Bio-Rad Aurum Ion Exchange Mini Kits User Manual

Aurum

™

Ion Exchange Mini Kits

and Columns

Instruction Manual

Catalog #
732-6710 Aurum AEX Mini Kits, 2 pk
732-6705 Aurum AEX Mini Kits, 10 pk
732-6706 Aurum AEX Mini Columns, 25 pk
732-6707 Aurum AEX Mini Columns, 100 pk

732-6711 Aurum CEX Mini Kits, 2 pk
732-6702 Aurum CEX Mini Kits, 10 pk
732-6703 Aurum CEX Mini Columns, 25 pk
732-6704 Aurum CEX Mini Columns, 100 pk

For technical service, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)

Table of Contents

Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Section 2 Components . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .6

Section 4 Necessary Supplies . . . . . . . . . . . . . . . . . . . . . .6

Section 5 Guidelines for Aurum Ion Exchange Columns . .8

Section 6 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Section 7 Troubleshooting Guide . . . . . . . . . . . . . . . . . . .12

Section 8 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Section 9 Ordering Information . . . . . . . . . . . . . . . . . . . .14

Section 1
Introduction

Proteomics Sample Fractionation

Sample preparation plays an ever-increasing role in proteomics research.
Selective separation techniques prior to multidimensional analysis, such as 2-D
gel electrophoresis, HPLC-MS, or HPLC-CE, can significantly improve the
chances of resolving complex protein mixtures into their individual proteins.
Among these techniques are anion exchange (AEX) and cation exchange
(CEX) chromatography. Aurum AEX and CEX columns allow rapid fractionation
of complex protein mixtures in only a few steps using the common technique
of ion exchange chromatography.

Ion Exchange Chromatography

Ion exchange chromatography can be used to concentrate and purify proteins
based on their ionic charge or isoelectric point (pI) at a given pH. At the
isoelectric point of a protein, its net charge is zero. At a pH higher than the pI
of the protein, the protein will be negatively charged and will bind to an AEX
resin. At a pH lower than the pI, the protein will be positively charged and will
bind to a CEX resin. When an ion is applied to an ion exchanger of opposite
charge, it is adsorbed to the resin, while neutral ions or ions of the same
charge are eluted in the flow-through fraction. Binding of the ions is reversible,
and adsorbed molecules are commonly eluted with salt or a change of pH.

Table 1. Choosing the appropriate column.

Resin Type Protein of Interest

AEX Acidic

CEX Basic

The choice of whether to use an anion or cation exchanger is determined
mainly by the pI, and the relationship between pH and the activity or stability of
the protein of interest. Once the type of ion exchanger is determined, the
choice of buffer and pH is also determined by the relationship between pH
and activity.

Resins and Buffers Description

Each Aurum AEX and CEX column contains 0.2 ml of UNOsphere
support, unique media based on a proprietary polymerization process. Both
the UNOsphere Q and S ion exchange media were developed to deliver
efficient protein capture and high binding capacity. The Aurum AEX and CEX

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™

Q or S

columns have binding and elution buffers included to afford minimal handling
and ease of use. The formulations for each of the buffers are outlined in
Table 2.

Table 2. Properties of Bio-Rad ion exchange resins.

Aurum CEX Aurum AEX

Resin type UNOsphere S UNOsphere Q

Binding capacity 12 mg IgG*/column 36 mg BSA*/column

Column bed volume 0.2 ml 0.2 ml

Binding buffer 20 mM sodium acetate, pH 5.0 20 mM Tris, pH 8.3

Elution buffer Binding buffer + 1.0 M NaCl Binding buffer+ 1.0 M NaCl

*Binding capacity determined using IgG (UNOsphere S) and BSA (UNOsphere Q). Capacity will differ
between proteins and will depend on the exact loading conditions.

cation support anion support

A broad range of buffer systems can be used with Aurum AEX and CEX
columns, depending on the protein of interest or specific process required.
The chemical stability and broad operating pH range of the UNOsphere ion
exchange media allow the use of a variety of buffers. Buffers commonly used
for AEX or CEX can be used in place of the provided buffers (see Table 3).

The best results are achieved when buffering ions that have the same charge
as the functional group on the ion exchanger are used, e.g., phosphate paired
with a cation exchanger, or Tris paired with an anion exchanger. As a general
rule, the pH of the experiment should fall within the buffering range of the
chosen buffer (see Table 3). This permits use of the lowest possible buffer
concentration while maintaining maximum buffering capacity. In most cases,
a buffer concentration of 20 mM is recommended.

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Table 3. Common buffers for ion exchange chromatography.

Cation Buffering Range Anion Buffering Range

Acetic acid 4.8–5.2 Bicine 7.6–9.0
Citric acid 4.2–5.2 Bis-Tris 5.8–7.2
HEPES 7.6–8.2 Diethanolamine 8.4–8.8
MES 5.5–6.7 Diethylamine 9.5–11.5
MOPSO 6.5–7.9 L-histidine 5.5–6.0
Phosphate 6.7–7.6 Imidazole 6.6–7.1
PIPES 6.1–7.5 Pyridine 4.9–5.6
TES 7.2–7.8 Tricine 7.4–8.8
Tricine 7.8–8.9 Triethanolamine 7.3–8.0

Tris 7.5–8.0

Sample Preparation

Proper adjustment of the sample pH and ionic strength is critical for consistent
and reproducible chromatography. For best results, the sample should be
exchanged into the loading buffer or diluted to the buffer’s concentration.
Buffer exchange can be accomplished using Micro Bio-Spin
#732-6221) or Bio-Spin

®

6 (catalog #732-6227) columns, Econo-Pac® Bio-Gel

™

6 (catalog

P-6 cartridges (catalog #732-0011), or Econo-Pac 10DG desalting columns
(catalog #732-2010). The correct product to use will be determined by the
volume of the sample. Always centrifuge or filter the sample (0.2–0.45 µm
filter) to remove particulates.

Use of Aurum Ion Exchange Columns to Concentrate Protein
Solutions

®

The Aurum AEX and CEX columns are not only purification tools but also can
be used to concentrate target proteins of interest in dilute solutions, a critical
factor for subsequent analyses such as 2-D electrophoresis. Protein solutions
of 0.1–0.2 mg/ml can be concentrated 30–70-fold using the appropriate resin
with little loss of protein.

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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

*BSA dissolved and washed in 20 mM Tris, pH 8.3; cytochrome c dissolved and washed in 20 mM
sodium acetate, pH 5.0

™

protein assay.

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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