Bio-Rad Affi-Gel Protein A Media User Manual

Econo-Pac

®

Protein A Kit

Protein A

Columns

Instruction

Manual

Catalog Number

732-2020
732-2022

For Technical Service

Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD

(1-800-424-6723)

Table of Contents

Section 1 Econo-Pac Protein A Kit..........................................1

1.1 Introduction .........................................................................1

1.2 Kit Components...................................................................1

1.3 Additional Items Required ..................................................2

1.4 Buffer Preparation...............................................................2

1.5 Sample Preparation..............................................................3

1.6 Standard Mouse IgG1Purification Procedure .....................3

1.7 Answers to Common Questions..........................................4

Section 2 Econo-Pac Protein A Columns ................................5

2.1 Introduction .........................................................................5

2.2 Buffer Considerations..........................................................6

2.3 Sample Preparation..............................................................6

2.4 Standard Mouse IgG1Purification Procedure .....................6

2.5 Column Performance...........................................................7

Section 3 References..................................................................8

Section 4 Ordering Information...............................................8

Section 1
Econo-Pac Protein A Kit

1.1 Introduction

The Econo-Pac protein A kit is based upon Bio-Rad’s Affi-Gel
protein A MAPS®II methods and contins everything necessary to
purify monoclonal antibodies from ascites or serum. Affi-Gel protein
A agarose consists of purified protein A coupled to crosslinked
agarose beads via chemically stable amide bonds. The Econo-Pac pro­tein A column contains 2 ml of Affi-Gel protein A with a total capac­ity of 10-14 mg mouse IgG1when used with the MAPS II buffers
(included).

The Econo-Pac protein A kit provides a simple and convenient
method for the purification of all IgG subclasses, including IgG1,
from ascites fluid. Sample preparation has been simplified by the
use of the Econo-Pac 10DG desalting columns. Binding, elution,
and regenerations steps can be completed with only one column vol­ume of buffer per step, making the total procedure fast and easy.

1.2 Kit Components

®

Econo-Pac protein A column One Econo-Pac column

Econo-Pac 10DG desalting
columns Four Econo-Pac 10DG

Binding buffer One bottle (471 g) of buffer

Elution buffer One bottle (25 g) of buffer

Regeneration buffer One bottle containing 400

Instruction manual

packed with 2 ml of Affi-Gel
protein A agarose.

desalting columns.

solids, reconstitution volume
= 1,500 ml.

solids, reconstitution volume
= 1,000 ml.

ml. CAUTION: Contains
methanol. DO NOT MOUTH
PIPETTE.

The Econo-Pac protein A kit contains a sufficient quantity of
reagents to purify approximately 300 mg of mouse IgG1.

1

1.3 Additional Items Required

Column rack The Econo-Pac 10 (12 place) acrylic rack is

Test tubes General purpose tubes for fraction collection

pH meter A pH meter is required to check the pH of

Mixer Standard laboratory magnetic stirrer and bar

Balance Standard laboratory scale for weighing out

Filters 0.22 micron filters for buffer preparation.

ideal for holding the Econo-Pac columns.
Other benchtop or lattice mount racks may
also be used.

are recommended.

the binding and elution buffers after reconsti­tution.

for buffer mixing.

buffer solids.

1.4 Buffer Preparation

Binding Buffer Preparation

The binding buffer is supplied as a premixed, preweighed solid.
Reconstitution and filtration are required prior to use. Dissolve 31.4 g
binding buffer solids in distilled, deionized water for a final volume
of 100 ml. (Use the full 471 g for 1,500 ml.) Stir for 10 minutes.
Filter through a 0.22 µm nylon filter and check the pH. The pH
should be 9.0 ± 0.2. If the pH is not in this range, adjust the pH with
10 N NaOH or 6 N HC1. Store buffer solids at room temperature.
Stoer reconstituted buffer at 4 °C. If desired, sodium azide may be
added to 0.05% (w/v).

Elution Buffer Preparation

The elution buffer is supplied as a preweighed, premixed solid.
Reconstitution and filtration are required prior to use. These salts
are hygroscopic. Any material in clumps should be broken up before
you weight the solids. Dissolve 2.2 g in distilled, deionized water for
a final volume of 100 ml (use the full 25 g for 1,100 ml). Stir for 10
minutes. Filter through a 0.22 µ m filter and check the pH. The pH
should be 3.0 ± 0.2. If the pH is not in this range, adjust the pH with
10 N NaOH or 6 N HC1. Store buffer solids at room temperature.
Reconstituted buffer should be stored at 4 °C, and if desired,
Thimerosal bacteriostat may be added to a final concentration of
one part per 10,000. The use of sodium azide in low pH buffers is haz­ardous.

2

1.5 Sample Preparation

Sample preparation is simplified by using the Econo-Pac 10DG
columns. Each Econo-Pac 10DG desalting column can process up to
3 milliliters of ascites fluid and the columns can be re-used. Two
Econo-Pac 10DG columns should be reserved for buffer exchange
at the end of the protein A purification protocol. It is also recom­mended that one Econo-Pac 10DG desalting column be used for
each (different) ascites to avoid cross contamination.

T o prepare up to 3 ml of ascites for purification on the Affi-Gel
protein a column:

1. Discard the buffer above the top frit of one Econo-Pac 10DG

column.

2. Add 20 ml of binding buffer to the column (fill to the top), and

snap off the bottom tip to start the column flowing.

3. Allow the buffer to drain to the top frit. The column will not run

dry. Flow will stop when the buffer level reaches the top frit.

4. Add 3.0 ml of ascites fluid to the column. If the sample is less

than 3.0 ml, add buffer to reach a total sample volume of 3.0

ml.

5. Allow the sample to completely run into the column. Discard

the first 3.0 ml eluted.

6. Add 4.0 ml of binding buffer to elute the ascites, while collect-

ing the 4.0 ml fraction from the column.

7. Wash the Econo-Pac 10DG column with 20 ml of binding buffer

if the column is going to be used again right away, or wash the

column with 20 ml of water containing 0.02% sodium azide for

storage.

1.6 Standard Mouse IgG1Purification
Procedure

1. Discard the buffer above the top frit of the Econo-Pac protein A

column.

2. Equilibrate the column with 10 ml binding buffer. Allow the

buffer to drain to the top frit. The column will not run dry . After

equilibration, the pH of the column effluent should be equal to

the pH of the binding buffer (pH 9.0).

3. Apply the prepared sample to the column. Maximum recom-

mended sample volume is 2.0 ml ascites fluid.

3

4. Wash the column with 20 ml binding buffer.

5. Elute the IgG with 10 ml of elution buffer. Elute the column

with an additional 20 ml of buffer to insure total removal IgG.

In most cases, the majority of the IgG will elute in the first 10 ml.

6. Wash the column with 10 ml of regeneration buffer, followed

by 10 ml of PBS containing 0.02% sodium azide for storage.

7. Set up an unused Econo-Pac 10DG column previously equili-

brated with 20 ml of PBS or other buffer suitable for storage of

immunoglobulin.

8. Apply 3 ml of the IgG-containing fraction collected in step 5 to

the prepared Econo-Pac 10DG column. Discard the first 3.0 ml

eluted.

9. Add 4.0 ml of appropriate buffer (see step 7) to the Econo-Pac

10DG column and collect the 4.0 ml fraction from the column.

This fraction contains the protein A purified IgG. For a more

precise collection method, elute the antibody with 8.0 ml of a suit-

able buffer and collect 1.0 ml fractions. Analyze the fractions to

determine which fractions to pool.

10. Repeat until the entire IgG-containing fraction has been buffer

exchanged.

11. The Econo-Pac 10DG column should then be washed with 20 ml

of deionized water. Store at room temperature with 0.02% sodi-

um azide for re-use.

1.7 Answers to Common Questions

1. Sensitivity of antibodies to low pH:

There are a few antibodies which are inactivated by low pH.

Inactivation can be avoided by collecting the eluted fractions

into a concentrated neutral buffer or buffer exchanging the

fraction using an Econo-Pac 10DG column. In cases of

extreme sensitivity, many immunoglobulins can be eluted at

pH 4-6 by adjusting the pH of the elution buffer with 10 N

NaOH.

2. Expected flow rate:

0.5-1.5 ml/min.

3. Column regeneration:

Regenerating the column after every use will increase the life-

time of the gel and help eliminate cross-over contamination of

IgG from one run to the next.

4

4. Shelf life to the Econo-Pac protein A kit:

The shelf life of the Econo-Pac protein A column is 1 year at

4 °C. The unreconstituted buffers are good for at least 1 year

when stored at room temperature. Reconstituted buffers can

be stored at 4 °C for approximately 2 weeks.

5. Purification of IgG from serum or tissue culture fluids:

In addition to ascites, serum and tissue culture samples are

suitable as samples for the Econo-Pac protein A kit. Serum

samples will require the same sample preparation and purifi-

cation procedures as ascites samples (as outlined in this manu-

al).

Tissue culture fluids should be concentrated to approximately

5 mg immunoglobulin/ml and then processed on an Econo-

Pac 10DG desalting column as described in this manual (or

diluted one to one with binding buffer). The purification pro-

cedure for the concentrated tissue culture fluid will then be

identical to the ascites procedure.

6. Purification of IgG other than mouse:

Human and rabbit IgG from serum (Bio-Radiations No. 53)

have been purified successfully with Affi-Gel protein A gel.

The protocol in this manual can be used without modification.

7. Purification of IgM with the Econo-Pac protein A kit:

Protein A will bind approximately 50% of all IgMs. The

purification of IgM using the Econo-Pac protein A kit will

therefore vary with each particular IgM sample.

Section 2
Econo-Pac Protein A Columns

2.1 Introduction

Bio-Rad’s Econo-Pac protein A columns are Econo-Pac 10
chromatography columns packed with 2 ml of Affi-Gel protein A gel
with an upper frit above the gel bed. Affi-Gel protein A consists of
purified protein A coupled to crosslinked agarose beads via stable
amide bonds. When used with the patented MAPS II buffer sys­tem, each column has a capacity of 10-14 mg mouse IgG1.

The Econo-Pac protein A columns provide a rapid and conve­nient way to purify monoclonal antibodies from small amounts of
ascites fluid. Binding, elution, and regeneration steps can be com-

5

pleted with only one column volume of buffer per step, making the
total procedure fast and easy.

2.2 Buffer Considerations

The affinity of IgG for protein A is not the same for all species.
Most mouse IgG1immunoglobulins have low affinity for protein A.
For this reason, the patented MAPS II buffer system has been devel­oped to optimize binding and recovery of mouse IgG1.1When Affi­Gel protein A is combined with the specially optimized MAPS II
buffer system, protein A capacity for mouse IgG1from ascites fluid
is 6-8 mg/ml gel. (This capacity is 8-10 times higher than that
obtained with published methods.
Econo-Pac protein A columns therefore have a mouse IgG1capac­ity of 10-14 mg per column.

An alternative binding buffer is 10 mM sodium phosphate, 0.15
M sodium chloride, pH 8.2. A typical elution buffer is 0.1 M sodi­um citrate, and a suitable regeneration buffer is 1.5 M sodium thio­cyanate.

1,2

) Using the MAPS II buffers, the

2.3 Sample Preparation

Bio-Rad’s Econo-Pac 10DG disposable desalting columns are
recommended for easy ascites or serum preparation with minimal
sample dilution. Each desalting column can prepare up to 3 milliliters
of raw ascites. The ascites sample is collected in 4 milliliters of
binding buffer, so sample dilution is minimized. An alternative is to
dilute ascites sample one to one with binding buffer , and then adjust
the pH to that of the binding buffer.

If your sample is tissue culture supernatant, it should be con­centrated to approximately 5 mg immunoglobulin/ml and then dilut­ed one to one with binding buffer.

2.4 Standard Mouse IgG Purification
Procedure

1. Discard the buffer above the top frit of the Econo-Pac protein

A column.

2. Equilibrate the column with 10 ml binding buffer. Allow the

buffer to drain to the top frit. The column will not run dry.

3. Apply prepared sample to the column. See “Column

Performance” for recommended sample volumes.

4. Wash the column with 20 ml binding buffer.

6

5. Elute the IgG with 10 ml of elution buffer. Elute the column

with an additional 20 ml of buffer to insure total removal of

IgG. In most cases, the majority of the IgG will elute in the first

10 ml. Prolonged exposure to acid pH should be avoided.

Neutralize immediately after elution. The Econo-Pac 10DG

desalting columns can be used to rapidly neutralize, desalt and

buffer exchange the eluate into a suitable buffer (e.g., PBS).

[Note: the use of the Econo-Pac 10 DG desalting columns effec-

tively eliminates the need for lengthy dialysis procedures required

when samples are to be analyzed by SDS-PAGE.]

6. Wash the column with 10 ml of regeneration buffer.

7. Wash the column with 10 ml of binding buffer for the next chro-

matography cycle or store in PBS containing 0.05% sodium

azide at 4 °C.

2.5 Column Performance

Packed gel Affi-Gel protein A
Bed volume 2.0 ml
Reservoir volume 30 ml
Colume capacity 10-14 mg IgG
Recommended sample

volume Maximum 2.0 ml ascites or serum
Packing buffer 10 mM sodium phosphate, 150

mM NaC1, pH 7.0, with 0.02%
sodium azide

1

Flow rate range 0.5-1.5 ml/min
Column material Polypropylene
Frit material Nominal 35 µm porous, polyethy-

pH range pH 2-11
Recommended storage 4 °C, with PBS containing 0.02%

lene

sodium azide

7

Section 3
References

1. Ey, P. L., Prowse, S. J. and Jenkins, C. R., Immunochemistry, 15, 429 (1978).

2. Begbee, W. L., Vanderlaan, M., Fong, S. S. N. and Jensen, R. M., Mol. Immunol., 20, 153

(1983).

Section 4
Ordering Information

Catalog
Number Product Description

732-2020 Econo-Pac Protein A Kit, includes one 2 ml Affi-

Gel protein A prepacked Econo-Pac column, 4
Econo-Pac 10 DG desalting columns, binding,

elution, and regeneration buffers.
732-2022 Econo-Pac Protein A Columns, 5
732-2010 Econo-Pac 10 DG Desalting Columns, 30
732-1015 Econo-Pac 10 Rack, 12 place

Other Affi-Gel Protein A Products from Bio-Rad:

153-6153 Affi-Gel Protein A Agarose, 5 ml
153-6154 Affi-Gel Protein A Agarose, 50 ml
153-6159 Affi-Gel Protein A MAPS II Kit, includes 5 ml Affi-

Gel protein A, binding, elution and regeneration

buffers, and 1 x 10 cm Econo-Column chromatog-

raphy column.
153-6160 Affi-Gel Protein A MAPS II Buffers, includes

binding, elution, and regeneration buffers.
153-6161 Affi-Gel Protein A MAPS II Binding Buffer, 5 liters
153-6162 Affi-Gel Protein A MAPS II Elution Buffer, 5 liters
153-6163 Affi-Gel Protein A MAPS II Regeneration Buffer,

5 liters

8

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