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
1
™
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.
2
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.
3
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%.
4
250 kD
150
100
75
Conalbumin
50
37
Ovalbumin
25
20
15
Cytochrome c
10
1234567
Fig. 1. Separation of three test proteins on Aurum AEX and CEX
columns.
The Aurum CEX column was equilibrated in 20 mM sodium acetate buffer, pH 5.0. The mixture of
three proteins (lane 3, 0.4 ml at 30 mg/ml total protein) was added to the column. The unbound
protein containing ovalbumin was collected (lane 4). The bound fraction containing conalbumin and
cytochrome c (lane 5) was eluted with 2 x 300 µl washes with 20 mM sodium acetate buffer, pH 5.0,
containing 1 M NaCl. The eluates from the washes were desalted and buffer exchanged using Micro
Bio-Spin 6 desalting columns (catalog #732-6221) and applied to an Aurum AEX column. The
unbound fraction containing cytochrome c was collected (lane 6), while the bound fraction containing
conalbumin (lane 7) was recovered with 2 x 300 µl washes of 20 mM Tris, pH 8.3, containing 1 M
NaCl. The fractions were assayed for protein using the Bio-Rad protein assay to determine protein
recovery. Purity was estimated from SDS-PAGE analysis on a 4–20% linear gradient Criterion
Tris-HCl gel (catalog #345-0033). The gel was stained with Coomassie Blue. Bio-Rad Precision Plus
™
Protein
standards (catalog #161-0373) are shown in lane 1.
™
5
Section 2
Components
The Aurum ion exchange products contain the following components:
2 pk 10 pk 25 pk 100 pk
Aurum AEX or CEX columns 2 10 25 100
Collection tubes, 2.0 ml 15 –* –* –*
Plastic test tubes, 12 x 75 mm 2 10 –
AEX or CEX binding buffer 15 ml 50 ml – –
AEX or CEX elution buffer 15 ml 50 ml – –
Micro Bio-Spin 6 columns, 4 20 – –
Tris buffer
Instruction manual 1 1 1 1
Protocol overview 1 1 1 1
*Order catalog #223-9430, EZ Micro
†
Order catalog #732-6714, Test tubes, 12 x 75 mm, 100/bag
™
test tubes, 2 ml, 500
†
–
†
Section 3
Storage Conditions
Solutions and columns should be stored at 4°C. Do not freeze. Shelf life is
12 months at 4°C.
Section 4
Necessary Supplies
• Microcentrifuge (≥10,000 x g)
6
AEX or CEX column
12 x 75 mm test tube
2.0 ml collection tube
(only included in 2 pk)
Fig. 2. Aurum ion exchange mini kit components
Micro Bio-Spin 6 column, Tris
7
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
(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).
8
®
Blue columns
• 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.
9
Section 6
Protocol
Please read the section “Guidelines for Aurum Ion Exchange Columns” before
proceeding.
1. Place an ion exchange column in a 12 x 75 mm test tube and allow the
resin to settle for at least 5 min.
2. Remove the cap and break off the tip from the bottom of the ion
exchange column. Return column to test tube.
3. Start gravity flow in the column and allow residual buffer to drain from the
column (approximately 2 min). If the column does not begin to flow, push
the cap back on the column and then remove it again to start the flow.
4. Once the residual buffer has drained, wash the column with 2 x 1 ml of
the appropriate Aurum AEX or CEX binding buffer using gravity flow.
Allow each wash to pass fully through the column and drain.
5. After the last wash, place the column in an empty 2.0 ml collection tube
and centrifuge for 10 sec at 1,000 x g in a microcentrifuge to dry resin
bed and frit. Do not overdry resin bed and frit. Discard the
collection tube.
6. Place the column in a clean 2.0 ml collection tube labeled “unbound”.
7. Load the sample in the appropriate buffer onto the column and allow the
sample to gravity filter through the column, collecting in the collection
tube. If the column does not begin to flow, push the cap back on the
column and remove it again to start the flow.
8. Place the column in another clean 2.0 ml collection tube.
9. Wash the columns with 0.3 ml of Aurum AEX or CEX binding buffer.
Unbound fractions can be combined for further analysis if desired.
Otherwise, unbound fractions can be discarded.
10. Place the column in a 2.0 ml collection tube and wash with 0.6 ml of the
appropriate Aurum AEX or CEX binding buffer. Centrifuge column for
20 sec at 1,000 x g in a microcentrifuge, collecting the eluate in the tube.
This wash can be discarded.
11. Place the column in a new 2.0 ml collection tube labeled “bound #1”.
12. Add 0.3 ml of the appropriate Aurum AEX or CEX elution buffer to the top
of the column and centrifuge for 10 sec at 1,000 x g.
13. Wash with an additional 0.3 ml of the Aurum AEX or CEX elution buffer
into the same “bound #1” tube. Centrifuge for 10 sec at 1,000 x g.
10
14. Place the column in a new 2.0 ml collection tube labeled “bound #2”.
15. Wash with 0.3 ml of the Aurum AEX or CEX elution buffer. Centrifuge for
10 sec at 1,000 x g.
Sample Preparation for IEF Using Micro Bio-Spin 6 Chromatography
Desalting Procedure
1. Invert the column sharply several times to resuspend the settled gel and
remove any bubbles. Snap off the tip and place the column in a 2.0 ml
collection tube. Remove the top cap. If the column does not begin to
flow, push the cap back on the column and remove it again to start the
flow. Allow the excess packing buffer to drain by gravity to the top of the
gel bed (about 2 min). Discard the drained buffer and place the column
back into the 2.0 ml tube.
2. Centrifuge for 4 min in a microcentrifuge at 1,000 x g to remove the
residual packing buffer. Discard the buffer.
3. Place the column in a clean 1.5 or 2.0 collection tube. Carefully apply the
sample (20–75 µl) directly onto the center of the column. Application of
more or less than the recommended sample volume may decrease
column performance.
4. After loading sample, centrifuge the column for 4 min at 1,000 x g.
5. Following centrifugation, the purified sample is now in the low ionic
strength binding buffer. Molecules smaller than the column’s exclusion
limit will be retained by the column.
6. Dispose of the used column.
Buffer Exchange
The gel in the Micro Bio-Spin columns is suspended in Tris buffer, pH 7.4.
The gel matrix is compatible with most aqueous buffers. Buffer exchange can
be achieved using the following procedure.
1. Follow steps 1 and 2 in the Desalting Procedure section.
2. Apply the new buffer in 500 µl aliquots. After each application of new
buffer, let the buffer drain out by gravity, or centrifuge the column for
1 min to remove the buffer. Discard buffer from collection tube. Repeat as
required. Three washes result in >99% of the buffer exchanged. Four
washes result in >99.9% of buffer exchanged.
3. Sample can now be applied to the column as directed in steps 3–6 in the
Desalting Procedure section.
11
Section 7
Troubleshooting Guide
Problem Possible Cause Possible Solution
Low protein Low levels of protein Increase load on column
concentration in sample
Concentrate sample in
SpeedVac
High protein High levels of protein Decrease the load
concentration in sample applied to column
Buffer does not flow Frit is clogged with Prefilter sample prior
through column sample debris to loading
Protein does not bind Incorrect column chosen CEX column is for
to column basic proteins; AEX
column is for acidic
proteins
Ionic detergents can Avoid using detergents
prevent sample from with opposing net
binding charges to that of ion
exchange column.
Detergents can be
removed using
Bio-Beads
macroporous beads
(catalog #152-3920)
®
SM-2
Ionic strength too high Dilute sample or desalt
pH not correct Check pH of loading
buffer
Protein does not elute Salt concentration too Increase salt
from column low in elution buffer concentration to 2 M
and/or change pH
Sample is dilute after Excess packing buffer Remove excess packing
centrifugation was not removed buffer by centrifugation
prior to loading sample
12
Section 8
Bibliography
• UNOsphere anion exchange support, Bio-Rad bulletin 2724
• UNOsphere Q technical data, Bio-Rad bulletin 2729
• UNOsphere cation exchange support, Bio-Rad bulletin 2669
• UNOsphere S technical data, Bio-Rad bulletin 2678
• UNOsphere Q and S ion exchange media instruction manual, Bio-Rad bulletin
4110109
• Sample preparation — A guide to methods and applications, Bio-Rad bulletin
1825
• 2-D electrophoresis for proteomics: A methods and product manual, Bio-Rad
bulletin 2651
• Colorimetric protein assays, Bio-Rad bulletin 1069
• Yamamoto S et al. (eds), Ion Exchange Chromatography of Proteins, Marcel
Dekker, New York (1988)
• Beynon RJ and Easterby JS, Buffer Solutions: The Basics, IRL Press at Oxford
University Press, Oxford, p 84 (1996)
• Burgett MW and Greenley LV, Cibacron Blue F3GA affinity chromatography,
Amer Lab 9, 74–85 (1977)
• Lindmark R et al., Binding of immunoglobulins to protein A and immunoglobulin
levels in mammalian sera, J Immunol Methods 62, 1–13 (1983)
• Craft G et al., Comparison of Cibacron Blue affinity based formats for depletion of
albumin from samples prior to two-dimensional gel electrophoresis, APAF poster
(2002)
13
Section 9
Ordering Information
Kits* Columns (Bulk)*
2 pk 10 pk 25 pk 100 pk
Aurum AEX mini 732-6710 732-6705 732-6706 732-6707
Aurum CEX mini 732-6711 732-6702 732-6703 732-6704
Aurum Affi-Gel Blue mini 732-6712 ------------ 732-6708 732-6709
Aurum serum protein mini 732-6713 732-6701 ------------ ------------
Micro Bio-Spin 6, Tris ------------- ----------- 732-6221 732-6222
*Kits include columns, buffers, and instructions. Columns include columns and instructions only.
Cibacron is a trademark of Ciba-Geigy Corp. Coomassie is a trademark of Imperial Chemical Industries
PLC. SpeedVac is a trademark of Savant Instruments, Inc.
14
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
(510) 741-1000
1-800-424-6723
4110137 Rev B
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