p. 4
In order to retain maximum b inding capacity of CN Br-activated Sep harose 4
Fast Flow prior to coupl ing the ligand, us e cold (0–4 °C ) solutions. T he
time interval betwe en washing and coup ling must be minim ised; therefore
preparations of all req uired solution s prior to coupling i s recommended.
1. Prepare the cou pling solution , i.e. dissolve the l igand to be coupled i n
a suitable coupling buf fer, e.g. 0.1 M NaHCO3 pH 8.3 conta ining 0.5 M
NaCl. For good coupling e fficiency avo id unnecessari ly dilute soluti ons
(Recommended rati o of volumes, coupli ng solution/m edium is 0.5:1). Th e
coupling pH depends on t he ligand. Norma lly pH in the range 7–9 is use d.
ppm sugar
400
300
200
100
0
010 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 fi ltrate after wa shing with dif ferent
medium volumes of cold 1 mM HC l.
2. CNBr-activate d Sepharose 4 Fast F low is supplied fre eze-dried wit h
sugar additives and is w ashed initiall y with 10–15 medium volume s of
cold 1 mM HCl, see Fig 1. Use sma ll wash portio ns (e.g. 1 medium vo lume)
and let the mixture equ ilibrate a few minute s during each washi ng step.
After washing, deter mine the exact me dium volume obtain ed using e.g.
centrifugation or PD -10 column (the medium vol ume may vary betw een
experiments).
3. Mix the washed m edium and coupling s olution. Adju st pH to the desired
value. To obtain good reprodu cibility it is wise t o adjust total reac tion
volume to a fixed value wi th coupling buf fer.
4. Coupling is no rmally very fast . At room temperatu re the reaction is
usually completed af ter 2–4 hours. If cou pling is perfor med at 4 °C, it can
be performed overn ight. It may be prac tical to follow the r eaction using
UV-absorbance measurements.