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™ Q or S 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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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 |
|
cation support |
anion support |
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.
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™ 6 (catalog #732-6221) or Bio-Spin® 6 (catalog #732-6227) columns, Econo-Pac® Bio-Gel® 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™ 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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