Gel filtration using ÄKTA™ start
Contents
1. Fig 3 UNICORN start process picture illustrating priming of the flow path f End the manual run Prepare Frac30 fraction collector a Fillthe outer row of holders with 15 ml tubes b Move the dispenser arm to the dispensing position c Insert the fractionation tubing into the tubing holder 29 1120 91 AA Connecting the column Connect the HiPrep 16 60 Sephacryl S 200 HR column to the system Fig 4 To avoid introducing air into the column connect the column drop to drop 8 Attach two column holders to the column holder rail on the right hand side of the instrument Fig 4 Image showing the gel filtration column being attached to the AKTA start instrument 9 Mount the column vertically on the column holders 10 Remove the G5 and G6 tubing Connect a 90 cm long PEEK tubing internal diameter 0 75 mm to the Manual injection valve port 1 Connect a 50 cm long PEEK tubing internal diameter 0 75 mm to the UV monitor 11 Start a manual run with 0 5 ml min flow rate Wait for the buffer to flow continuously from the 90 cm long tubing and then start filling the top part of the column with the buffer When the top part of the column is filled with buffer connect the tubing from the Manual injection valve to the top part of the column 12 Remove the column transport device and connect the 50 cm long tubing from the UV to the column outlet 13 End the manual run Note Save the transport device for2. G9 Crema eee irea Pi ren raro rosee DORIA MOFA A Nanny Panne er rip ra a Ll pn el pus ne Che hace AMO E EP ICATION Immer Cowen a Cowerted ia carr Comelec yy Dein Fig 7 UNICORN start Process picture image showing Sample inject message screen 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 29 1120 91 AA 3 28 After manually switching the position acknowledge the message by clicking Confirm and continue The sample is automatically injected from the loop on to the column 29 After the required volume of sample is injected a prompt appears on the screen depicted in Fig 8 AA as pe yee aveu poe Joh tip Y ras x uw m a Po cr P wen JO messen rem _ a a Dra Shed eck AMEE MPUCATION Ma weer Caron a Cowerted in cortal Comel ty Denk Fig 8 UNICORN start Process picture showing sample inject message prompt following sample injection 30 Manually turn the injection valve to LOAD position 31 After manually switching the injection valve position acknowledge the message by clicking Confirm and continue 32 Upon completion of the method the run ends automatically Typical result 33 In the Evaluation module of UNICORN start double click on the GF 01 result to open the file A representative ch
3. GE Healthcare Life Sciences Gel filtration using AKTA start Training cue card This protocol will help you understand the practical principles of gel filtration by taking you step by step through the purification of a Bovine Serum Albumin BSA and cytochrome C cyt C mixture Requirements e KTA start system e Frac30 fraction collector e Computer installed with UNICORN start 1 0 control software e Gel filtration buffer GF Buffer 25 mM sodium phosphate pH 7 5 150 mM NaCl Prepare at least 500 ml of buffer e Sample 5 mg ml BSA and 2 5 mg ml cytochrome C in GF Buffer Prepare 5 ml of sample e Column HiPrep 16 60 Sephacryl S 200 HR 120 ml e 2mlSample loop e Fraction tubes 15 ml tubes Checklist e Ensure that the PC Connection Cable is connected between the connector marked as PC Connection at the back of AKTA start and a USB port on the computer e Ensure the Frac30 fraction collector is connected to the AKTA start instrument e Ensure the pump tube is properly inserted in the pump head and the pump cover is closed properly e Ensure there is no column connected in the flow path while preparing the system for a run e Ifthe system or column is stored in ethanol wash with water prior to starting the run Fig 1 AKTA start instrument with Frac30 fraction collector and UNICORN start software Preparing the system 1 Place the bottle containing the GF Buffer in the buffer tray on to
4. olume 1 2 CVt Sample Apply sample using loop application Sample Volume 2 0 ml Elution and Isocratic elution 0 B 1 0 CV fractionation Fixed volume fractionation Fractionation Volume 4 ml 0 15 MPa 15 bar 22 psi t CV Column volumes Starting the run 21 Click the Method run from the UNICORN Start System Control toolbar 22 This opens the Select method dialog box Fig 6 23 For User defined find and select the GF 01 method in the folder pane 24 Click the OK button to start the selected method run This opens the Start protocol dialog 25 Review the Variable List and change method parameters if required and then click Next to proceed to the next page 26 Specify a result name e g GF 01 and then click Start to start the run Selection Criteria 9 User defined Pre defined Folder name System Last modified Created b S HCEJWNOKZ1 E DefaultHome 2014 04 23 17 14 System B 0000000000001 722536 2014 04 29 17 31 Default a AIEX BSA 0000000000000 2014 04 29 17 52 Default al GF 01 0000000000000 2014 05 14 16 50 Default Fig 6 UNICORN start Select method dialog box During the run 27 When prompted on the screen as depicted below in Fig 7 manually turn the injection valve to INJECT position _ _ _ ___ _ _ _ _ _ _ _ __ _ _ De Yen yv Jere Tom tip iras an un o gt a lt ann By ewes m
5. orage see HiPrep 16 60 Sephacryl S 200 HR instructions Check your knowledge Q1 How can you load your sample on the column using KTA start a Viathe pump b Via a sample loop c All of the above Q2 What is the maximum sample volume that can be applied on a gel filtration column and still achieve maximum resolution a 5 of total column volume b 2 of total column volume c 0 1 of total column volume Q3 Why should the Sample inject message be acknowledged as soon as the Manual injection valve has been manually turned a To ensure that the injection mark coincides with the injection event b To ensure that the injection of sample is performed c All of the above Q4 Which predefined methods are there in the UNICORN start Method Editor Anion Exchange Desalting Gel Filtration Affinity Cation Exchange All of the above 7 Oo A290 70 Q5 Which gradient elution types can be set in the Elution and Fractionation phase Linear Linear and Step Step Isocratic All of the above panca 06 Where can the result file be viewed after the run is completed a Inthe Evaluation module b Onthe display c There is no result file 29 1120 91 AA 5 Answers 1 C 2 B 3 A 4 F 5 E 6 A 6 29 1120 91 AA Ordering information Product Quantity Code number HiPrep 16 60 Sephacryl S 200 HR 1x5 ml 17 1166 01 Sample Loop 1x2 ml 18 1114 02 Column Holder 1 28 9562 82 PEEK t
6. p of the instrument 2 Immerse buffer inlet A in the bottle containing the GF buffer 3 Place the waste bottle on the right side of the instrument Note The waste tubing from Wash valve Manual injection valve and Outlet valve should be inserted into the waste bottle as shown in Figure 1 4 Power ON the AKTA start instrument Note Enable Frac30 from the Fraction collector screen in the Settings and service screen menu if not previously enabled 5 Start the computer and launch UNICORN start and connect to AKTA start 29 1120 91 AA 1 6 7 2 Prime the entire flow path buffer tubing to outlet fractionation tubing with GF Buffer to ensure the tubing is filled with GF buffer before starting the chromatography run a Place the fractionation tubing in a waste container b From UNICORN start System control module click Manual run P Manual Run c The Manual run dialog box will open d Set the flow rate to 5 ml min and click Ok Fig 2 Flow Rate 5 ml min 0 5 5 E Zero flow rate Save result as LA Fig 2 UNICORN start Manual run settings dialog with flow rate set to 5 ml min e Fromthe process picture set the Wash valve to position Column and Outlet valve to position Fraction collector Fig 3 Prime the entire flow path for 2 minutes see green highlighted part in the process picture Note Ensure that there is no column in line before switching the wash valve
7. romatogram for the chromatography run is shown in Figure 9 mAU 4444444444 4444 14441 20 10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 ml Fig 9 Representative chromatogram from UNICORN start Evaluation module Equilibration phase is not depicted in this chromatogram 4 29 1120 91 AA 34 From the Evaluation module a pdf report of the chromatogram can be generated and peak integration curve and chromatogram comparisons can be performed For details refer to the UNICORN start user manual Troubleshooting High back pressure e Column clogged Clean the column according to instructions Make sure the sample has been centrifuged and or filtered through a 0 45 um filter e System clogged Replace the column with a piece of tubing Check pressure using water at a flow rate of 5 ml min If backpressure is more than 0 3 MPa 3 bar 43 5 psi clean system according to instructions in the manual No separation e Check that the correct column is used e Check that the inlet tubing from each buffer is connected to the correct inlet port e Run the purification at lower flow rates e Check the volume of sample that was loaded Too much sample can lead to decreased resolution If that is the case use a smaller sample volume System maintenance and storage For detailed description of maintenance and storage see AKTA start operating instructions Storage of column For detailed description of column st
8. subject to the terms and conditions of sale ofthe company within GE Healthcare which supplies them A copy ofthese terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29 1120 91 AA 07 2014
9. ubing id 0 75 mm 1 16 1 18 1112 53 Reference information Document KTA start System cue card KTA start Maintenance cue card KTA start Operating instructions UNICORN start 1 0 User manual HiPrep 16 60 amp 26 60 Sephacryl High Resolution instructions Related literature Product Application notes Purification of N terminal histidine tagged protein using KTA start Purification of GST tagged protein using KTA start Purification of antibodies using KTA start and HiTrap Protein G HP column Depletion of albumin from serum samples using KTA start Training cue cards Desalting using KTA start Affinity purification using KTA start Anion exchange purification using KTA start Code number 29 0240 42 29 0240 43 29 0270 57 29 0602 44 28 4026 53 Code number 29 0642 77 29 0642 98 29 0643 02 29 0642 95 29 1094 91 29 1150 58 29 1107 59 29 1120 91 AA 7 For local office contact information visit www gelifesciences com contact www gelifesciences com akta GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden imagination at work GE GE monogram and imagination at work are trademarks of General Electric Company KTA HiPrep HiTrap Sephacryl and UNICORN are trademarks of General Electric Company or one of its subsidiaries 2014 General Electric Company All rights reserved First published Jul 2014 All goods and services are sold
10. use when storing the column Loading sample 14 Ensure that the 2 ml sample loop is connected to the Manual injection valve ports 2 and 5 15 Ensure the Manual injection valve is in LOAD position as illustrated in Figure 5 Wash the sample loop with 10 ml GF Buffer with a syringe through port 3 of the Manual injection valve LOAD INJECT N Fig 5 Image showing Manual injection valve in LOAD position and sample loop attached to ports 2 and 5 16 Pre fill the loop with 2 5 ml sample Note e in order to avoid sample drainage the syringe should not be removed after filling the sample loop e It is recommended to overload the loop to make sure that the loop is completely filled e For high resolution separation in gel filtration a sample volume from 0 5 to 4 of the total column volume is recommended depending on the type of medium used For most applications the sample volume should not exceed 2 to achieve maximum resolution Creating the method 17 Select New method in the UNICORN start Method Editor module 18 Select the Predefined method for Gel Filtration 19 Set the parameters for the method as shown in Table 1 20 Save method as GF 01 Table 1 UNICORN start method overview Method flow Method settings Method settings Column type HiPrep 16 60 Sephacryl S 200 HR Pressure limit 0 15 MPa Flow rate 0 5 ml min Column Volume 120 ml Prime and Prime equilibration Equilibration V
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