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