1. Competitive media for protein complex analysis using OptiPrep™ Sucrose and glycerol

2. 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

3. Oligomerization of  -Amyloid Peptide Ward, R.V., et al (2000) Biochem. J., 348, 137-144 Fraction numberIncubation time at 35° 30 min 0 min 18 h 18 days

4. Plasma lipoprotein analysis using OptiPrep™ Competitive media Potassium bromide + sodium chloride

5. Density (in potassium bromide) and size of human plasma lipoproteins NameDensity (g/ml)Size (nm) Chylomicrons<0.9575-1200 VLDL0.95-1.00630-80 LDL1.006-1.06318-25 HDL1.063-1.215-12

6. 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

7. 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/ml VLDL KBr 1.063 g/ml LDL 1.21 g/ml KBr HDL

8. 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

9. Lipoprotein fractionation strategy I Blood Plasma 2000g 20 min 100,000g 30 min LDL VLDL HDL 350,000g 2.5-3.0h Saline Chylomicrons OptiPrep + 1/5th vol. of

10. Harvest gradient into equal volume fractions P Gilson Fraction Collector 96-well plate

11. Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector MB

12. 1 h2 h 3 h 0 h Fraction Number 791113155 Density (g/ml) 13 1.0 1.1 1.2 TLN 100; 350,000g

13. 13579111315 8 6 4 2 0 1.6 1.2 0.8 0.4 0 Cholesterol (mM) TAG (mM) Fraction numberbottomtop Typical fractionation in 12% iodixanol in the TLN100 From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, Experimental Methods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.

14. Fraction number Comparison of lipoprotein banding in 12% iodixanol gradient (TLN100): plasma from two subjects From: Higgins JA, Graham, JM & Davies I (2001) In Atherosclerosis, Experimental Methods and Protocols (ed Drew AF) Humana Press, Totowa, NJ pp 37-49.

15. Lipoprotein fractionation strategy II LDL VLDL HDL 350,000g 2.5-3.0h 6% or 9% iodixanol Saline Blood Plasma 2000g 20 min 100,000g 30 min Chylomicrons + 1/5 th vol. of OptiPrep

16. 0.20 0.10 1.30 1.20 1.10 1.000 44 Fractions umoles/fraction Density (g/ml) Cholesterol profiles of three subjects in 9/12% iodixanol self-generated gradients in TLN100 rotor From Sawle, A, Higgins, MK, Olivant, MP,and Higgins, JA (2002) J. Lipid Res. 43, 335-343.

17. 340,000g/3h 1D Gel Scanner Fractionation and analysis of Coomassie blue stained lps in NVT65 From Davies, IG and Griffin BA (2001) British Hyperlipidaemia Association Edinburgh, July 2001; Atherosclerosis (2001) 159, 247-252. 9% 12% 3 ml 8 ml

18. 1 2 3 Density Reference scale 1 2 3 0.28 0.33 0.42 0.53 Analysis of Coomassie-blue stained LDL band From Davies, IG and Griffin BA (2001) British Hyperlipidaemia Association Edinburgh, July 2001; Atherosclerosis (2001) 159, 247-252.

19. 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

20. 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