Bio-Rad Aurum Ion Exchange Mini Kits - Manual (Page 10)

Section 5
Guidelines for Aurum
Ion Exchange Columns
• All samples should be clarified before application to Aurum ion exchange
columns. This can be accomplished using a 0.45 µm syringe filter or by
centrifuging the sample at 10,000 x g for 5 min.
• For effective use of the Aurum ion exchange columns, the samples need to
be adjusted to the approximate pH and ionic strength of the respective
binding buffers. This can be accomplished in several ways. Depending
upon the buffer of the sample, it may be sufficient to dilute the sample in
the Aurum binding buffer. A second method would be desalting and buffer
exchange using desalting columns. For small sample volumes, use Micro
Bio-Spin or Bio-Spin desalting columns, and for larger volumes use
Econo-Pac cartridges. A third alternative would be dialysis against the
corresponding binding buffer.
• Sample loading will depend on the protein concentration of the sample and
the protein capacity of the Aurum AEX or CEX column.
• If necessary, dilute samples can be concentrated using a SpeedVac
concentrator or a lyophilizer.
• Knowledge of the pI of a protein of interest allows selection of resin type
and buffer pH for either selective binding to or release from the resin.
• See Table 3 for buffer recommendations for the different Aurum AEX and
CEX columns.
• Serum samples — Serum can be diluted 1:4 in Aurum binding buffer and
then 0.4–0.6 ml applied to the ion exchange column. Using the
recommended buffers, albumin will pass through the CEX column while
binding to the AEX column. To selectively remove albumin and IgG from
serum prior to ion exchange chromatography, use the Aurum serum protein
mini kit (catalog #732-6701), which can remove >90% of these two major
proteins. To remove only the albumin, use Aurum Affi-Gel
® Blue columns
(catalog #732-6712).
• E. coli lysate — For a typical E. coli lysate, dilute the lysate 1:4 in the
appropriate binding buffer and load 0.4–0.8 ml on the ion exchange
column.
• Tissue culture samples — Clarified tissue culture samples should be diluted
or the buffer exchanged in binding buffer. For removal of bovine serum albumin
added to culture media, use Aurum Affi-Gel Blue columns (catalog #732-6712).
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