Bio-Rad Aurum Ion Exchange Mini Kits - Manual (Page 6)

Table 4. Enrichment of typical proteins with Aurum AEX and CEX
columns.
Initial Solution Final Solution
Resin Protein Volume Concentration Enrichment Recovery
AEX BSA 30 ml 0.1 mg/ml 70–fold 90%
CEX Cytochrome c 30 ml 0.2 mg/ml 60–fold 95%
Bovine serum albumin (BSA) and cytochrome c were dissolved in buffer* and 30 ml loaded on an
Aurum AEX or CEX column, respectively, in 1 ml aliquots. No protein was detected in the unbound
fractions. The columns were eluted with 2 x 300 µl washes* containing 1 M NaCl. Protein
concentrations were determined using the DC
™ protein assay.
*BSA dissolved and washed in 20 mM Tris, pH 8.3; cytochrome c dissolved and washed in 20 mM
sodium acetate, pH 5.0
Ability to Purify Proteins of Differing pl
Three proteins, ovalbumin (MW 45,000, pI 4.6), conalbumin (MW 77,000,
pI 6.9), and cytochrome c (MW 12,000, pI 10.7), were completely separated
using Aurum CEX and AEX columns in tandem. The respective unbound and
bound fractions were analyzed by 4–20% SDS-PAGE (Figure 1). The three
proteins were equilibrated in 20 mM sodium acetate buffer, pH 5.0, and
applied to the CEX column. At this pH, the ovalbumin (pI 4.6) passed through
the column while the conalbumin (pI 6.9) and cytochrome c (pI 10.7) remained
bound to the column. The bound fractions were then eluted with elution buffer,
desalted, buffer exchanged into 20 mM Tris, pH 8.3, and then applied to the
AEX column at pH 8.3. The conalbumin remained bound while the cytochrome c
was recovered in the unbound fraction. All three proteins were recovered with
an estimated purity of >95% and a total recovery of >90%.
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