep 17 2009, 01:13 AM said:

Thank you so much for sharing but I also have a step by step protocol, it's not difficult. However, my problem is before the first step you mentioned. I need to isolate the membrane fraction of my strain and I don't know which method is applicable to continue the downstream steps for the GC. Base on my reading, for the fatty acid determination, they used the sucrose gradient (but this method is quite old, since 1980s, and it's difficult to perform). The alternative method is using ultracentrifugation but in this case i don't know other fatty acids can contaminate the membrane fraction or not. Anyhow, I will use one of them, if it's not good, I have to change, but it's better to have a good understanding.

The critical element is saponification - hydrolysis of the ester linkages to liberate the fatty acid.

GeorgeWolff on Sep 18 2009, 04:26 AM said:

Yes, I agree .
How about the ratio of cytosolic free fatty acids to membrane fatty acid? If the membrane fatty acids are the major one, maybe I don't need to separate them.

I have a step in my protocol which says I must add 5% NaCl to my derivitized sample. Then I'm supposed to dry under gas and disolve in organic solvent (ethyl acetate).
I have two questions about this

1) The Dried sample creates salt crystals which are then unable to disolve.

2) I was under the impression that chlorinated samples should be avoided for any samples going into a Gas Chromatograph?

Do you perhaps have any answers to these Quasimondo (or anyone else)?

Stephan on Sep 25 2009, 02:08 PM said:

I don't have an answer for that because I'm studying how to use this method. In fact, my protocol is different, I have to add some droplets of SATURATED NaCl to the solution until the two phases separated and use the top phase as the final fatty acids solution for determination by GC.

ARG!! I just realized that the person who gave me the protocol left out the critical step of leaving the NaCl behind. I was thinking that that was the problem but I was following my protocol blindly. If only this thread was around earlier!!
thanks anyway.