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 PeptideWard, R.V., et al (2000) Biochem. J., 348, 137-144
Fraction numberIncubation timeat 35°
30 min
0 min
18 h
18 days
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
2000g
20 min
Plasma
1.0 ml saline (1.006 g/ml)
100,000g
30 min
Chylomicrons (<0.95 g/ml) removed
Fractionation of human plasma lipoproteins by sequential flotation (II)
Chylomicron-free plasma (1.03 g/ml)
100,000g
20 h
saline
>>1.03 g/ml
1.006 g/mlVLDL
KBr
1.063 g/mlLDL
1.21 g/ml
KBr
HDL
Fractionation of human plasma lipoproteins in a NaCl/KBr gradient
Discontinuous
Plasma (1.21 g/ml)
1.15 g/ml
1.063 g/ml
1.006 g/ml 200,000g
24 h
Continuous
VLDL
LDL
HDL
Lipoprotein fractionation strategy I
Blood Plasma
2000g
20 min100,000g
30 min
LDLVLDL
HDL350,000g
2.5-3.0h
Saline
Chylomicrons
OptiPrep
+ 1/5thvol. of
Harvest gradient into equal volume fractions
P
Gilson FractionCollector
96-well plate
Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector
MB
1 h 2 h 3 h0 h
Fraction Number7 9 11 13 155
Density (g/ml)
1 31.0
1.1
1.2
TLN 100; 350,000g
1 3 5 7 9 11 13 15
8
6
4
2
0
1.6
1.2
0.8
0.4
0
Ch
oles
tero
l (m
M)
TA
G (
mM
)
Fraction numberbottom top
Typical fractionation in 12% iodixanol in the TLN100
From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, ExperimentalMethods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.
Fractionnumber
Comparison of lipoprotein banding in 12% iodixanolgradient (TLN100): plasma from two subjects
From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, ExperimentalMethods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.
Lipoprotein fractionation strategy II
LDLVLDL
HDL
350,000g
2.5-3.0h
6% or 9%iodixanol
Saline
Blood Plasma
2000g
20 min100,000g
30 min
Chylomicrons
+ 1/5th vol. of OptiPrep
0.20
0.10
1.30
1.20
1.10
1.000
44 Fractions
um
oles
/fra
ctio
n
Den
sity
(g/
ml)
Cholesterol profiles of three subjects in 9/12% iodixanolself-generated gradients in TLN100 rotor
From Sawle, A, Higgins, MK, Olivant, MP,and Higgins, JA (2002)J. Lipid Res. 43, 335-343.
340,000g/3h
1D Gel Scanner
Fractionation and analysis of Coomassie blue stained lpsin NVT65
From Davies, IG and Griffin BA (2001) British Hyperlipidaemia AssociationEdinburgh, July 2001; Atherosclerosis (2001) 159, 247-252.
9%
12%3 ml
8 ml
12 3
DensityReferencescale
12
3
0.280.33 0.42
0.53
Analysis of Coomassie-blue stained LDL band
From Davies, IG and Griffin BA (2001) British Hyperlipidaemia AssociationEdinburgh, 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