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Fig 4. Binding capacity of human IgG, HSA and human transferrin at different pH´s
on HiTrap Q HP, 1 ml.
4. Apply the sample solution to the column with a pump or a syringe, at a
flow rate equal to the flow rate to be used in the purification method.
Collect fractions and continue sample application until the column is
saturated.
5. Wash the column with 5–10 column volumes start buffer or until no
material appears in the effluent.
6. Elute bound proteins with 3–5 column volumes of elution buffer (start
buffer with 1 M NaCl) and collect eluate.
7. Analyse fractions and eluates from steps 4 and 6 for the specific protein
and determine the breakthrough profile (sample concentration as a
function of the amount of sample applied). The dynamic capacity is the
amount that can be applied without any significant breakthrough. The
total capacity for the specific protein is determined from step 6.