Transcript Lipoprotein profiling in self-generated iodixanol gradients

Competitive media for protein
complex analysis using
OptiPrep™
• Sucrose and glycerol
ANALYSIS OF PROTEIN
COMPLEX FORMATION AND
PROTEIN OLIGOMERIZATION
• Non-covalent bonds between protein
molecules influenced by tightly-bound
water molecules around amino acid side
chains
• Low water activity (high osmolality) of
sucrose and glycerol gradients leads to loss
of these tightly-bound water molecules
Oligomerization of -Amyloid Peptide
Ward, R.V., et al (2000) Biochem. J., 348, 137-144
Incubation time
at 35°
0 min
30 min
18 h
18 days
Fraction number
Plasma lipoprotein analysis
using OptiPrep™
• Competitive media
• Potassium bromide + sodium
chloride
Density (in potassium bromide) and size
of human plasma lipoproteins
Name
Density (g/ml)
Size (nm)
Chylomicrons
<0.95
75-1200
VLDL
0.95-1.006
30-80
LDL
1.006-1.063
18-25
HDL
1.063-1.21
5-12
Fractionation of human plasma
lipoproteins by sequential flotation (I)
Blood
Plasma
2000g
100,000g
20 min
30 min
1.0 ml saline (1.006 g/ml)
Chylomicrons (<0.95 g/ml) removed
Fractionation of human plasma
lipoproteins by sequential flotation (II)
KBr
saline
100,000g
20 h
VLDL
1.006 g/ml
>>1.03 g/ml
Chylomicron-free plasma (1.03 g/ml)
HDL
1.21 g/ml
LDL
KBr
1.063 g/ml
Fractionation of human plasma
lipoproteins in a NaCl/KBr gradient
Continuous
Discontinuous
VLDL
1.006 g/ml
1.063 g/ml
200,000g
LDL
24 h
1.15 g/ml
Plasma (1.21 g/ml)
HDL
Lipoprotein fractionation strategy I
Blood
Plasma
Chylomicrons
2000g
100,000g
20 min
VLDL
LDL
HDL
30 min
Saline
350,000g
2.5-3.0h
+ 1/5th
vol. of
OptiPrep
Harvest gradient into equal
volume fractions
Gilson Fraction
Collector
P
96-well plate
Labconco Auto Densi-flow linked to a
Gilson FC205 Fraction Collector
MB
Density (g/ml)
TLN 100; 350,000g
0h
1h
2h
3h
1.2
1.1
1.0
1
3
5
7
9
11
Fraction Number
13
15
8
1.6
6
1.2
4
0.8
2
0.4
0
0
1
3
bottom
5
7
9
11
Fraction number
13
TAG (mM)
Cholesterol (mM)
Typical fractionation in 12% iodixanol in the TLN100
15
top
From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, Experimental
Methods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.
Comparison of lipoprotein banding in 12% iodixanol
gradient (TLN100): plasma from two subjects
Fraction
number
From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, Experimental
Methods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.
Lipoprotein fractionation strategy II
Blood
Plasma
Chylomicrons
2000g
100,000g
20 min
VLDL
LDL
HDL
30 min
350,000g
2.5-3.0h
Saline
6% or 9%
iodixanol
+ 1/5th vol. of OptiPrep
Cholesterol profiles of three subjects in 9/12% iodixanol
self-generated gradients in TLN100 rotor
0.20
1.20
0.10
1.10
0
1.00
44 Fractions
From Sawle, A, Higgins, MK, Olivant, MP,and Higgins, JA (2002)
J. Lipid Res. 43, 335-343.
Density (g/ml)
umoles/fraction
1.30
Fractionation and analysis of Coomassie blue stained lps
in NVT65
8 ml
9% 340,000g/3h
12%
1D Gel Scanner
3 ml
From Davies, IG and Griffin BA (2001) British Hyperlipidaemia Association
Edinburgh, July 2001; Atherosclerosis (2001) 159, 247-252.
Analysis of Coomassie-blue stained LDL band
1
3
2
Density
Reference
scale
0.42
0.33
0.28
0.53
2
1
3
From Davies, IG and Griffin BA (2001) British Hyperlipidaemia Association
Edinburgh, July 2001; Atherosclerosis (2001) 159, 247-252.
Advantages of the new technology
• Lipoproteins close to their native state
• Self-generated gradients simple and
reproducible
• Close to 100% recovery of lipids
• Identification and quantitation of the
principal LDL subclasses
• Potential for adaptation of techniques to
HDL and VLDL subfractionation
Publications database for
macromolecules and
macromolecular complexes
• OptiPrep (since 1994) over 100
• Nycodenz® (since 1984) approx 100
• Using either the OptiPrep Applications CD or the
website:
• www.axis-shield-density-gradient-media.com
• Follow the instructions to access the relevant Index
• Click on the macromolecule or macromolecular
complex of interest