Upload
kristin-barber
View
236
Download
2
Embed Size (px)
DESCRIPTION
Discontinuous gradient by underlayering C A B D
Citation preview
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