Bio-Rad Aurum Ion Exchange Mini Kits - Manual (Page 11)

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• Bound proteins are eluted by increasing the salt concentration to 1 M NaCl.
For some separations, varying the pH of the elution buffer in addition to its
salt concentration may be advantageous.
• Aurum AEX and CEX columns show some differences in elution
characteristics when eluted sequentially with different elution volumes.
Table 5 shows the protein recovery in each of the three sequential elutions
from both columns. Protein is more tightly bound to the CEX column, so
larger elution volumes are required.
Table 5. Protein recovery for AEX and CEX elution washes.
Elution Volume
200 µl 300 µl 400 µl
Column type AEX CEX AEX CEX AEX CEX
% Protein Recovery — 84 60 82 73 86 85
1st Wash
% Protein Recovery — 14 31 14 23 11 13
2nd Wash
% Protein Recovery — 2 9 4 4 3 2
3rd Wash
BSA was bound to the AEX column in 20 mM Tris, pH 8.3, and eluted with 20 mM Tris, pH 8.3,
containing 1 M NaCl. IgG was bound to the CEX column in 20 mM sodium acetate, pH 5.0, and
eluted with 20 mM sodium acetate, pH 5.0, containing 1 M NaCl.
• The unbound fractions from the ion exchange columns can be used directly
for 2-D gel electrophoretic analysis, provided the protein concentration is
sufficient. For 2-D analysis, the bound fractions will require desalting.
Micro Bio-Spin 6 columns (catalog #732-6221) are recommended.
• For 2-D electrophoretic analysis, see bulletin 2651, 2-D Electrophoresis for
Proteomics: A Methods and Product Manual.
• Fractions can be analyzed for protein using the Bio-Rad protein assay
(catalog #500-0006, Bradford method) or the DC protein assay
(catalog #500-0116). See bulletin 1069, Colorimetric Protein Assays.
• If pI of protein is unknown — We recommend running an IEF gel (catalog
#345-0072) to determine its pI. If the sample is not pure, we recommend
visualizing the protein using an activity assay or identifying the protein with
an immunoblotting assay. If the amino acid composition is known, the
theoretical pI can be computed using databases available on the Internet,
such as at www.expasy.org.
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