Competitive media for protein complex analysis using OptiPrep ™

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

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

5. Density (in potassium bromide) and size of human plasma lipoproteins

6. 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 (I)

7. saline KBr VLDL 100,000g 1.006 g/ml 20 h >>1.03 g/ml Chylomicron-free plasma (1.03 g/ml) LDL HDL 1.063 g/ml 1.21 g/ml KBr Fractionation of human plasma lipoproteins by sequential flotation (II)

8. Continuous Discontinuous VLDL 1.006 g/ml 200,000g LDL 1.063 g/ml 24 h 1.15 g/ml HDL Plasma (1.21 g/ml) Fractionation of human plasma lipoproteins in a NaCl/KBr gradient

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

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

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

12. Density (g/ml) 0 h 1 h 2 h 3 h 1.2 1.1 1.0 1 3 5 7 9 11 13 15 Fraction Number TLN 100; 350,000g

13. Typical fractionation in 12% iodixanol in the TLN100 1.6 8 6 1.2 TAG (mM) Cholesterol (mM) 0.8 4 0.4 2 0 0 1 3 5 7 9 11 13 15 bottom Fraction number 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.

14. 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.

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

16. Cholesterol profiles of three subjects in 9/12% iodixanol self-generated gradients in TLN100 rotor 1.30 0.20 1.20 Density (g/ml) umoles/fraction 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.

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

18. Analysis of Coomassie-blue stained LDL band 1 3 2 Density Reference scale 0.42 0.33 0.28 0.53 2 3 1 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