1. 1- Making Density Gradients Pre-formed discontinuous gradients Pre-formed continuous gradients Self-generated gradients 2- Density Gradient Harvesting

2. Discontinuous gradient by overlayering B A C

3. Discontinuous gradient by underlayering C A B D

4. top bottom density Diffusion of a discontinuous gradient

5. Rapid formation of continuous from discontinuous gradient Discontinuous Continuous 45-60 min

6. Two-chamber device for continuous gradients AB M T SB P

7. The Gradient Master B

8. Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time Density (g/ml) Fraction Number 13579 1.07 1.09 1.11 1.13 2 min 2.5 min 3.0 min

9. Swinging-bucket rotor axis of rotation At rest g Spinning g

10. Fixed-angle and vertical rotors Fixed-angle Vertical g 1g

11. Axis of rotation Vertical Swinging-bucket Sedimentation path length of rotors

12. Sample/gradient in vertical rotor Axis of rotation AB

13. Self-generated gradients At 360,000g av iodixanol molecules will start to sediment g time

14. Self-generated gradient strategy 1g1g 1g1g 350,000g

15. Near-vertical rotors are best of all

16. Iodixanol self-generated gradient requirements Rotors with a short sedimentation path length ~ 17 mm RCF of 180,000-350,000g av

17. Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at 365,000g Fraction Number Density (g/ml) 13579111315 1.00 1.05 1.10 1.15 1.20 1.25 1.30 3 h 15% iodixanol 1 h 3 h 1 h 20% iodixanol

18. Fraction Number 357911 Density (g/ml) 1 1.0 1.1 1.2 1.3 15% (170,000g) 30% (170,000g) 15% (353,000g) 30% (353,000g) Effect of RCF and [iodixanol] in Beckman VTi65.1 (3 h)

19. Removal of banded material using a syringe

20. Collecting a band from a sealed tube

21. P Gilson Fraction Collector 96-well plate Harvest gradient into equal volume fractions by tube puncture

22. Upward displacement P Perfluorodecalin P

23. P 2 P 3 P 4 Labconco Auto Densi-flow collects from meniscus B P 1 C M EP

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