1- Making Density Gradients Pre-formed discontinuous gradients Pre-formed continuous gradients...

1- Making Density Gradients• Pre-formed discontinuous gradients• Pre-formed continuous gradients• Self-generated gradients

2- Density Gradient Harvesting

Discontinuous gradient by overlayering

BA C

Discontinuous gradient by underlayering

CA B D

top bottom

density

Diffusion of a discontinuous gradient

Rapid formation of continuous from discontinuous gradient

Discontinuous

Continuous

45-60 min

Two-chamber device for continuous gradients

A B

M

TSB SBP

The Gradient Master

B

Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of

timeDensity (g/ml)

Fraction Number1 3 5 7 9

1.07

1.09

1.11

1.132 min2.5 min3.0 min

Swinging-bucket rotoraxis of rotation

At restg

Spinning g

Fixed-angle and vertical rotors

Fixed-angle Vertical

g

1g

Axis ofrotation

Vertical

Swinging-bucket

Sedimentation path length of rotors

Sample/gradient in vertical rotor

Axis of rotation

A B

Self-generated gradients • At 360,000gav iodixanol

molecules will start to sedimentg

time

Self-generated gradient strategy

1g 1g

350,000g350,000g

Near-vertical rotors are best of all

Iodixanol self-generated gradient requirements

• Rotors with a short sedimentation path length ~ 17 mm

• RCF of 180,000-350,000gav

Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at

365,000g

Fraction Number

Density (g/ml)

1 3 5 7 9 11 13 151.00

1.05

1.10

1.15

1.20

1.25

1.30

3 h

15% iodixanol1 h 3 h

1 h20% iodixanol

Fraction Number3 5 7 9 11

Den

sity

(g/m

l)

11.0

1.1

1.2

1.315% (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 FractionCollector

96-well plate

Harvest gradient into equal volume fractions by tube

puncture

Upward displacement

P

Perfluorodecalin

P

Perfluorodecalin

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