p. 16
Fig 4. Binding capacity of huma n IgG, HSA and human t ransferrin a t different pH´s
on HiTrap Q HP, 1 ml.
4. Apply th e sample solutio n to the column with a p ump or a syringe, at a
flow rate equal to the f low rate to be used in th e purificati on method.
Collect fraction s and continue samp le application un til the column is
saturated.
5. Wash the co lumn with 5–10 column volum es start buff er or until no
material appears in the effluent.
6. Elute bou nd proteins with 3–5 co lumn volumes of elu tion buffer (s tart
buffer with 1 M NaCl) a nd collect eluat e.
7. Analys e fractions and e luates from steps 4 a nd 6 for the specif ic protein
and determine the brea kthrough profi le (sample conce ntration as a
function of the amoun t of sample applied ). The dynamic ca pacity is the
amount that can be applied without any significant breakthrough. The
total capacity for the spe cific protein is de termined from st ep 6.