Biology Reference
In-Depth Information
For efficient separation and to obtain sharp bands of nonpolar lipids, a two-step
separation system is used. First, lipids are separated on a TLC plate using light
petroleum/diethyl ether/acetic acid (35:15:1; per vol.) as a solvent system until
the front reaches 2/3 of the height of the plate. After drying the plate, the separation
is continued in the same direction using light petroleum/diethyl ether (49:1; v/v) as a
second solvent system until the front reaches the top of the plate. On the dried plate,
ergosterol and ergosteryl esters can be quantified by densitometric scanning with a
TLC scanner (CAMAG) at 275 nm. Ergosterol can be used as standard for both com-
pounds. To quantify the amount of ergosteryl esters, the value calculated from the
densitometric analysis has to be multiplied by factor 1.67 because the average
molecular mass of ergosteryl esters compared to ergosterol is enhanced by this fac-
tor. For irreversibly staining of nonpolar lipids, the TLC plate is incubated in a char-
ring solution of 0.63 g MnCl
2
4H
2
O, 60 mL water, 60 mL methanol, 4 mL conc.
sulfuric acid for 10 s followed by heating at 105
C. The staining intensity depends
on the heating time, which should be at least 30 min. The scanning of bands for non-
polar lipid quantification should be performed shortly after charring, because the
spot intensity bleaches out with time. SE and TG visualized as described above
can be quantified by densitometric scanning with a TLC scanner (CAMAG) at
400 nm. Triolein is used for the quantification of TG, whereas cholesteryl oleate
can serve as standard for SE.
For the analysis of diacylglycerols (DG), an aliquot of LD is extracted as described
above and solved in chloroform/methanol (2:1; v/v). A 1-2
g protein equivalent of
m
the LD fraction and 0.5, 1, 2, and 5
g of diolein standard are spotted onto a TLC plate.
Since DG and ergosterol show similar Rf-values with solvent systems described above,
they should preferentially be separated in an ascending manner using chloroform/ac-
etone/acetic acid (45:4:0.5; per vol.) as a solvent system. After drying and irreversibly
staining the lipids as described above, DG can be quantified by densitometric scanning
with a TLC scanner (CAMAG) at 400 nm using diolein as standard.
m
2.2.3.3
Phospholipid analysis
For a separation of individual phospholipids a two-dimensional TLC separation system
is recommended. Therefore, a LD equivalent to 0.1-0.2 mg protein is extracted, lipids
are solved in chloroform/methanol (2:1; v/v) and applied as single spot to a TLC
plate (10
10 cm) approximately 1-1.5 cm distant from one corner of the plate. Phos-
pholipids are separated using chloroform/methanol/25%ammonia (65:35:5; per vol.) as
a first solvent system for approximately 50 min. After drying the TLC plate, lipids are
further separatedusing chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5;
per vol.) as a solvent systemfor the seconddimension for another 50 min. Phospholipids
are visualized on the dried TLC plate by staining with iodine vapor in a saturated
chamber for some seconds. Prior to phosphate determination, the iodine vapor has to
be removed by heating the plate after moistening with deionized water.
Phosphate determination of individual phospholipids is carried out as described
by
Broekhuyse (1968)
. Spots are scrapped off and transferred into a phosphate
free glass tube with ground neck. 1, 2, 4, and 6
LofaK
2
HPO
4
solution
m
