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

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Presentation transcript:

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

Discontinuous gradient by overlayering B A C

Discontinuous gradient by underlayering C A B D

top bottom density Diffusion of a discontinuous gradient

Rapid formation of continuous from discontinuous gradient Discontinuous Continuous min

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

The Gradient Master B

Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time Density (g/ml) Fraction Number min 2.5 min 3.0 min

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

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

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

Sample/gradient in vertical rotor Axis of rotation AB

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

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

Near-vertical rotors are best of all

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

Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at 365,000g Fraction Number Density (g/ml) h 15% iodixanol 1 h 3 h 1 h 20% iodixanol

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

Removal of banded material using a syringe

Collecting a band from a sealed tube

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

Upward displacement P Perfluorodecalin P

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

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