Biomedical Engineering Reference
In-Depth Information
3.6.1 Cyanogen Bromide Activation
of Polysaccharide-Based Supports
Caution!
Cyanogen bromide (BrCN, bromocyan) is extremely
toxic. Work in a hood. Deactivate cyanogen bromide-containing
solutions thoroughly before wasting.
Cyanogen bromide is a solid with a high vapor pressure at room
temperature; therefore, it is recommended to use a commercially
available solution of BrCN in acetonitrile.
Equipment which was in contact with cyanogen bromide and
washings has to be decontaminated with alkaline potassium per-
manganate solution before cleaning and disposal.
A2M Na
2
CO
3
in de-ionized water
Solutions/Reagents
B .0M BrCN in acetonitrile
This protocol describes the activation of an agarose gel, e.g.,
Sepharose 4B. Other polysaccharide-based supports are handled
analogously.
Wash the agarose gel on a sintered-glass filter with about 5 vol.
of de-ionized water. Suck off remaining water until a solid but not
dry cake is obtained. Mix 10 g of this gel with 20 ml of Soln. A and
cool in an ice bath to 0
◦
C. Do not use a magnetic stirrer!
In a hood add 2 ml Soln. B quickly to the ice-cold suspension,
mix, suck off after 2 min, and wash with 150 - 200 ml ice-cold water.
For a high degree of activation it is important to maintain the
temperature low and to work quickly.
Use the activated gel immediately for coupling (e.g., Proto-
col 3.6.2), because the reactive groups hydrolyze fast.
As a variant use 1-cyan-4-dimetylaminopyridinium-tetrafluoro-
borate (CDAP) instead of BrCN. This reagent is not so hazardous
and the degree of activation is easier to control.
A
30% acetone (v/v) in de-ionized water
Solutions/Reagents
B
60% acetone (v/v) in de-ionized water
C
30% acetone (v/v) in de-ionized water
D
acetone/0.1 N HCl 1:1 (v/v)
Ten grams of Sepharose are sucked off on a sintered-glass funnel.
First wash with 3 vol.water,thenwith3vol. 30% acetone in water
(v/v), and then with 3 vol. 60% acetone (v/v) in water. Suspend
the gel in 10 ml of 60% acetone and cool to 0
◦
C. Add the needed
amount CDAP dropwise (cf. Table 3.9) followed by the respective
amount of triethylamine (TEA) to the stirred gel suspension. Suck
offthegelafter2min and wash with a tenfold volume of Soln. D.
The activated gel is stable for 1 h.
