p. 5
5. Wa sh away exess lig and with at leas t 5 medium volumes o f coupling buf fer.
6. After coupli ng, non-reacte d groups on the medi um should be blocke d.
Transfer the medium to 0.1 M Tris-HCl b uffer pH 8.0 or 1 M etha nolamine
pH 8.0. Let it stand for 2 hour s.
7. Wash the co upled medium usi ng alternate low a nd high pH.
Recommended buffe rs are 0.1M acetate buf fer pH 3-4 containin g 0.5 M
NaCl and 0.1 M Tris-HCl buffer pH 8 –9 containing 0.5 M Na Cl. A suitable
procedure could be 3x1 medi um volume Tris HCl buf fer followed by
3×1 volumes acetate buf fer. Repeat this cycle 3– 6 times.
8. The coup led medium is now re ady for use. To prevent micro bial growth,
store in 20% ethanol for ex ample.
3. Column packing guidelines General column packin g guidelines for Sep harose Fast Flow b ased media.
3.1 Recommended columns
Lab-scale columns
• TricornTM 5/20 (5 mm i.d.) for bed volu mes up to 0.55 ml at bed hei ghts up
to 2.8 cm
• Tricorn 5/50 (5 mm i.d.) for b ed volumes up to 1.1 ml at b ed heights up to
5.8 cm
• Tricorn 10/20 (10 mm i.d.) for bed vo lumes up to 2.2 ml at bed h eights up
to 2.8 cm
• Tricorn 10/50 (10 mm i.d.) for bed v olumes up to 4.5 ml at be d heights up
to 5.8 cm
• Tricorn 10/100 (10 mm i.d.) for bed vo lumes up to 8.5 ml at be d heights up
to 10.8 cm
• XK 16/20 (16 mm i.d.) for bed volum es up to 30 ml at bed heigh ts up to
15 cm.
• XK 26/20 (26 mm i.d.) for bed volumes up to 80 ml at bed height s up to
15 cm.