Bio-rad Aurum User Manual

Aurum

™

Serum Protein Mini Kit

Instruction Manual

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 Kit Components . . . . . . . . . . . . . . . . . . . . . . . . .2

Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .2

Section 4 Necessary Supplies . . . . . . . . . . . . . . . . . . . . . .2

Section 5 Guidelines for Using the

Aurum Serum Protein Mini Kit . . . . . . . . . . . . . . . .4

Section 6 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Section 7 Troubleshooting Guide . . . . . . . . . . . . . . . . . . . .8

Section 8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Section 9 Ordering Information . . . . . . . . . . . . . . . . . . . .10

Section 1
Introduction

The Aurum serum protein mini kit contains Micro Bio-SpinTMcolumns filled with
a mixture of Affi-Gel
the simultaneous removal of both albumin and immunoglobulin (IgG) in a single
step from serum or plasma samples prior to two-dimensional (2-D)
polyacrylamide gel electrophoresis (PAGE).

Proteins in serum and other biological fluids are difficult to resolve by 2-D
PAGE, largely due to the abundance of serum albumin and IgG. In human
serum, albumin constitutes 50–70% of the total protein and IgG constitutes
10–25%. The presence of these proteins obscures other proteins in the gel
and limits the amount of proteins in the serum that can be resolved by 2-D
analysis. Furthermore, these proteins have wide pI and molecular weight
ranges, further reducing resolution and masking many proteins of potential
interest. Theoretically, removing 90% of the albumin and IgG from serum
should reduce the protein load by 70–80%, thus allowing the application of
3–4 times more serum and at the same time significantly improving the
resolution of polypeptide spots on 2-D gels.

Affi-Gel Blue affinity gel is a beaded, cross-linked agarose gel with covalently
attached Cibacron Blue F3GA dye. It has a capacity for albumin binding of
greater than 11 mg/ml. Affi-Gel Blue gel has been utilized to separate and
purify a number of different serum and plasma proteins (Gianazza and Arnaud
1982, Herman and Roberts 1980) and has been used as a first step in the
purification of serum proteins by removing the major serum constituent,
albumin (Burgett and Greenley 1977). The major advantage of Affi-Gel Blue in
the current application is its high albumin capacity. This product has been
used to rapidly remove albumin from multiple human serum samples prior to
2-D analysis (Rengarajan et al. 1996). The disadvantage of this ligand is its
possible lack of specificity, based on its numerous applications. Although the
binding of serum proteins other than albumin and IgG cannot be excluded, the
current product has been developed to minimize nonspecific adsorption.
Binding conditions have been optimized to saturate the matrix binding sites
with the very high-affinity albumin molecules.



Blue and Affi-Gel protein A. This resin blend allows for

Protein A, from Staphylococcus aureus, has the property of binding with high
specificity the Fc region of IgG molecules (Kronvall and Williams 1969). When
coupled to agarose beads, protein A has been extensively used to bind and
purify IgG species from various mammalian species (Lindmark et al. 1983).
Affi-Gel protein A has a binding capacity of greater than 15 mg human IgG/ml
gel.

1

Section 2
Kit Components

The Aurum serum protein mini kit contains the following
components:

Aurum serum protein columns (filled and capped) 10

12 x 75 mm plastic test tubes 10

2.0 ml collection tubes with caps 30

Yellow column tips 10

Aurum serum protein binding buffer 50 ml

Instruction manual 1

Protocol overview 1

Section 3
Storage Conditions

Solutions and columns should be stored at 4ºC. Do not freeze. Shelf life
is 12 months at 4ºC.

Section 4
Necessary Supplies

• Microcentrifuge (≥10,000 x g)

2

2.0 ml collection tube

12 x 75 mm test tube

Aurum serum protein column

Fig. 1. Aurum serum protein mini kit

Yellow column tip

3

Section 5
Guidelines for Using the Aurum Serum
Protein Mini Kit

• Serum samples should be clarified before application to Aurum serum
protein columns.

• High salt concentrations (>200 mM) should be avoided due to
interference with albumin removal. Salt can also interfere with subsequent
IEF analysis. High salt samples can be dialyzed against a low-salt buffer
(25 mM phosphate, pH 7.0).

• For serum samples containing a high concentration of albumin, reduce
the sample load on the resin from 200 µl to 150 µl. The load may need to
be further optimized.

• For serum samples with low concentrations of albumin, increase the
sample load from 200 µl to 250 µl. An alternative would be to dilute the
initial sample 1/3 with binding buffer and apply 200–250 µl. The load may
need to be further optimized.

• The bound albumin and IgG can be recovered from the Aurum serum
protein columns and analyzed. For 1-D analysis, elute the column with
500 µl of Laemmli sample buffer (Bio-Rad catalog #161-0737). For 2-D
analysis, elute the column with 500 µl of ReadyPrep
extraction reagent 3 (Bio-Rad catalog #163-2104).

TM

sequential

• If necessary the albumin/IgG-depleted samples can be concentrated
using a SpeedVac or lyophilizer.

• Albumin/IgG depleted serum samples, if not analyzed immediately, should
be stored at -20ºC.

• For 2-D electrophoresis analysis, see bulletin 2561, 2-D Electrophoresis
for Proteomics. A Methods and Product Manual.

4

Section 6
Protocol

Please read Section 5, “Guidelines for Aurum Serum Protein Mini Kit” before
proceeding.

1. Place an Aurum serum protein 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 Aurum
serum protein column. Return serum protein column to test tube.

3. Start gravity flow in the column and allow residual buffer to drain from the
column (approximately 2 min).

4. Once the residual buffer has drained, wash the column with 2 x 1 ml of
Aurum serum protein 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 20 sec at 10,000 x g in a microcentrifuge to dry resin
bed and frit. Do not overdry resin bed and frit. Discard the collection
tube.

6. Place a yellow column tip on the bottom of the column to stop any
residual buffer flow from column. Place the column in a clean 2.0 ml
collection tube labeled “unbound”.

7. In a separate 2.0 ml collection tube, prepare sample to be purified by
diluting 60 µl of plasma or serum with 180 µl of Aurum serum protein
binding buffer. Mix by inverting tube several times.

8. Add 200 µl of the diluted serum sample to the top of the resin bed. Allow
sample to penetrate column matrix.

9. Gently vortex the column. Repeat at 5 and 10 min. Allow column to sit an
additional 5 min for a total incubation time of 15 min.

10. Remove yellow tip from column and return to 2.0 ml collection tube.

5

11. Centrifuge the column for 20 sec at 10,000 x g in a microcentrifuge,
collecting the eluate in the “unbound” 2.0 ml collection tube.

12. Remove the column and tube together from the centrifuge and wash the
resin with 200 µl of the binding buffer. Replace tube and column in
centrifuge.

13. Centrifuge column for 20 sec at 10,000 x g in a microcentrifuge, collecting
the eluate in the same “unbound” tube, which contains the albumin- and
IgG-depleted serum sample.

14. The treated sample is ready for IEF analysis. For serum, this procedure
typically yields protein concentrations around 1.5–2.0 mg/ml as determined
by the modified Lowry method (Bio-Rad DC protein assay, catalog #500-

0112).

6

Aurum™Serum
Protein Mini Kit

Protocol Overview

Column Setup

1. Place serum protein column in a test tube
for 5 min to allow resin to settle.

2. Remove cap and break tip from column
and return to test tube to start gravity
flow in column.

3. Wash column with 1 ml of serum protein
binding buffer using gravity flow. Repeat.

4. Place column in empty 2.0 ml collection tube
and centrifuge for 20 sec at 10,000 x g to
dry resin bed. Discard collection tube.

5. Place a yellow column tip on bottom of column
and place into a clean 2.0 ml collection tube
labeled “unbound”.

1 ml

binding
buffer

2x

Sample Binding and Purification

6. In a separate tube, prepare sample by diluting
60 µl serum or plasma with 180 µl of
serum protein binding buffer.

7. Add 200 µl of diluted serum to top of
resin bed in column.

8. Gently vortex column and repeat after
5 and 10 min. Allow column to sit

an additional 5 min.

60 µl

serum

200 µl

diluted
serum

Collection of Purified Samples

9. Remove yellow tip from column and return
to tube. Centrifuge column for 20 sec at
10,000 x g, collecting protein fraction in
“unbound” collection tube.

10. Using same collection tube, wash column
with 200 µl of serum protein binding buffer.

11. Centrifuge column for 20 sec at 10,000 x g,
collecting protein fraction in same “unbound”
collection tube. Discard serum protein column.

12. The combined fractions contain the albumin­and IgG-depleted serum or plasma sample.
The sample is now ready for gel analysis.

200 µl

binding
buffer

Fig. 2. Aurum serum protein mini kit protocol overview

180 µl

binding
buffer

Vortex

3x

7

Section 7
Troubleshooting Guide

Problem Possible Cause Possible Solution

Low protein Low levels of protein or Increase serum load
concentration albumin in sample on column

Concentrate treated
sample in SpeedVac

High protein High level of albumin in Decrease the serum
concentration/ sample load applied to
insufficient albumin column
removal

8

Section 8
References

Burgett MW and Greenley LV, Cibacron Blue F3GA affinity chromatography,
Amer Lab 9, 74-85 (1977)

Gianazza E and Arnaud P, Chromatography of plasma proteins on
immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction,
Biochem J 203, 637-641 (1982)

Herman CA and Roberts R, Purification and immunological characterization
of human myocardial MB creatine kinase, Anal Biochem 106, 244-252 (1980)

Kronvall G and Williams RC Jr, Differences in anti-protein A activity among
IgG subgroups, J Immunol 103, 828-833 (1969)

Lindmark R et al., Binding of immunoglobulins to protein A and
immunoglobulin levels in mammalian sera, J Immunol Methods 62, 1-13
(1983)

Rengarajan K et al., Removal of albumin from multiple human serum
samples, BioTechniques 20, 30-32 (1996)

* Cibacron is a trademark of CIBA-Geigy Corp.

9

Section 9
Ordering Information

Catalog # Description

732-6713 Aurum Serum Protein Mini Kit, 2 pk, includes

2 serum protein columns, 2 clear 12 x 75 mm polystyrene
tubes, 6 sample collection tubes, 10 column tips, 15 ml
binding buffer, protocol overview, instructions

732-6701 Aurum Serum Protein Mini Kit, 10 pk, includes

10 serum protein columns, 10 clear 12 x 75 mm polystyrene
tubes, 30 sample collection tubes, 10 column tips, 50 ml
binding buffer, protocol overview, instructions

10

Bio-Rad Laboratories, Inc.

2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
[phone]

1-800-424-6723

4110021 Rev B