Profinia Protein Purification System Instruction Manual Rev C

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Legal Notices No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage or retrieval system, without permission in writing from Bio-Rad Laboratories, Inc. Bio-Rad reserves the right to modify its products and services at any time. This user guide is subject to change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors or omissions, or for any damages resulting from the application or use of this information. Purification and preparation of fusion proteins and affinity peptides containing at least two adjacent histidine residues may require a license under US patent 5,284,933 and US patent 5,310,663, including foreign patents (assignee: Hoffmann-La Roche). Expression and purification of GST fusion proteins may require a license under US patent 5,654,176 (assignee: Chemicon International). Benzonase is a trademark of Merck KGAA Corp. Excel, Microsoft, Windows 2000 and XP are trademarks of Microsoft Corporation. Pentium is a trademark of Intel Corporation. Teflon and Tefzel are trademarks of E.I. du Pont de Nemours and Co. Vaseline is a trademark of Unilever Supply Chain Inc. Copyright © 2007 by Bio-Rad Laboratories, Inc. All rights reserved.

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Caution!

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Please read all safety-related information before operating the Profinia system. This instrument is for laboratory use only. Always connect the power supply to a 3-prong, grounded AC outlet using the AC power cord provided with the Profinia instrument. Do not use an adaptor to connect to a 2-terminal outlet. The right-side back panel is easily removable for access to user-serviceable parts. Never remove any other outer casings of the Profinia instrument. Call your local Bio-Rad office for instrument service. Do not operate the Profinia instrument in extreme humidity (>95%), or in a condensing environment. When taking the Profinia instrument into a cold room, the unit can be operated immediately. However, when removing the unit from the cold room, let it warm up to room temperature for a minimum of 2 hours before switching on the power. This avoids damage from possible condensation. The Profinia instrument is not designed for use with flammable liquids. Flammable liquids should never be placed in either the buffer or sample inlets of the instrument. This product conforms to Class A standards for Electromagnetic Emissions intended for laboratory equipment applications. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or on the same circuit as those appliances. The user should be aware of this potential and take appropriate measures to avoid interference. This Bio-Rad instrument is designed and certified to meet EN-61010 safety standards; EN-61010-certified products are safe to use when operated in accordance with this instruction manual. This instrument should not be modified or altered in any way. Alteration of this instrument will void the manufacturer’s warranty, void EN-61010 certification, and create a potential safety hazard for the user. Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modifications of the instrument not performed by Bio-Rad or an authorized agent.

Table of Contents Page

Section 1

Profinia Protein Purification System Description ...1

1.1 1.2 1.3 1.4 1.5

Profinia System Overview ...1 Key Features of the Profinia System ...1 System Components and Optional Accessories...2 Related Consumables and Products...4 About This Guide ...4 1.5.1 Conventions...4

Section 2

Installation Procedures...5

2.1 2.2

System Installation...5 First-Time Use of the Profinia System...8 2.2.1 Preparing the Instrument for Cleaning...8 2.2.2 Selecting the First-Time Cleaning Procedure ...9 2.2.3 Setting the Local Date and Time ...10 2.2.4 Validating the System ...10

Section 3

Key Terms and Definitions...14

3.1 3.2 3.3 3.4 3.5

Bio-Rad Methods ...14 Program Methods ...14 Toolbar Navigation Buttons...15 System Status Indicator Definitions...16 Alphanumeric and Numeric Keypads ...17 3.5.1 Alphanumeric Keypad...17 3.5.2 Numeric Keypad ...17

Section 4

Profinia System Quick Guide ...19

4.1 4.2

Profinia Installation...19 Profinia System Quick Guide ...23

Section 5

Home Screen Overview ...27

Section 6

Bio-Rad Methods...29

6.1 6.2 6.3

Selecting Method System ...32 Selecting Method Type and Options ...32 Selecting Sample Options...34 6.3.1 Selecting Sample Flow Rate and Wash Time ...34 6.3.2 Selecting Sample Incubation Time...35 Entering Run and Sample Information ...36 6.4.1 Adding or Editing Run and Sample Information...38 6.4.2 Adding Lot Number Tracking Information...38 Installing Reagents, Sample Tubes, and Fraction Tubes...39 6.5.1 Loading Buffers...41 6.5.2 Installing Sample Tubes...44 6.5.3 Installing Fraction Collection Tubes...45 Installing Cartridges ...46 6.6.1 Preparing for Cartridge Installation...46 6.6.2 Setting Up Cartridge Positions for Priming ...47 6.6.3 Priming the Cartridge Lines...48 6.6.4 Installing Cartridges ...48 6.6.5 Installing the Sample Loop (Desalting-Only Methods) ...52 Starting the Run...54

6.4

6.5

6.6

6.7

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6.8

6.9

6.10

6.7.1 Viewing Steps...55 6.7.2 Viewing Real-Time Data With Profinia Software...56 6.7.3 Clearing System Memory to Start the Run ...57 Monitoring Run Progress ...58 6.8.1 Run in Progress Screen...58 6.8.2 Peak Detection ...59 6.8.3 Collecting the Purified Sample ...61 6.8.4 Canceling or Pausing a Run ...62 6.8.5 Completing the Run ...62 Preparing System for End-of-Run Cleaning ...62 6.9.1 Preparing for Line Cleaning ...62 6.9.2 System Cleaning in Progress...63 Performing End-of-Run Procedures ...63 6.10.1 Exporting Data to a Computer...64 6.10.2 Performing Final Run Procedures ...64

Section 7

Program Methods...68

7.1 7.2 7.3

Selecting Method System ...69 Selecting Method Type and Options ...70 Entering Run and Sample Information ...71 7.3.1 Adding or Editing Run and Sample Information...73 7.3.2 Adding Lot Number Tracking Information...73 Editing and Saving a Program Method...74 7.4.1 Editing a Program Method ...74 7.4.2 Saving a Program Method ...77 Installing Reagents, Sample Tubes, and Fraction Tubes...78 7.5.1 Preparing and Installing Reagents and Samples for the Purification Run ...80 Installing Cartridges ...80 7.6.1 Installing the Sample Loop (Desalting-Only Methods) ...81 Viewing Real-Time Data With Profinia Software...83 Starting the Run...83 7.8.1 Viewing Steps...84 7.8.2 Running the Manual Prime Function ...84 7.8.3 Clearing System Memory to Start Run ...85 Monitoring Run Progress ...85 7.9.1 Peak Detection ...85 7.9.2 Collecting Purified Sample ...86 7.9.3 Completing the Run ...86 Preparing System for End-of-Run Cleaning ...86 7.10.1 Preparing for Line Cleaning ...86 7.10.2 System Cleaning in Progress Screen...86 Performing End-of-Run Procedures ...86 7.11.1 Exporting Data to a Computer...87 7.11.2 Performing Final Run Procedures ...87

7.4

7.5

7.6 7.7 7.8

7.9

7.10

7.11

Section 8

Saved Methods...89

8.1 8.2 8.3

Retrieving Saved Methods ...89 Selecting Saved Methods ...90 Running Saved Methods...90

Section 9

Data and Utilities ...92

9.1

Exporting Data & Real-Time Data Acquisition ...92 9.1.1 Exporting Data to USB Portable Memory Device ...92

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9.2

9.3

9.1.2 Real-Time Data Transfer to Profinia Software ...93 System Utilities ...93 9.2.1 Calibrating Touch Screen ...94 9.2.2 Checking Pump Flow Rate ...94 9.2.3 Calibrating UV Monitors ...95 9.2.4 Calibrating Conductivity Monitor...96 9.2.5 Adjusting Peak Detection Sensitivity ...97 9.2.6 Calibrating pH Probe...97 9.2.7 Calibrating Air Detection System...98 9.2.8 Calibrating Pressure ...98 Diagnostic/Maintenance Functions ...98 9.3.1 Displaying Firmware Versions...99 9.3.2 Setting and Operating Instrument Manually ...99 9.3.3 Setting Time and Date ...104 9.3.4 Cleaning Sample Lines ...105 9.3.5 Cleaning Fraction Lines ...106 9.3.6 Cleaning Buffer Lines...107 9.3.7 Washing System With Ethanol...108 9.3.8 Cleaning Inlet and Outlet Lines ...109 9.3.9 Cleaning Pump Check Valves...110 9.3.10 Washing System With NaOH ...111 9.3.11 Cleaning Inline Check Valve ...112 9.3.12 Affinity Cartridge Cleaning in Place ...112 9.3.13 Desalting Cartridge Cleaning in Place...113 9.3.14 Selecting End of Sample/Reagent Detection (Air Sensors) ...114 9.3.15 Selecting Method Temperature...115 9.3.16 Select Display Contrast...116

Section 10 Profinia Purification Kits...117 10.1

10.2 10.3 10.4 10.5

Introduction...117 10.1.1 Background ...117 10.1.2 Product Information...117 10.1.3 Equipment and Materials Required ...118 10.1.4 Storage Conditions ...118 Native IMAC Purification and Buffer Kits ...118 Denaturing IMAC Purification and Buffer Kits ...120 GST Purification and Buffer Kits...122 Desalting Buffer and Separation Kits...123

Section 11 Connecting External Components...125 11.1 11.2

11.3 11.4

pH Monitor ...125 Larger Buffer, Sample, and Fraction Containers ...128 11.2.1 External Buffer Containers ...128 11.2.2 External Sample Containers ...129 11.2.3 External Fraction Collection Containers ...130 Profinia Cooling Accessory ...130 Connecting non-Bio-Rad Cartridges...132

Section 12 Software...133 12.1 12.2 12.3

Operating Requirements...133 Software Structure ...133 Transferring Data to Profinia Software ...134 12.3.1 Real-Time Data Transfer...134 12.3.2 Portable Memory Device Data Transfer ...135

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12.4

12.5

12.6 12.7 12.8

Viewing Run Data Files...135 12.4.1 Opening Files in Profinia Software ...135 12.4.2 Chromatogram Overview ...136 12.4.3 Eliminating Run Parameters Displayed ...136 12.4.4 Customizing Chromatogram Display Options...136 Reports...139 12.5.1 Standard Reports...139 12.5.2 Custom Reports...139 Exporting Chromatograms ...140 Exporting Data Files...140 Help Information ...140

Section 13 Care and Maintenance ...141 13.1

13.2

Cleaning the Instrument ...141 13.1.1 Daily Cleaning...141 13.1.2 Weekly Cleaning...141 13.1.3 Storage...141 13.1.4 System Cleaning Procedures...142 13.1.5 Cleaning Touch Screen ...143 Maintenance ...144 13.2.1 Pump Check Valves ...145 13.2.2 Pump Seals ...149 13.2.3 UV Monitor Lamp...153 13.2.4 UV Monitor Flow Cells ...158 13.2.5 Buffer and Sample Inlet Tubes...161 13.2.6 Tubings and Fittings...161 13.2.7 Inline Filter ...166 13.2.8 Power Conditioning...167

Appendix A

Profinia System Specifications...168

Appendix B

Profinia System Ordering Information...169

Appendix C

Profinia System Troubleshooting ...172

Appendix D

Profinia System Error Messages ...176

Appendix E

Sample Preparation ...177

Appendix F

Sample Preparation, Application, and Analysis Tips ...179

Appendix G

Profinia System Annual Maintenance ...184

Appendix H

Profinia Instrument Cleaning and Maintenance Log ...186

Appendix I

Profinia Protein A and G Method Manual ...187

Appendix J

Profinia Profinity eXact Method Manual ...194

Appendix K

Profinia Program Methods Planning Worksheets...202

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Section 1 Profinia™ Protein Purification System Description 1.1 Profinia System Overview The Profinia protein purification system is an automated chromatography system that provides a simple solution for the purification and desalting of milligram amounts of affinitytagged proteins. The Profinia system is easy to operate, allowing users to concentrate on solving the questions posed by science, rather than spending time performing separation techniques. In the Profinia system, the cartridges, tubes, fittings, and Bio-Rad methods have all been optimized and are built in, so no expertise in chromatography is required to set up the instrument or perform affinity purification. The Profinia system requires minimal user interaction; a user-friendly touch screen provides guidance through all setup, purification, and run completion procedures. The yield and purity of proteins obtained using the Profinia system are comparable to those from more complicated or labor-intensive manual methods such as gravity-flow chromatography. Users have two options for running purification methods: Bio-Rad methods with automated templates for common GST, IMAC, Protein A and G purifications, Profinity eXact purification or program methods created by editing the Bio-Rad method templates. Both Bio-Rad and program methods include automatic desalting (not all Bio-Rad methods include desalting) as well as automatic system and cartridge cleaning. Both types of methods also provide a data record of results that can either be viewed and recorded at the end of the run, exported to a computer with Profinia software for data review, or viewed in real time on a computer with Profinia software. A variety of purification, buffer, and starter kits are available for use with the Profinia instrument. Profinia purification kits include cartridges, concentrated reagents, and buffers, and are optimized for use with Bio-Rad methods. Profinia buffer kits include all concentrated reagents necessary to run a Bio-Rad method. Profinia starter kits are available for GST and native IMAC methods. These kits include cartridges, concentrated reagents, and control lysate to perform a Bio-Rad method. All kit components are clearly labeled to show where they should be placed in the instrument, and formulations of all buffers are listed for use in reports. Buffer and cartridge kits, optimized methods, and instrument automation ensure high quality and reproducible purification purity and yield, along with significant time savings. From setup to purification runs to exportable data files, the Profinia system is intuitive and easy to operate, allowing researchers at any level of chromatography expertise to move quickly and efficiently through purifying affinity-tagged proteins to downstream protein-based experiments.

1.2 Key Features of the Profinia System •

Integrated system design - no need to assemble tubing and fittings or pack chromatography columns

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Easy instrument setup - plug in the instrument and load reagents

•

Simple system operation - automated purification and desalting of affinity-tagged proteins

•

Automatic calculations - estimated purified protein yield and concentration displayed at end of run

•

Touch-screen interface - guided instrument and method setup; displays current run information

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Built-in cleaning and storage steps - automated system cleaning ensures continued peak instrument performance

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Purification kits - affinity and desalting cartridges, buffers, and solutions optimized for Bio-Rad methods

•

Optional cooling accessory - samples and fractions of purified samples stay cold without keeping the instrument in a cold room or using a cold box

•

Optional Profinia software - real-time data and customized reports can be displayed, used for basic protein comparisons and analysis, and incorporated into lab notebooks and presentations

1.3 System Components and Optional Accessories The Profinia protein purification system components (see Figure 1.1) are: Profinia instrument - shipment box #1 Profinia accessory kit - shipment box #2; includes: •

Profinia buffer bottle starter pack - 4 x 125 ml and 4 x 250 ml empty bottles

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Profinia buffer lids - pack of eight lids

•

Profinia lids for 15 ml sample conical tubes - pack of two lids

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Profinia lids for 50 ml sample conical tubes - pack of two lids

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Profinia waste/diluent (water) bottle pack - 2 x 2 L bottles

•

Profinia cleaning tray - placed in the fraction collection area to collect liquid by products from line cleaning and system wash procedures

•

Inline filter - 12 replacement filters

Profinia starter kit - shipment box #3; choice of native IMAC or GST starter kit, includes 1 ml affinity cartridge, 10 ml desalting cartridge, vial of Profinia control lysate, and a complete buffer set for native IMAC or GST purification

Profinia instrument

IMAC or GST starter kit

Cleaning tray Waste and diluent (water) bottles Buffer and sample lids

Buffer bottles

Inline filter

Fig. 1.1. Profinia purification system components.

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The following accessories and components can be ordered separately for use with the Profinia system: •

Profinia control lysate - dual-tag (histidine and GST) protein that can be used to validate the performance of the Profinia system

•

Profinia software - provides data reporting and basic analysis of run results

•

Cooling accessory - holds two 15 ml or 50 ml conical tubes (holds two samples and/or two purified protein fraction tubes), and is designed to keep samples or purified protein cooled below 4°C when a cold room environment is not available

•

Profinia purification kits - prepackaged cartridges, buffers, and reagents, for use with Bio-Rad methods. Bottles and accessories are clearly labeled to indicate positioning in the Profinia instrument (see Table 1.1)

•

Profinia buffer kits - prepackaged buffer and reagents for use with Bio-Rad methods; cartridges are not included. Bottles and accessories are clearly labeled to indicate positioning in the Profinia instrument (see Table 1.1)

•

Bio-Scale™ Mini cartridges - IMAC, GST, Protein A cartridges (1 ml and 5 ml), Profinity eXact cartridges (1 ml and 5 ml), and desalting cartridges (10 ml and 50 ml)

•

Desalting sample loops - required for use in desalting methods only; available in 2 ml and 10 ml sizes. This accessory is not required for Bio-Rad methods that include affinity and desalting (for example, native IMAC and desalting or GST and desalting)

•

Bottle starter pack - replacement empty buffer bottles (4 x 125 ml and 4 x 250 ml buffer bottles, eight buffer lids)

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Waste/diluent bottle set - replacement waste and diluent bottles with caps and tubing

Table 1.1. Types of Profinia purification and buffer kits.

Method Native IMAC*

Denaturing IMAC

GST**

Desalting

Purification Kit 1 ml or 5 ml affinity cartridge plus 10 ml or 50 ml desalting cartridge configurations 1 ml or 5 ml affinity cartridge configurations; desalting does not apply to this kit 1 ml or 5 ml affinity cartridge plus 10 ml or 50 ml desalting cartridge configurations 10 ml or 50 ml cartridge configurations

* IMAC = immobilized metal affinity chromatography. ** GST = glutathione S-transferase.

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Buffer Kit Buffers and reagents for native IMAC and desalting methods Buffers and reagents for denaturing IMAC methods Buffers and reagents for GST and desalting methods Buffers and reagents for desalting-only methods

1.4 Related Consumables and Products The following related products are available from Bio-Rad (see Appendix B for a list of ordering information and catalog numbers): •

Detection antibodies - used for histidine (His) and glutathione S-transferase (GST)

•

Bacterial/Lysis/Extraction reagent - chemical lysis buffer used to prepare sample lysates; without the need for sonication. This reagent is not used in the instrument

•

Profinia control lysate - lyophilized sample prepared with a His- and GST-tagged protein from E. coli

•

Profinity eXact Antibody Reagent - a mouse monoclonal antibody used to detect expression of the target protein

The following related products are available from other vendors: •

Conical tubes - 15 ml and 50 ml, for use in the sample and fraction collection positions

Note: Not all tubes made by all vendors will fit properly in the Profinia instrument; Bio-Rad recommends the following vendor tubes: 50 ml, VWR catalog #21008-178 and #20171-029 and BD Biosciences catalog #352098; 15 ml, VWR catalog #20171-025 and BD Biosciences catalog #352097.

1.5 About This Guide 1.5.1 Conventions The following messages in bold are used to highlight critical information and reminders throughout this document: •

Warning - indicates an action that can result in an irretrievable loss of sample or data

•

Note - serves as a reminder, repeating relevant information from previous sections or offering advice helpful to achieving desired results

•

Important - indicates information critical to achieving optimal results

Touch-screen button commands are indicated by initial capitalization, for example: Touch the Select button. Screen names are indicated by initial capitalization, for example: You will proceed to the Data/Utilities screen. Functions located within menus on touch screens are indicated by initial capitalization and enclosed in double quotation marks, for example: Highlight the “System Ethanol Wash” option.

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Section 2 Installation Procedures 2.1 System Installation The Profinia™ purification system is shipped in three boxes. The largest box contains the instrument wrapped in foam-insert protective packaging; the smaller boxes contain all the additional components sold as part of the Profinia system. Warning: Use care to follow the instrument unpacking procedures described below. Improper handling of the instrument can cause damage. To unpack and set up the Profinia system: 1. Open the smaller boxes containing the system components and make sure all parts were received (see section 1.3 for a list of all components included with the Profinia purification system). 2. Set the larger box containing the Profinia instrument on the floor with the arrows pointing up (Figure 2.1).

Fig. 2.1. Profinia instrument box.

3. Use scissors or wire cutters to cut the plastic straps. 4. Use a large screwdriver or similar tool to remove the staples (if present) along the sides, near the bottom of the box.

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Fig. 2.2. Three boxes containing Profinia instrument and components.

5. Use the handles cut into the side of the top section of the box to lift it off the instrument (Figure 2.3). 6. Remove the protective packaging from the instrument (Figure 2.3).

Fig. 2.3. Upper box and protective packaging removed from the top of the Profinia instrument.

Note: Do not grasp the instrument by the top cover; damage to the instrument can result from mishandling. Note the “No Lift” labels that hold the top cover in place. 7. Use the handles on the left and right rear of the instrument to lift it onto the benchtop (Figure 2.4).

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Fig. 2.4. Using lift handles to remove the instrument from packaging.

8. Connect the power cable to the back of the instrument (see Figure 2.5 for location of power cord connection). 9. Install the diluent (water) and waste bottle tubing by unscrewing the shipping plugs from the deionized (Di) water and waste ports on the back of the instrument; connect the diluent and waste bottle tubing to these ports (Figure 2.5).

Waste port Di water port

Lift handles

Power cable connection

Fig. 2.5. Rear instrument view showing the location of the power cable connection, lift handles, and Di water and waste ports. Water and waste bottles are shown properly connected to the ports.

Note: The 2 L bottles packaged with the accessories include both “Water” and “Waste” labels. Determine which bottle will be used for which function, then use a permanent marker to cross out the other label. This will avoid confusion and possible contamination.

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2.2 First-Time Use of the Profinia System Before using the Profinia system, perform buffer, sample, and fraction line cleaning procedures to flush the system and remove any air trapped in the lines. 2.2.1 Preparing the Instrument for Cleaning a.

Buffer tray area

Cartridge holders

Waste bottle holder

Sample loading area

Diluent (water) bottle holder

Fraction collection area

b.

Fig. 2.6.a. Profinia system ports without bottles and tubes; 2.6.b. Profinia cleaning tray placement in the fraction collection area.

Note: For detailed instructions on installing reagent bottles, sample tubes, fraction tubes, and using cartridge connections, see section 6.4 and 6.5. To prepare the Profinia system for cleaning prior to its first use: 1. Remove the plastic bag covering the Profinia instrument and remove the “No Lift” stickers from the top cover. 2. Turn the instrument around so the front faces you, and turn the system on using the switch on the lower right in front of the diluent (water) bottle. 3. Open the instrument top cover. Remove the plastic caps from the ends of the buffer and sample tube lines.

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4. Fill the four 125 ml and three of the 250 ml buffer bottles provided with the instrument to capacity with deionized water, and place them in buffer positions 1 through 7. 5. Fill one buffer bottle with 100 ml of 20% ethanol and place it in buffer position 8. 6. Fill two 50 ml conical tubes with 25 ml of deionized water each, and place them in the sample 1 and 2 (S1 and S2) positions. Note: Tubes containing water must be placed in both sample positions during line cleaning, even after performing a run with only one sample (line cleaning only). 7. Locate the cartridge holder fittings at the left center of the instrument. Secure the upper cartridge holder fittings to the lower cartridge holder fittings by sliding the upper fittings down until they contact the lower fittings. Tighten the black lock ring on each fitting by turning it clockwise; do not overtighten (see section 6.5 for more details). 8. Place the cleaning tray in the fraction collection position (Figure 2.6). 9. Fill the diluent bottle with deionized water and place it in the right-side bottle position, with the tube in place. 10. Make sure the waste bottle is empty and place it in the left-side bottle position, with the tube in place. Note: Make sure the waste bottle is empty before running any method or cleaning procedure. 2.2.2 Selecting the First-Time Cleaning Procedure To flush the system and prepare it for first use: 1. Make sure the water and ethanol solutions are placed as described in steps 4 through 6 above, and the cleaning tray is in place, as described in step 8 above. 2. Touch the Data/Utilities button in the lower toolbar of the system home screen (see section 5 for information on home screen functions). 3. Touch the arrow buttons to scroll through the list of functions and highlight “Clean All Inlet and Outlet Lines” (Figure 2.7). Select Start to perform this procedure. A new screen will display the progress toward completion. When the cleaning procedure is complete, the system will return to the Data/Utilities screen.

Fig. 2.7. Clean all Inlet and Outlet Lines screen.

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It is recommended that this function be perfomed a second time prior to first use to remove any air that might have entered the system. The system has now been flushed with ethanol and water, and is ready for use. 2.2.3 Setting the Local Time and Date During purification runs, sample elution time is shown according to the system clock time as set by the user. To set the system time and date: 1. Touch Data/Utilities from the system home screen. 2. Touch the Diag/Maint Functions button. 3. Touch Time and Date Setup. 4. Touch the arrows adjacent date and time settings to adjust as desired (Figure 2.8); touch OK when finished to retain new settings and return to the Diag/Maint Functions screen.

Fig. 2.8. Set Time & Date screen.

2.2.4 Validating the System Performing an initial validation run of the Profinia system is recommended. This will allow you to familiarize yourself with the set up, operation, and expected results of a purification run. Use the starter kit provided with the instrument; if you need a new starter kit or additional Profinia control lysate, see Appendix B for ordering information. The starter kit includes a His/GST dual-tag control protein lysate to be used for this validation run. The results are characterized in this section. The dual-tag control lysate can be used with both the native IMAC and GST purification methods. To prepare and use the control lysate: 1. Prepare the sample lysate by reconstituting it with 12 ml of 1x IMAC or GST lysis buffer (dilute 6 ml of IMAC or GST lysis buffer 1:1 with 6 ml of deionized water to make 12 ml of a 1x solution). This yields enough lysate for two of the 1 ml affinity runs. 2. Transfer 6 ml of resuspended sample to a 15 ml conical tube.

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3. Place the tube in the sample 1 position and select the “1 Sample” option in the appropriate run set-up screen. 4. Prepare the buffers and reagents as described in the starter kit instruction manual. 5. Follow the set-up steps in section 4 (quick guide) or in section 6 (Bio-Rad methods programming) of this manual to select the method and load the buffers, sample, cartridges, and fraction tubes. 6. Compare your results to the following expected results for the IMAC or GST purification of the control lysate. Expected Results for IMAC Purification of the Control Lysate

Fig. 2.9. Typical chromatogram of the native IMAC control lysate purification with desalting.

Gel lanes 1 2

3

4

5

6

MW 250 150 100 75 50 37

25 20

15 10

Fig. 2.10. Typical gel electrophoresis results of the native IMAC control lysate purification. Lane 1, Precision Plus™ MW standard; 2, load; 3, flow-through; 4, wash 1; 5, wash 2; 6, eluted protein.

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Fig. 2.11. Typical Experion™ system electropherogram of the purified fraction of the native IMAC control lysate purification.

Expected Results for GST Purification of the Control Lysate

Fig. 2.12. Typical chromatogram of the GST control lysate purification with desalting.

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MW

Gel lanes 1 2

3

4

5

6

250 150 100 75 50 37

25 20

15

10

Fig. 2.13. Typical gel result of the GST control lysate purification. Lane 1, Precision Plus™ MW standards; 2, skip; 3, load; 4, flow-through; 5, wash; 6, eluted protein.

Fig. 2.14. Typical Experion system electropherogram of the purified fraction of the GST control lysate purification.

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