ÄKTA™start - GE Healthcare Life Sciences
Contents
1. 0 04 mAU al x n Gu 0 00 8 0 Set B 0 50 ml min 0 00 MPa Start peak collection B Flow path details for flow of 100 buffer B through Outlet valve to waste Accumulated Volume 0 00 MPa 0 50 ml min C Flow path details during gradient operation at specified B concentration C3 PRS AutoZero 0 00 MPa 1 00 B Set B 0 50 ml min D Flow path details Outlet valve collection Note Fraction collector is disabled AKTA start System Cue Card 29 0240 42 AC E Flow path details fraction collection Note Fraction collector is enabled Colum pee U J 0 00 MPa 5 00 B Set B 0 Tube number 0 50 ml min Wash valve Start Fractionation ml F Flow path details while loading Sample through the Pump lee eed 0 00 MPa I a 0 Tube number 0 50 ml min Getting help AKTA start Instrument Display Help e Accessible from every screen on the Instrument Display by tapping the question mark located in the upper right corner e The Display Help text provides information about the content of the current screen or refers to more detailed documentation UNICORN start Online Help e Dialog descriptions of instructions needed to operate UNICORN start and analyze chromatography run data AKTA start System Cue Card 29 0240 42 AC e Accessible within UNICORN start by pressing F2. Import Result to UNICORN start The result file stored on the USB stick can be imported to UNICORN start for analysis printing and generating a report UNICORN start File Import Import Result from USB stick e Locate USB drive e Locate and select the result file Select multiple files using Shift or Ctrl keys Open Select destination folder for result Note The result files will be saved in the GE folder which is automatically created by the instrument once the USB memory stick is plugged in Only 10 results can be stored in the GE folder To save further results transfer the result files to another folder PC or rename the GE folder Quick start methods Quick start contains ready to run methods like Affinity lon exchange Gel filtration and Desalting The run parameters are pre set in the method Details of sample volume need to be provided before starting the run e If required the run parameters can be changed Edit run in Run view screen when the run is in progress e Use appropriate columns as indicated in the template names E g use 1 ml HiTrap column in case of selecting the Quick start AC IEX step 1 ml HiTrap or 5 ml column when selecting AC IEX step 5 ml HiTrap Quick start workflow Method run Quick start Select the desired technique g Set Sample volume E Tick the checkbox Save Result to USB Edit run to alter run parameters Ex
3. and B concentration sustained e Pause pauses the run by stopping the pump e End terminates the current run Run view UV absorbance Oo mAU Conductivity mS cm Flow rate 0 mi min Pressure MPa Tube no Edit run Operation from UNICORN start System Control module is used to start view and control arun The instrument is controlled via simple clicks on the flow path depicted in the process picture e g to turn the valves set flow rates change B concentration and start stop fractionation Evaluating arun Save Result as BMP image The system generates a BMP file of the result which can be viewed under any OS that has an image viewer supporting BMP The chromatogram is plotted as UV abs mAU vs Volume ml along with Fractionation marks Note BMP file is generated only for the last 4 hours of run Evaluating a run using UNICORN start Evaluation module of UNICORN start is used to manage and evaluate the results from chromatography runs For instructions to evaluate results that are generated using AKTA start refer to the UNICORN start 1 0 User Manual Save Result to USB memory stick Connect USB stick to KTA start Eg Manual run Method run mo 6 4 Select chromatography method for method run In the Run parameters or Manual run screens tick the checkbox Save Result to USB Import and view Result file on UNICORN start or BMP image on any other OS
4. 1 or using the Help menu e The online help covers the system control method editor and result evaluation functions in UNICORN start The user documentation available with AKTA start is listed in section Reference information Reference information Ordering information For ordering information visit www gelifesciences com AKTA Selecting protocols columns and buffer Method Column supported Volume ml AC 120 HiPrep 16 60 Sephacryl S 500 HR 120 A HiTrap Desalting H 5 3 5 HiPrep 26 10 Desalting 5 il IRONS 120 Zo 120 11 Buffer selection Buffer types needed For AC e Binding buffer e Elution buffer e Wash buffer optional For DS e One buffer type per run For IEX e Binding buffer e Elution buffer e Wash buffer optional FON GE e One buffer type per run AC buffer suggestions for His tagged proteins suggested buffer 50 mM Tris HCl pH 7 5 0 5 M Nacl 20 40 mM imidazole If performing binding using HisTrap or HiTrap Chelating wash 50 mM Tris HCl pH 7 5 0 5 M NaCl 100 mM imidazole 50 mM Tris HCl pH 7 5 0 5 M NaCl 500 mM imidazole 1 The imidazole concentration is protein dependent step elution AC buffer suggestions for GST tagged proteins If performing suggested buffer binding using 50 mM Tris HCl pH 7 5 150 mM GSTrap 4B HP or FF Nacl 1 mM DTT step elution 50 mM Tris HCl 10 mM reduced gluthathione pH 8 DS buffer suggestions If p
5. AKTA start System Cue Card Contents System overview 2 System preparation 4 Column preparation 6 Startingarun 6 Monitoringarun 7 Quick start methods 8 Flow charts 10 Getting help 11 Reference information 11 System Overview AKTA start is a simple and modern liquid chromatography system intended for preparative purification of proteins at laboratory scale The system can be used for a variety of research purposes to fulfill the needs of the users in the academia and in the life sciences industry AKTA start is intended for research use only and should not be used in any clinical procedures or for diagnostic purposes The System Cue Card describes how to prepare the system for a chromatography run start and monitor a run and evaluate the result System overview Part Description KTA stort Frac30 Fraction collector PC with UNICORN start software Main features of AKTA start e KTA start is operated in two modes 1 using Instrument Display touch screen and 2 using UNICORN start e KTA start is offered with a dedicated Fraction collector Frac30 e Quick start methods available for easy purification of common tagged untagged proteins e Method templates available for Affinity Chromatography AC lon Exchange Chromatography IEX Gel Filtration GF and Desalting DS e Predefined methods available for cleaning the system flow path flow cells UV and Conductivity and testing the s
6. Method Seting wO amma GEPSCTESTPC r Prane and E qitu oton Bdeastione DELS prem Marsi X ID Predetned Methods imn Eaiecton O Aray Oromatogacty Sangto Apg esten Shem by lechay boy Dine a S n type ead TA v Select the cohn vina wo R100 395593 0 Guha ond Crectecaton Select the perene im gre cokamn 0 W po o X Prime and Eoetibration z E AKTA start System Cue Card 29 0240 42 AC e Evaluating and analyzing the results from KTA start Tgp naus pR ToS E m G fe ide View Integrate Opentisns Took Help Hd ST A renege J tar Peni Tatie a womi Oratart Concerten Owon 1 Sangaften Mernse Oron Cameos Module Features Provides an intuitive and easy to use interface to control KTA start System Control Performs and monitors manual chromatography runs Performs and monitors automated predefined or user defined chromatography method runs Performs system performance method runs Method Editor Provides the flexibility to automate the chromatography runs Allows creation of methods from predefined chromatography templates like Affinity Gel Filtration lon Exchange and Desalting Gives flexibility to create a customized method by dragging and dropping the chromatography phases such as Prime and Equilibration Sample Application Elution and Fractionation etc Allows exporting methods to a USB flash drive for importing them in
7. eden www gelifesciences com AKTA GE and GE monogram are trademarks of General Electric Company KTA GSTrap HiPrep HiTrap HisTrap MabSelect MBPTrap StrepTrap Superloop SuRe and UNICORN are trademarks of General Electric Company or one of its subsidiaries TALON is a registered trademark of Clontech Laboratories Inc All other third party trademarks are property of their respective owners Any use of UNICORN is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request UNICORN start 2013 2014 General Electric Company 2013 2014 General Electric Company All rights reserved First published Sep 2013 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29 0240 42 AC 07 2014
8. erforming suggested buffer Buffer exchange 50 mM Tris HCl pH 8 0 desalting IEX buffer suggestions suggested buffer depends on the pl of the protein 50 mM Tris HCl pH 8 0 20 mM MES pH 6 0 50 mM Tris HCl pH 8 0 1 M Nacl 20 mM MES pH 6 0 1 M NaCl If performing binding to AIEX binding to CIEX elution from AIEX elution from CIEX GF buffer suggestions If performing suggested buffer Buffer exchange a suitable buffer e g 50 mM size exclusion Tris HCl pH 7 5 150 mM NaCl chromatography Health and Safety Declaration Form Please complete the checklist in the On Site Service Health amp Safety Declaration Form or the Health amp Safety Declaration Form for Product Return or Servicing depending on whether the instrument is going to be serviced on site or returned for service respectively Copy the form you need from AKTA start Operating Instructions Section 10 4 Health and Safety Declaration Form or print it from the PDF file available on the User Documentation CD User documentation for AKTA start Documentation Book PDF Web Online Operating Instructions e z Maintenance Manual System Cue Card Maintenance Cue Card e m UNICORN start 1 0 User e z Manual UNICORN start Online Help DisplayHelp o o o e For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 SE 751 84 Uppsala Sw
9. it UNICORN start System control Method run i Pre defined Quick start AC IEX DS GF KTA start System Cue Card 29 0240 42 AC The pre set run parameters for each technique are listed in the table below Parameter AC step DS IEX step IEX step IEX IEX 5ml i 53 ml 1ml 5 ml gradient gradient HiPrep i i 1ml 5ml ml Flow rate ml min Pressure Limit MPa Save Result to USB ml Volume CV Volume CV Elution Volume TP CV Hold at 100 B CV Volume CV Volume ml the results will be saved only if the USB memory stick is inserted before opening the method variable parameter Note The sample can be loaded only through Pump B Quick start methods description 100 The ready to run methods available with KTA start are presented below The length of each step or phase is indicated on the diagrams as column volumes CV so S 2 2 2 Sample variable volume to be set by the user S Bg T E z Pump wash a E Fractionation P for AC IEX 5 ml Buffer B then 5 ml of Buffer A wash a G 3 eea ce for DS GF 5ml of Buffer A wash l l 5 CV 15CV 5 CV 5CV AC Step 1 ml 5 ml HiTrap Affinity Chromatography e Commonly used for purification of tagged proteins e g DS 5 ml HiTrap 53 ml HiPrep Desalting Buffer Exchange His tag GST tag Mab tagged proteins etc Chromatography e The bound proteins are eluted in a single
10. itch the Injection valve to Load position when prompted by the message below Tap Continue Turn the Manual Injection Valve to Inject position to inject the sample and then tap Continue Sample inject Turn the Manual Injection Valve to Load position and then tap Continue 3 Note The system is in hold state while injecting the sample from loop To ensure that the injection mark coincides with the injection event acknowledge the message immediately after the action is performed Note For AC IEX methods when loading the sample through a loop it is advisable to empty the loop with 3x the loop volume to achieve good sample recovery Cold room operations Preparation 1 Place the AKTA start instrument in the cold room 2 Allow the instrument to stabilize at the temperature of the cold room 3 Tighten all connections and pump DM water through the system to check for leaks 4 Tighten any leaking connector Removal from cold room Switch off the instrument and disconnect power cable Loosen all connections to prevent them sticking when the system returns to room temperature Allow the instrument to stabilize at room temperature Tighten all connections and pump DM water through system to check for leaks Column preparation The Column preparation method is used for e Preparing a new column e Equilibrating the column Note Equilibrate columns before starting a new run Instruction 1 Im
11. merse the buffer inlet tubing into the buffer used in the chromatographic run 2 Connect the column in the flow path Operation from the Instrument Display Method run m g Ww Y Prepare system Column preparation Set Run parameters to suit your requirement Exit Operation from UNICORN start System control Pre defined System preparation SY f Column preparation Starting a run Chromatography runs can be performed either by running predefined methods or by using manual operations Perform a method run The following method types are available with AKTA start to perform chromatography runs e Quick start e Templates e User defined Follow the workflows below to run predefined methods Operation from the Instrument Display AkTAstart Cty A A Select method e Quick start e Templates e User defined Set Run parameters to Suit your requirement d dn p lt a q ki m g Operation from UNICORN start End System control d p Method run Pre defined Select method e Quick start e Templates e User defined methods Ok Start KTA start System Cue Card 29 0240 42 AC Perform a manual run Follow the workflows below to run the system manually Operation from the Instrument Display AKTA start Manual run E Set Run parameters to suit your requirement Operation from UNICORN
12. nnection at the back of AKTA start and a USB port on the computer Launch UNICORN start and connect to KTA start Connect Frac30 to AKTA start Note Frac30 should not be connected or disconnected when KTA start is switched ON Instruction 1 Connect the Frac30 Cable between the intended ports at the back of Frac30 and AKTA start 2 Switch on AKTA start 3 Enable connection of Frac30 from Instrument Display Fractionation For many purification schemes it is important to collect fractions of the eluent AKTA start offers the fractionation options presented in the table below Fractionation options Instrument configuration KTA start UNICORN start Frac30 e Fixed volume fractionation e Peak fractionation Level based Slope based UNICORN start Level based Prepare the Fraction collector Instruction Place a sufficient number of collection tubes 1 5 ml 5 ml 15 ml in the Bowl assembly Fit the outlet tubing into the tubing holder 1 Fit the tubing holder into the corresponding port on the Dispenser arm 2 Gently move Dispenser arm to dispensing position 3 Enable Frac30 from Instrument Display Home gt Settings and service gt Fraction collector gt Enable Frac Set the delay volume Home gt Settings and service gt _ System gt Delay volume setting Calibration Calibrate the modules in the order described below to ensure optimal performance of the s
13. rating Instructions Instruction 1 Depending on column dimension choose the appropriate location to place the column The column holder rails are indicated in the image below Attach an appropriate column holder to the column holder rail on the instrument Mount the column on the union connector if the column type requires a union Fix the column to the column holder then connect the tubing to the top and to the bottom of the column Sample application The sample can be applied to the column in two ways e via Pump The sample can be applied directly from a sample container through the Sample valve as shown in the image below It allows application of large volume of sample gt 5 ml and facilitates unattended operation A e via Loop A sample loop allows the injection of small sample volumes 25 ul 5 ml onto the column For AKTA start System Cue Card 29 0240 42 AC larger sample volumes 10 150 ml use a Superloop The loop has to be connected to the Injection valve ports 2 and 5 as depicted below INJECT LOAD LOAD INJECT eee To apply the sample from a loop e Fill the loop with sample through port 3 of the Injection valve in Load position e Switch the Injection valve to Inject position when prompted by the message below Tap Continue The sample is loaded onto the column only when the Injection valve is in Inject position during the run Sample inject N e Sw
14. start UNICORN start System control r Manual run Manual through Instructions process picture Export a method from UNICORN start Follow the workflow below to export a method from UNICORN start to a USB memory stick UNICORN start E Method editor Select method to export Select several methods using Shift or Ctrl keys File Export Export method to USB stick Select USB stick location x y Save For detailed instructions on exporting methods refer to the UNICORN start 1 0 User Manual AKTA start System Cue Card 29 0240 42 AC Import a method to AKTA start Transfer the UNICORN method to USB memory stick then perform the following operations to import a method The imported methods are saved in User defined methods menu on the AKTA start Connect USB stick to KTA start L Create method i USB import Select method to import Import mail Monitoring a run The user can monitor control the ongoing run either from the KTA start Instrument Display or from UNICORN start Operation from the Instrument Display The following options are available from Run view screen e View real time values UV absorbance Conductivity Flow rate Pressure Tube number e Graph view the chromatogram e Edit run edit the run parameters of ongoing run e Hold temporarily holds the run with set flow rate valve positions
15. step using e The proteins are eluted in a single step using single single elution buffer elution buffer e Make sure to load the recommended sample volume AKTA start System Cue Card 29 0240 42 AC 9 B 100 50 gt Pump wash Equilibration Sample Elution Fractionation collection 3 CV 1 2CV IEX Step 1 ml 5 ml HiTrap lon Exchange Chromatography The bound proteins are eluted in a single step using single elution buffer B 100 50 Fractionation collection Pump wash Equilibration Sample Wash unbound Re equilibration 5 CV 5 CV 5 CV 5 CV IEX Gradient 1 ml 5 ml HiTrap lon Exchange Chromatography The bound proteins are eluted using two buffers with linear increase in the concentration of buffer B over a specified time followed by a step with 100 B concentration B 100 F Pump wash Equilibration Sample Wash unbound Re equilibration Fractionation collection 5 CV 5 CV 20 CV 5 CV 5 CV GF 16 60 HiPrep Gel Filtration Chromatography e The proteins are eluted in a single step using single elution buffer e The column needs to be pre equilibrated before the start of the run B 100 50 Pump wash Equilibration oO O E 5 O n Ww Fractionation collection 0 2 CV 1 2 CV 10 Flow charts The active flow path is depicted in Green color A Flow path details for flow of 100 buffer A through Outlet valve to waste
16. to AKTA start Allows viewing and presentation of results including creation of PDF reports Evaluation Allows various evaluation operations on curves and chromatograms including comparison peak integration etc Allows importing results from AKTA start and exporting results to other formats Allows administration of the UNICORN start database for backup restore archive and retrieve operations Adminis tration Allows reviewing of UNICORN start and system logs Safety Read AKTA start Operating Instructions before using the instrument System preparation Preparation workflow Instruction 1 Place the buffer bottles A amp B in the buffer tray and immerse the buffer inlet tubing A amp B in the respective buffer bottles 1 Place the sample inlet tubing in the sample container or prefill the sample loop with the sample 2 Place all the waste tubing in the waste bottle 3 Make sure the pump tubing is installed in the Pump Fit the collection tubing to the Fraction collector Switch on the instrument Calibrate the instrument modules ca Connect a PC to AKTA start Note Before connecting the computer to AKTA start install the UNICORN start software on the computer For instructions refer to the UNICORN start 1 0 User Manual Instruction 1 Connect power to the computer and monitor and start the computer Connect the PC Connection Cable between the connector marked as PC Co
17. ystem For instructions refer to AKTA start Operating Instructions Instruction Wash the entire flow path of the instrument with demineralized DM water then calibrate the modules A calibration guide for when to calibrate the modules is available in AKTA start Maintenance Cue Card Display Touch Screen calibration Pressure Sensor Zero Offset Pump Note At delivery the Flow rate calibration instruments are pre calibrated Based on the results of the system performance test the user can decide if re calibrations are required Conductivity Temp sensor calibration Cond flow cell calibration AKTA start System Cue Card 29 0240 42 AC Prefill the flow path Cleaning prefilling the flow path including all inlets and outlets is performed using the system methods listed below For instructions on how to prime the sample inlet tubing and sample loop refer to AKTA start Operating Instructions section 5 7 Sample application For instructions on how to perform system methods refer to AKTA start Maintenance Cue Card e Pump wash A Pump wash B prime buffer A amp B inlets e Washout fractionation tubing clean fractionation tubing e Column preparation prepare equilibrate a column e System cleaning clean the flow path using 1 M NaOH and DM water The flow path can also be prefilled by using a manual run Connect a column For detailed instructions on how to connect a column refer to AKTA start Ope
18. ystem performance Instrument Display home screen AKTA start Create method Settings and service The AKTA start home screen displays four options for the user to select and perform operations from Method run run predefined methods Manual run start and control the system in real time Create method create edit import and delete methods Settings and service configure calibrate troubleshoot and diagnose modules Instrument illustration The illustration below shows the locations of the modules placed on the wet side of the instrument The modules have the following functions e To deliver the liquid in the system flow path and divert the flow as required e To monitor the UV absorbance and conductivity of the liquid 8 9 10 Part Description Part Buffer valve Mixer Description Wash valve Injection valve 4 Pump Conductivity Monitor KTA start System Cue Card 29 0240 42 AC Sample valve E UV Monitor Frac30 Fraction collector Part Description Part Description UNICORN start overview UNICORN start is a software package for e Controlling the KTA start instrument arm ate me 1 Mube number Stort Froctisaation ml Manual Run Boc Nowstch Connection Connected in control Controlled by DefsukGHCE ZICKSRI e Creating and editing methods L Method idto Aifinity Chromatography fesd Onn oe cars a Ded r Phase Library ax s
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