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
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