M. Johan Broekman
hanbroekman at csi.com
Wed Sep 15 19:11:47 EST 1999
"Paul S. Brookes." wrote:
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> The most commonly used lipid extractions are Folch and Bligh & Dyer, both
> original papers were sometime in the 50's/60's. Folch (my version of) is as
> follows..... add 5 volumes of 2:1 chloroform/methanol to your aqueous sample,
> shake/homogenise/mix, leave 20 minutes, filter into separating funnel and add
> 2.5 volumes 0.73% NaCl. Shake, leave 20 minutes. Draw off bottom organic
> layer thru' sodium sulphate. Add another 1 volume of chloroform to top layer
> and shake/leave to settle again. Repeat this wash another 2 times, combining
> all the extracts in a round bottom flask, and filtering them all thru' the
> Na2SO4, then reduce the whole extract in a rotavap' to about 0.5ml. Throw some
> BHT (say 0.01%) into your 2:1 C/M as antioxidant for good measure. This
> will give you a total lipid extract, so you'll have to do silicic acid column
> chromatography to extract the polar lipids (i.e. mainly phospholipids).
> Basically, load up a glass column with 1g silicic acid (300 mesh, make sure its
> been dried at 300 C), and run the sample in about 5ml chloroform. Then run
> 10ml CHCl3 to elute non-polar lipids. Then elute the polar fraction with 10ml
> MeOH. and reduce in a rotavap. This also removes some of the BHT so add more
> to prevent oxidation. To get at specific PL head group classes you'll have
> to do TLC or HPLC.
> Dr. Paul S. Brookes. (brookes at uab.edu)
> UAB Department of Pathology, G004 Volker Hall
> 1670 University Blvd., Birmingham AL 35294 USA
> Tel (001) 205 934 1915 Fax (001) 205 934 1775
> The key to success is to have low expectations
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For another opinion:
I never liked the Folch method, mainly because the volumes get so big.
I always used a slightly modified Bligh and Dyer method, which I believe
we described initially in some detail in our 1971 paper on platelets (ie
a tissue that is very dispersed in aqueous buffer, not at all like solid
tissue). The reference is:
P. Cohen, M. J. Broekman, A. Verkley, J. W. Lisman, and A. Derksen.
Quantification of human platelet inositides and the influence of ionic
environment on their incorporation of orthophosphate-32P. J.Clin.Invest.
We found it necesssary to add lots of salt and EDTA to get the
polyphosphoinositides and PA extracted (may not be necessary for you).
Silicic acid column chromatography is very good for preparative purposes
when large quantities/volumes of lipids are used, or if there are very
large quantities of neutral lipids which have to be separated from polar
lipids. If this is not the case (you should know more about your
tissue), it is a waste of time and effort (IMHO).
The TLC system described in the above article now should work with
commercial TLC plates not easily available then, but they should have no
Ca-containing binder. Visualization is by iodine vapor (in an empty TLC
tank put some I2 crystals and marvel at the purple vapor; experiment
with time of exposure, a few minutes is sufficient usually). For FA
determinations we sprayed with a rhodamine 6G. We never used
antioxidant, I believe because it interfered horribly with our gas
chromatography FA determinations. However, it is absolutely essential
to use very high grade (GLC or better) solvents and to dry samples under
high purity nitrogen gas and keep them under N2 at all times, especially
when spotting the TLC plates (see gadget I designed and described in:
M. J. Broekman. Endogenous phosphatidylinositol 4,5-bisphosphate,
phosphatidylinositol, and phosphatidic acid in stimulated human
platelets. Meth.Enz. 169A:415-430, 1989.)
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