Biomedical Engineering Reference
In-Depth Information
Depending on target stability or further steps of purification,
especially with respect to the sometimes severe elution conditions,
it has to be decided whether the interested protein will be adsorberd
to the HIC matrix or stay in solution.
The HIC medium with the optimal properties is chosen for
batch or column absorption.
Sample application is done analogously to ion exchange chro-
matography (see Sect. 3.4.3).
3.5.2 Elution
As described for IEC, elution is done by a stepwise or a continu-
ous change of buffer composition. The mildest elution buffer is an
aqueous buffer with low ionic strength, e.g., 20 mm Tris-HCl.Ifit
is not successful, desorb with a chaotropic solvent, e.g., 2 M potas-
sium rhodanide (thiocyanate), 2.5 M guanidinium hydrochloride,
up to 7 M urea, or with increasing concentrations of methanol or
acetonitrile. Especially the use of rhodanide or urea may be ac-
companied by a chemical modification of amino acid side chains,
which disturbs amino acid analysis.
3.5.3 Regeneration
Regenerate the HIC medium after elution as follows:
Wash the gel with one bed volume of ddH
2
O. Then wash w ith
one bed volume each of 33, 67, and 95% ethanol (v/v) in ddH
2
O.
Continue with one bed volume n-butanol, 95, 67, 33% ethanol and
at least five bed volumes of ddH
2
O. Finish the regeneration by
equilibration with starting buffer.
Store the gel in a refrigerator in the presence of 0.02% sodium
azide.
References
Kennedy RM (1990) In: Deutscher MP (ed.) Guide to protein purification.
Academic Press, San Diego, p 339
3.5.4 Analytical HPLC of Hapten-Protein Conjugates
Reversed-phase (rp-) or hydrophobic (HIC) HPLC is an excellent
tool for analysis of proteins and peptides. Especially phenyl media,
e.g., TosoHaas TSK-Gel Phenyl-5PW-RP, fit well
A
0.1% trifluoroacetic acid (TFA) (v/v) in ddH
2
O
Solutions/Reagents
B
0.05% TFA (v/v) in acetonitrile (HPLC grade)
Use an HPLC apparatus with gradient system, pre-column and
5
0.5-cm separation column is used. Equilibrate the column with
Soln. A at flow rate of 0.8 - 1.0 ml
×
/
min.
