GE 71-5000-15 AD - Manual (Page 5)

p. 5
5. Wash away exess ligand with at least 5 medium volumes of coupling buffer.
6. After coupling, non-reacted groups on the medium should be blocked.
Transfer the medium to 0.1 M Tris-HCl buffer pH 8.0 or 1 M ethanolamine
pH 8.0. Let it stand for 2 hours.
7. Wash the coupled medium using alternate low and high pH.
Recommended buffers are 0.1M acetate buffer pH 3-4 containing 0.5 M
NaCl and 0.1 M Tris-HCl buffer pH 8–9 containing 0.5 M NaCl. A suitable
procedure could be 3x1 medium volume Tris HCl buffer followed by
3×1 volumes acetate buffer. Repeat this cycle 3–6 times.
8. The coupled medium is now ready for use. To prevent microbial growth,
store in 20% ethanol for example.
3. Column packing guidelines
General column packing guidelines for Sepharose Fast Flow based media.
3.1 Recommended columns
Lab-scale columns
• Tricorn
TM
5/20 (5 mm i.d.) for bed volumes up to 0.55 ml at bed heights up
to 2.8 cm
• Tricorn 5/50 (5 mm i.d.) for bed volumes up to 1.1 ml at bed heights up to
5.8 cm
• Tricorn 10/20 (10 mm i.d.) for bed volumes up to 2.2 ml at bed heights up
to 2.8 cm
• Tricorn 10/50 (10 mm i.d.) for bed volumes up to 4.5 ml at bed heights up
to 5.8 cm
• Tricorn 10/100 (10 mm i.d.) for bed volumes up to 8.5 ml at bed heights up
to 10.8 cm
• XK 16/20 (16 mm i.d.) for bed volumes up to 30 ml at bed heights up to
15 cm.
• XK 26/20 (26 mm i.d.) for bed volumes up to 80 ml at bed heights up to
15 cm.