Bio-Rad Profinia Protein Purification System Kits User Manual
For technical support, call your local Bio-Rad office or, in the U.S.,
call 1-800-4BIORAD (1-800-424-6723)
Profinia™Native IMAC
and GST Starter Kits
Instruction Manual
(Step-by-Step Guide to
Profinia Operation)
Table of Contents
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Section 1 Introduction............................................................1
1.1 Background ....................................................................1
1.2 Product Information..........................................................1
1.3 Storage Conditions ..........................................................2
Section 2 Native IMAC and GST Starter Kits ........................2
2.1 Native IMAC Component Descriptions ............................2
2.2 GST Component Descriptions..........................................3
Section 3 Equipment and Materials ......................................5
3.1 Profinia Instrument and Accessories ................................5
Section 4 Setup and Running of the Profinia
IMAC Starter Kit ....................................................5
4.1 Reagent Preparation........................................................5
4.2 Instrument Startup, User Interface, and
Method Selection ............................................................6
Section 5 Setup and Running of the Profinia
GST Starter Kit ....................................................10
5.1 Reagent Preparation......................................................10
5.2 Instrument Startup, User Interface, and
Method Selection ..........................................................11
Section 6 Software and Analysis ........................................12
6.1 Viewing Starter Kit Data ................................................12
6.2 Analysis of Purified Protein ............................................13
Section 7 Shutdown, Cleaning, Storage Sequence,
and Data Export ..................................................14
Section 8 Frequently Asked Questions and
Troubleshooting ..................................................15
Section 9 Legal Notices ......................................................16
Section 10 Ordering Information ..........................................17
Section 1
Introduction
1.1 Background
The Profinia protein purification system includes a complete line of
consumables to use with an automated, user-friendly instrument for the
purification of immobilized metal affinity chromatography (IMAC) and
glutathione-S-transferase (GST) affinity-tagged proteins. The system has
been designed to use standard purification buffers, resins, and reagents
packaged into an easy-to-use kit. The two most common purification
applications, native IMAC and GST, have been packaged into starter kits
that contain all components necessary to purify a control affinity-tagged
protein. The buffers are supplied as concentrates and are diluted by the
instrument; no predilution is required. Profinia IMAC and GST starter kits
can be used after instrument installation and setup to familiarize the user
with the instrument or when troubleshooting purification problems. See
Section 10 for starter kit information and the complete list of Profinia products.
1.2 Product Information
Profinia starter kits are used to get started with the Profinia protein
purification system. The kits also help diagnose proper operation of the system. The Profinia starter kits contain a lyophilized 51 kD dual-tagged protein
that contains an N-terminal GST sequence tag and a C-terminal tag with
histidine (His) residues.* Once resuspended in diluted equilibration buffer,
the lysate yields sufficient protein for two independent purifications. Each
kit contains a complete set of buffers and solutions (affinity, desalting,
cleaning, and storage solutions), reagents (glutathione), and cartridges (1 ml
affinity paired with a 10 ml desalting) required for two affinity purifications
only. However, additional Bio-Scale™Mini IMAC or GST cartridges can be
purchased separately, as the buffers provided in the starter kits are
sufficient to perform 10 purification runs. See Proper Storage of Solutions
and Kits on page 15. Table 1 summarizes the reagents contained within the
two starter kits available for the Profinia system.
* The C-terminal histidine tag contains alternating histidine units and is not recognized by
6x histidine antibodies.
1
2
Table 1. Profinia IMAC and GST starter kits.
Kit Catalog # Solutions Reagents Cartridges
Purification Desalting Storage
Buffers Buffer Solutions Lysate Glutathione Affinity Desalting
Native IMAC 620-0229
✔✔✔✔ ✔✔
starter kit
GST starter kit 620-0230 ✔✔✔✔ ✔ ✔✔
1.3 Storage Conditions
Profinia starter kits are shipped at room temperature and can be safely
stored for short periods (up to 2 weeks) at room temperature. For long-term
storage, the kits should be stored at 4°C. The individual components and
outside kit box labels provide exact expiration dates.
Section 2
Native IMAC and GST Starter Kits
2.1 Native IMAC Component Descriptions
The affinity buffers provided in the native IMAC kit are formulated from
potassium salts and buffers, and contain increasing concentrations of
imidazole for optimized washing and eluting of His-tagged proteins. Native
IMAC buffers are used for proteins soluble in
E. coli
lysates and that have
accessible tags containing histidine residues. Table 2 provides a list of
buffer compositions. The native lysis buffer is used for preparing
sample lysates; is not used in the instrument. This buffer can be used for
subsequent purifications of target protein outside of the starter kit
applications.
The IMAC cartridges are shipped in 20% ethanol and are packed with
1 ml Profinity™IMAC resin. The desalting cartridges are provided in
20 mM Bis-Tris, pH 6.5 with 0.05% azide as a preservative. The control
lysate is provided as a lyophilized sample prepared from 50 ml
E. coli
culture. When resuspended in 12 ml of 1x IMAC lysis buffer, ~4 mg of
tagged target protein can be purified from a 5 ml sample load. All
components supplied with the native IMAC starter kit are shown in Table 2.
Table 2. Formulations for buffers and solutions provided in the
native IMAC starter kit.
Profinia Solution Supplied Concentrated Formulation Working 1x Formulation Volume Position #
As
Native IMAC 2x 600 mM KCl, 100 mM KH
2PO4
, 300 mM KCl, 50 mM KH2PO4,
lysis buffer 10 mM imidazole, pH 8.0 5 mM imidazole, pH 8.0 125 ml N/A
Native IMAC 2x 600 mM KCl, 100 mM KH
2PO4
, 300 mM KCl, 50 mM KH2PO4,
wash buffer 1 10 mM imidazole, pH 8.0 5 mM imidazole, pH 8.0 125 ml 1
Native IMAC 2x 600 mM KCl, 100 mM KH
2PO4
, 300 mM KCl, 50 mM KH2PO4,
wash buffer 2 20 mM imidazole, pH 8.0 10 mM imidazole, pH 8.0 100 ml 2
Native IMAC 2x 600 mM KCl, 100 mM KH
2PO4
, 300 mM KCl, 50 mM KH2PO4,
elution buffer 500 mM imidazole, pH 8.0 250 mM imidazole, pH 8.0 100 ml 3
Desalting buffer 5x 685 mM NaCl, 13.5 mM KCl, 137 mM NaCl, 2.7 mM KCl,
21.5 mM Na
2
HP04, 4.3 mM Na2HP04,
40.5 mM KH
2
HP04, 8.1 mM KH2P04, pH 7.4 200 ml 4
pH 7.0 (pH 7.4 upon dilution)
Cleaning solution 1 2x 1,000 mM NaCl, 500 mM NaCl,
100 mM Tris, pH 8.0 50 mM Tris, pH 8.0 125 ml 5
Cleaning solution 2 4x 2,000 mM NaCl, 500 mM NaCl,
400 mM NaOAc, pH 4.5 100 mM NaOAc, pH 4.5 125 ml 6
Storage solution 2x 4% C
6H5CH2
OH 2% C6H5CH2OH
(benzyl alcohol) (benzyl alcohol) 200 ml 7
2.2 GST Component Descriptions
The lysis and wash buffers contained in the GST kit are formulated
from sodium salts, phosphate buffers, and EDTA, and provide optimized
binding, washing, and eluting of GST-tagged proteins. EDTA is included
as a chelating compound that protects against metalloproteases, which can
be present in
E. coli
lysates. GST fusion proteins must be in a native
conformation prior to purification. The buffers used in this system are
designed for the purification of proteins that are soluble in
E. coli
lysates
and that have accessible, native GST sequence tags. Table 3
provides a list of buffer compositions. The lysis buffer is used for preparing
sample preparations; it is not used in any of the instrument ports. This
buffer can be used for subsequent purifications of target protein outside of
the starter kit applications.
The GST cartridges are shipped in 20% ethanol and are packed with
1 ml Profinity GST resin. The desalting cartridges are provided in 20 mM
Bis-Tris, pH 6.5 with 0.05% azide as a preservative. The vial of lyophilized
lysate is described in sections 1.2 and 2.1, and when resuspended in 12 ml
1x GST lysis buffer, provides adequate sample for two independent
purification runs.
3
Table 3. Formulations for buffers and solutions provided in the GST
kits.
Profinia Supplied
Solution AsConcentrated Formulation Working 1x Formulation Volume Position #
GST 2x 300 mM NaCl, 20 mM 150 mM NaCl, 10 mM Na
2
HPO4,
lysis buffer Na
2
HPO4, 10 mM EDTA, 5 mM EDTA, pH 7.4 125 ml N/A
pH 7.4
GST 2x 300 mM NaCl, 20 mM 150 mM NaCl, 10 mM Na
2
HPO4,
wash buffer Na
2
HPO4, 10 mM EDTA, 5 mM EDTA, pH 7.4 200 ml 1
pH 8.0
GST 2x 40 mM glutathione, 200 mM Tris, 20 mM glutathione, 100 mM Tris,
elution buffer 10 mM EDTA, pH 8.0 5 mM EDTA, pH 8.0 100 ml 3
Desalting 5x 685 mM NaCl, 13.5 mM KCI, 137 mM NaCl, 2.7 mM KCl,
buffer 21.5 mM Na2HPO4, 40.5 mM 4.3 mM Na2HPO4, 8.1 mM
KH
2
HPO4, pH 7.0 KH2PO4, pH 7.4 200 ml 4
(pH 7.4 upon dilution)
Cleaning 2x 1,000 mM NaCl, 500 mM NaCl, 50 mM Tris,
solution 1 100 mM Tris, pH 8.0 pH 8.0 125 ml 5
Cleaning 4x 2,000 mM NaCl, 500 mM NaCl,
solution 2 400 mM NaOAc, pH 4.5 100 mM NaOAc, pH 4.5 125 ml 6
Storage 2x 4% C
6H5CH2
OH 2% C6H5CH2OH
solution (benzyl alcohol) (benzyl alcohol) 200 ml 7
4
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