Sedimentation slides

choppedspleenMécanique

21 févr. 2014 (il y a 3 années et 6 mois)

45 vue(s)

Sedimentation etc

Topic 3 Part 1

Biophysics

General Principles

f v

F (like
mg)

Sphere:
f
0

= 6
ph
R

Other particle: get r = f/
f
0

and f = r f
0

Example


prolate with a = 3 and b = 2

Sedimentation

1
-

r

is the buoyancy factor ~ 1
-

r
o
/
r

(if
r
o

>
r

then it floats).

s = sedimentation coefficient, [s] = Svedberg, 1 x 10
-
13

sec = 1 Svedberg.

Density of medium,
r

= m
o
/

V, V = volume.


is specific volume,
volume/mass of substance in solution (V/m)

v


s
r
v
f
N
ρ
v
1
M
2
A




v
v
Determining s

Analytical Centrifuge

This instrument scans
absorption along the centrifuge
cell as a function of time


giving concentration.

Determining s

v
b
= dr
b
/dt =

2
sr
b
, where the subscript b signifies the
boundary (so r
b

is the boundary between solvent
and solution).

So plot ln(r
b
) vs t and get slope which is equal to

2
s.


Determining s

Diffusion blurs boundary

D = RT/N
A
f,


More on s


Depends on temperature and viscosity so
define for standard conditions


s depends on M and f (shape) so if know M
can get f (shape information/stokes radius)

Density Gradient used for
separation

Real data on hemoglobin free and
bound to haptoglobin

Electrophoresis

Now have
F

= Ze
E
, fv = ZeE.

Mobility, U = v/E = Ze/f

Sphere: U = Ze/(6
ph
R)

Movement on a Gel



2
0
tD
2
Ft
x
x
e
tD
1
2
N
t
,
x
C











π
This is a gaussian centered around x = x
o

+

Ft

With rms of , velocity =

F,



= 1/(6
ph
r) with r = radius and
h

= viscosity

D =

k
B
T = diffusion constant












t
x
C
x
F
x
x
t
x
C
D
t
t
x
C
2
2
,
,
,









tD
Running DNA on a Gel


Closed small plasmids give discrete bands


Long DNA tunnels and separation goes as
1/mass


Can get screening from lots of cations


2
-
D electrophoresis good for large pieces of
DNA


Genome project used Saenger method

Running Plectonemic helices


L = T + W


For DNA at several Kbp,
helices run by writhe and
hence (for constant twist), L


Topoisomers for DNA of
defined length give gaussian
band due to different energies


Wang Paper


http://www.cbs.dtu.dk/staff/dave/roanoke/
supercoil.jpg