GE 71-5000-15 AD - Manual (Page 4)

p. 4
In order to retain maximum binding capacity of CNBr-activated Sepharose 4
Fast Flow prior to coupling the ligand, use cold (0–4 °C) solutions. The
time interval between washing and coupling must be minimised; therefore
preparations of all required solutions prior to coupling is recommended.
1. Prepare the coupling solution, i.e. dissolve the ligand to be coupled in
a suitable coupling buffer, e.g. 0.1 M NaHCO
3
pH 8.3 containing 0.5 M
NaCl. For good coupling efficiency avoid unnecessarily dilute solutions
(Recommended ratio of volumes, coupling solution/medium is 0.5:1). The
coupling pH depends on the ligand. Normally pH in the range 7–9 is used.
ppm sugar
400
300
200
100
0
0 10 20 30 40 50 60
A-method
A-method: Add portions of 4
column volumes cold 1 mM HCl,
stir and immediately remove the
liquid.
B-method: Add portions of 1
column volume cold 1 mM HCl,
stir for approx. 5 min and then
remove the liquid.
B-method
Gel vol.
Fig 1. The content of sugar in the filtrate after washing with different
medium volumes of cold 1 mM HCl.
2. CNBr-activated Sepharose 4 Fast Flow is supplied freeze-dried with
sugar additives and is washed initially with 10–15 medium volumes of
cold 1 mM HCl, see Fig 1. Use small wash portions (e.g. 1 medium volume)
and let the mixture equilibrate a few minutes during each washing step.
After washing, determine the exact medium volume obtained using e.g.
centrifugation or PD-10 column (the medium volume may vary between
experiments).
3. Mix the washed medium and coupling solution. Adjust pH to the desired
value. To obtain good reproducibility it is wise to adjust total reaction
volume to a fixed value with coupling buffer.
4. Coupling is normally very fast. At room temperature the reaction is
usually completed after 2–4 hours. If coupling is performed at 4 °C, it can
be performed overnight. It may be practical to follow the reaction using
UV-absorbance measurements.