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MEA User Manual

1. Stop 00 00 Capacity mL 0 2000 Intake lt Expel gt Controller version CAPTURE 1 3 0 1108 41302530U PURIFY Port ENRICH Protocol Then press OK which will pop up the editor dialog Edit method Volume after selected Capacity mL step Line number Intak Version 1 7 9 Mar 7 2013 57 Copyright 2013 PhyNexus Inc All Rights Reserved Press the Add button to insert new method steps New steps may be edited after they have been added see below Steps may be re ordered either individually or in groups by selecting the steps to be re ordered and using the Move Up and Move Down buttons A single step or a group of steps may be cut copied or re inserted using the Cut Copy and Paste buttons these work as most Windows users would expect 6 2 How to edit an existing method Select Method gt Edit to pop up the editor dialog An example is shown below Edit method Volume after selected Capacity mL step Line number BSE 1 Parameters if an Home fAuto Controlle aaa Blowout Home Controller Comment Comment Equilibrate PhyTip Columns gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt Position row 1 1 Protocol Air Gap 10uL psc Loadtips Position row 7 1 Down 4230 Protocol Equil 200uL psc Comment Comment Lapture 555222222
2. 1 MEA Purification System Manual PhyNezus Inc 3670 Charter Park Drive Suite A San Jose CA 95136 www phynexus com Version 1 7 9 For Software Revision 3 0 1 March 7 2013 Operating Instructions for PhyTip MEA Personal Purification System Introduction In order to extend the benefits of PhyTip columns for protein purification and enrichment PhyNexus has developed the PhyTip MEA Personal Purification System as the platform of choice for fully automated protein purification and enrichment When used with the PhyNexus Operating Software this system offers validated procedures that maximize the purification and enrichment efficiency of the various chromatography resins available for PhyTip columns In combination with the fully programmable positioning of the 12 channel system and the ability to place samples PhyTip columns wash stations and elution plates in any available position the MEA Personal Purification System is an ideal automated system for small scale protein purification Version 1 7 9 Mar 7 2013 2 Copyright O 2013 PhyNexus Inc All Rights Reserved These operating instructions are divided into the following sections SECTION A 1 UNPACHKING 15 6 Sete ISSUES eerie nba ES 2 SETTING UP THE SYSTEM tene ne IU ROT t 3 USING THE SYSTEM entrent i e ert ere ere i era eee eite es SECTION B F INTRODUCTION rete EROR Uer rie E S
3. EVEN IF SELLER HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES OR LOSSES PhyNexus Inc Tel 408 267 7214 Version 1 7 9 Mar 7 2013 74 Copyright 2013 PhyNezus Inc All Rights Reserved
4. 1 1 CONVENTIONS 1 2 DEFINITIONS 1 3 COMPATIBILITY 2 1 OPEN PROTOCOL FILE 2 2 EKECUTE PROTOCOL 2 2 1 Check serial port 2 2 2 Check capacity setting 2 2 3 Start protocol execution 2 3 VERIFY PROTOCOL 3 1 OPEN METHOD 3 2 EKECUTE METHOD 4 MANUAL COMMANDS e eH eie e ETHER SU Wee 4 RESET VOLUME 4 2 STOPPING MANUAL COMMANDS 5 1 HOw CREATE NEW PROTOCOL 5 2 HOW TO EDIT AN EXISTING PROTOCOL 5 3 CREATE PROTOCOL STEP 5 4 EDIT PROTOCOL STEP 5 5 SAVE PROTOCOL TO FILE gt 6 1 HOw TO CREATE A NEW METHOD 6 2 HOW TO EDIT AN EXISTING METHOD 6 3 CREATE METHOD STEP 6 4 EDIT METHOD STEP 6 5 SAVE METHOD TO FILE APPENDIX A DEVELOPER COMMANDS eee nennen nennen rennen APPENDIX METHOD FILE FORMAT APPENDIX C FLOW RATE LIMITS aese esros APPENDIX D FLOW RATE IMPLEMENTATION eene eene eene APPENDIX E REGISTRY SETTINGS ette eter eee tere ecce CLEANING INSTRUCTIONS SAFETY NOTICES caue endi te e UR T D HO DRE DRESD RRRRERRR Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved HOW TO OPEN AND EXECUTE A PROTOCOL eene nennen eene HOW TO OPEN AND EXECUTE A METHOD 5 44 eene PROTOCOL EDITING visi ees METHOD EDITING ei eeu tibolone e e
5. 9 7 2013 21 Copyright 2013 PhyNezus Inc Rights Reserved Command Home Auto Controller Parameters if any Home Controller Execute Protocol IMAC Air Gap Position 1 1 Loadtips Position 7 1 Down 4225 Execute Protocol IMAC Capture Position 4 1 Down 4280 Execute Protocol IMAC Wash1 Position 5 1 Down 4280 Execute Protocol IMAC Wash2 Position 8 1 Down 4225 Execute Protocol IMAC Elution Position 1 1 Down 4100 Eject Home Auto Controller This method shows the automated movement of the 12 channel system for 1 row of samples In detail this can be broken down as follows 1 Homing the system Command Home Auto Controller Parameters if any Home Controller Execute Protocol IMAC Air Gap Position 1 1 Home Auto Controller moves the robotics tower to the Home position full left position Home Controller performs a zeroing of the 12 channel system followed by a resetting of the system to zero Version 1 7 9 Mar 7 2013 Copyright 2013 PhyNexus Inc All Rights Reserved 22 Execute IMAC Air Gap executes a stored protocol which in this case is setting the 12 channel system prior to attachment of PhyTip columns The 12 channel system withdraws air into the syringes to create an air buffer in the system that will later be used to push liquid out of the PhyTip columns
6. chiller positions 7 amp 8 it is possible to maintain both the pre purified material and the purified sample at sub ambient temperatures 2 Preparing wash buffer s for Purification process Refer to the Product Insert that is included with each Affinity resin for details on each buffer used in the purification process Some buffers are shipped as concentrates and will require dilution before use 3 Prepare Elution Buffer for Enrichment process Refer to the Product Insert that ships with each Affinity resin for details on the Elution Buffer used in the enrichment process If using Protein A or Protein G or ProPlus affinity resins it is recommended to add Neutralization Buffer at the end of the Enrichment step in order to return the sample to physiological pH A separate Neutralization step should be incorporated into the automated purification process of the MEA see standard methods For the 2004 system once the elution buffer has been added to the elution plate the user has the option of placing the PhyTip Cover over the plate see separate instructions Place the PhyTip Holder over the plate in order to retain the plate in position during the final elution process 4 Load the appropriate Method File To load the appropriate Method File go to File on the user screen Enter Open and the choice of Protocol or Method will be shown Enter Method and hit OK This will take you to folders containing the preprogrammed method files avai
7. for the first wash step Down 4280 moves the robotics tower down to predefined position of 4280 assumes use of PhyNexus recommended plates with known dimensions for the wash step Execute Pro A Wash 1 implements the stored protocol for the first wash step This protocol like Capture will control the flow rate volume pause and number of cycles involved in this step Position 5 Row 1 moves the robotic tower up to the fully extended zero position and then moves it to position 5 and row in readiness for the next wash step Down 4280 moves the robotics tower down to predefined position of 4280 assumes use of PhyNexus recommended plates with known dimensions for the next wash step Execute Pro A Wash 2 implements the stored protocol for the second wash step This protocol like Wash 1 will control the flow rate volume pause and number of cycles involved in this step Position 8 Row 1 moves the robotics tower to the full extend position Position 0 and then moves it over to position 8 row 1 ready for the elution step Down moves the robotics tower down to predefined position of 4225 assumes use of PhyNexus recommended plates with known dimensions for the elution step Execute Pro A Elution implements the stored protocol for the elution step This protocol like Capture will control the flow rate volume pause and number of cycles involved in this step Note at the end of this step the last expel step is greater volume than the
8. instrument deck The instrument deck can be configured in a number of different ways but the standard configuration is as follows On the left there are two positions for Tip boxes and to the right of these there is enough space to hold 5 plates standard SBS and one reagent reservoir Position 1 PhyTip Box Position 2 PhyTip Box Position 3 Plate position Plate 1 Position 4 Plate position Plate 2 Position 5 Plate position Plate 3 Position 6 Reservoir position Position 7 Plate position Plate 4 Position 8 Plate position Plate 5 If necessary the positions of these plates can be changed by moving the attached pins and replacing them in the appropriate positions This maintains perfect alignment of each plate in each position during MEA operation The chiller requires its own power supply When on the chiller will maintain the last two positions under the deck Positions 7 amp 8 at a temperature of approximately 4 6 C This temperature can be modified by turning the control knob on the right side of the instrument Position 1 represents the coldest setting and position 5 the warmest setting available as demonstrated below Position Approximate Temperature C 1 2 8 3 12 4 16 5 20 Version 1 7 9 Mar 7 2013 10 Copyright 2013 PhyNexus Inc All Rights Reserved For a standard purification set up Position 1 PhyTip Columns new unused For the MEA system these shoul
9. row of samples If using this program as a template just cut and paste the entire page and replace row 1 with row 2 etc Version 1 7 9 Mar 7 2013 29 Copyright 2013 PhyNezus Inc Rights Reserved Example 3 Optimization Method The MEA can be used 85 very rapid optimization platform to assess the optimum conditions for purifying protein samples Below is an experimental design format as an example with Ni IMAC resin Capture Prepare the standard 96 well sample plate and program the MEA as follows For the Capture protocol are programmed so that Rows A C E amp G have 2 Capture cycles and Rows B D F amp H have 4 Capture cycles Column z ommo o m gt l irvin po Jo 3 A r 5 3 TN 3 invio 3 5 Purify The wash process for purification can be optimized when using Ni IMAC resin by varying the concentration of the Imidazole in the wash buffer In this example rows A amp B have 0 mM I
10. specific set of protocols being used Position 6 Row 1 automatically moves the robotics tower to the full extent Position 0 and then moves it over to position 6 the Neutralization Buffer Reservoir Down 4070 moves the robotics head with Tips down into the neutralization buffer This is a predefined position of 4070 in readiness for removing the Neutralization buffer Execute ProA Intake 5uL executes the stored protocol that implements aspiration of 5 UL of neutralization buffer into the 12 LTS Tips Position 8 Row 1 automatically moves the robotics tower to the full extent Position 0 and then moves it over to position 8 row the row that contains the purified protein Down 4140 moves the robotics head with Tips with neutralization buffer down to the predefined position of 4140 in readiness for adding the Neutralization buffer Execute ProA Expel 25uL executes the stored protocol that implements the dispense of 25 UL of neutralization buffer into row 1 Position 2 Row 1 automatically moves the robotics tower to the full extent Position 0 and then moves it over to position 2 row 1 the original tray for the Rainin Tips Down 4100 moves the robotics head with Tips down to a predefined position of 4100 in readiness for removing the Tips from the 12 channel system Eject removes the tips into their original position Home Auto Controller takes the robotics tower back to its safe starting position and completes the purification of 1
11. the message by inserting the escape sequence For example the following line pops up a message box containing the lines Hello and World pause Hello nWorld 4 Comment ignored lt comment gt Comments are ignored by the app when the method is ezecuted but can be useful for documenting methods 5 Home home Version 1 7 9 Mar 7 2013 64 Copyright 2013 PhyNezus Inc All Rights Reserved 6 The following method steps are provided as instructions to the auto controller 1 Blowout blowout Home auto home Position tray to specified step Position lt step gt The step must be between 0 and 39100 Move down towards tray specified number of steps down lt number of steps gt The number of steps must be between than 0 and 4725 Load tips load Move up to retracted position up Eject tips eject Turn external pump on off external pump on off Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 65 Appendix C Flow rate limits Flow rates are limited to a range which is dependent on capacity as indicated by the following table Capacity Minimum flow rate 20 uL min Maximum flow rate 17 1 mL min 20 uL min 85 7 mL min 20 uL min Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 63 7 mL min 66 Appendix D Flow rate implementation The 12 channel controller does no
12. to build in an automatic comment or prompt between actions The following method steps are auto controller instructions Home Autocontroller returns the robotics tower and Controller to registered Home position Down moves the robotics tower with 12 channel system down to designated step position 0 is the highest position above the deck and 4725 is the position closest to the deck Up moves the robotics tower with Controller up to the designated step position 0 is the highest position above the deck and 4725 is the position closest to the deck External Pump operates an external pump when fitted via the rear serial port allowing for the automated addition of reagents or use of a vacuum Rotate rotates the vertical Controller Position moves the robotics tower with 12 channel system to the desired x axis location on the deck There are a total of 39 100 positions Version 1 7 9 7 2013 59 Copyright 2013 PhyNezus Inc Rights Reserved Position Row Load Tips Eject XMOV Load Version 1 7 9 Mar 7 2013 along the x axis moves the robotic tower with 12 channel system to the desired Position 1 8 and specific Row 1 8 This command assumes the use of plates that have 8 rows e g standard 96 well plates certain 48 well plates etc loads a row of 12 PhyTip columns onto the Controller This also incorporates the function of going down to the tips loading the tips and moving the
13. volume Manual methods programming The MEA software not only includes standard Methods and Protocols that can be simply modified but also a feature that allows the user to calculate and program specific positions on the deck The standard windows screen shows five basic headings File Protocol Method Options Help File is the standard New Open Select Default Method Save Save as found in most Windows applications Protocol contains Execute Edit Execute begins the loaded Protocol and Edit allows the user to Edit the loaded Protocol Method contains Execute Verify Edit Execute begins the loaded Method Verify checks that the Method can be operated and Edit allows the user to Edit the loaded Method Options contain Communications S yringe Calibration Manual Positioning Submethods Perform Dry Run Communications sets the COM port for the Version 1 7 9 7 2013 39 Copyright 2013 PhyNezus Inc Rights Reserved MEA and recognizes the serial number of the 12 channel system Syringe verifies the 12 channel system type 200 or 1000 Manual Positioning allows control of the MEA tower Submethods organize and store labware definitions Perform Dry Run is used to run the MEA movements but disables the 12 channel functions Help shows the About information regarding the system To access the ability to specify x z positioning of the 12 channel system press Shift Alt this will add two new functions to the O
14. 0 4000 8000 40 8000 12000 50 The move wait cycle delay is computed as follows delay msec 60000 x volume increment mL desired flow rate mL min The number of cycles is simply the desired volume divided by the volume increment rounded up so that at least one cycle is performed For example suppose a 100 uL intake step needs to be made at 300 uL min In this case 50 cycles will be performed each withdrawing 2 uL at motor speed 1 with a 400 msec wait until the next cycle Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 68 Appendix E Registry Settings The app uses the Windows registry for storing settings from session to session The registry key for the app is as follows and found under HREY CURRENT USER so that different users get different settings Software VB and VBA Program Settings PhyNexus PumpControl 2 Settings are stored under the following subkeys Settings Subkey name Serial communications Serial Syringe settings Syringe MRU list MRU Browse directory protocol and method General Version 1 7 9 7 2013 69 Copyright 2013 PhyNezus Inc Rights Reserved Cleaning Instructions To clean the exterior of this instrument complete the following steps e Turn off and unplug the instrument Wipe the instrument exterior surfaces only using a towel dampened with laboratory grade cleaning agent
15. 2160 2060 2135 2135 1000 10 2350 2350 2350 2325 2325 1000 20 2660 2660 2660 2575 2575 1000 40 2660 2660 2660 2575 2575 1000 80 2660 2660 2660 2575 2575 1000 160 2660 2660 2660 2575 2575 1000 320 2980 2980 2980 2970 2970 250 Micro Plate PCP 96 00 10 Column Resin Bed Volume Volume Position Position Position Position Position Position pL pL 3 4 5 6 7 8 250 transfer tip 4070 250W transfer tip 200 5 4280 4280 4280 4225 4225 200 20 4280 4280 4280 4225 4225 1000 10 2705 2705 2705 2650 2650 1000 20 2705 2705 2705 2650 2650 1000 40 2705 2705 2705 2650 2650 1000 80 2705 2705 2705 2650 2650 1000 160 2705 2705 2705 2650 2650 1000 320 Not recommended Not recommended The system defaults to automatically load tips with the Loadtips command The user should NOT program a down 4210 and follow this with a Loadtips command D Reagents 1 Preparing samples for Capture Purify Enrich It is recommended that prior to the process of purification and enrichment by PhyTip columns all samples be checked for particulates and clarity If the final solution to be Version 1 7 9 7 2013 19 Copyright 2013 PhyNezus Inc Rights Reserved purified appears cloudy or contains particulates it is recommended to spin the samples down one more time at 3 000 g for 5 minutes Samples can be placed in any position However the default methods all have samples placed in position 7 With the
16. 2222222000222222000002222 Cut Position row 7 2 Down 4230 Protocol Capture 200uL no blowout psc Comment Comment lt lt lt lt Wash PhyTip Columns gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt gt Position row 7 3 Down 4230 Protocol Wash 1 200uL psc Calculate Position row 7 4 Intake Down 4230 Protocol Wash 2 200uL psc Comment Comment Position row ne lt i m Edit Move Down T Copy Cancel Es See below for a description of creating and editing method steps Version 1 7 9 Mar 7 2013 58 Copyright O 2013 PhyNexus Inc All Rights Reserved 6 3 Create method step To create a new method step press the Add button Note If no method step was selected in the editor window then the new step is placed at the end of the method If any steps were selected then the new step is placed at the end of the selected steps The Move Up or Move Down button may be used to move the new step if desired Select the type of method step desired this will cause the entry field s at the bottom of the dialog to change appearance as needed Enter the parameter needed as follows Execute executes a stored protocol e g Capture Audible Alert introduces an audible alert at the end of an action Home controller homes the 12 channel system to a zero volume Pause for Operator builds in an automatic pause in the method Comment allows operator
17. 7 2013 72 Copyright 2013 PhyNexus Inc Rights Reserved AWARNING CONTACT WITH DANGEROUS VOLTAGES CAN CAUSE DEATH OR INJURY COVER TO BE REMOVED ONLY BY TRAINED SERVICE PERSONNEL A AVERTISSEMENT TOUT CONTACT AVEC LES HAUTES TENSIONS PEUT ENTRAINER LA MORT OU DES BLESSURES SEVERES CE A WARNING HIGH LEAKAGE CURRENT ENSURE PROPER GROUNDING A AVERTISSEMENT COURANT DE FUITE LEV FORNIR UNE MISE A LA TERRE EFFICACE WARNING If the MEA is used in a manner not specified by PhyNexus Inc the protection provided the equipment may be impaired MEA Power Supply Input MEA Power Supply Output Pipettor Power Supply Input AC Voltage and Current DC Voltage and Current AC Voltage and Current 100 240V 100 240V 47 63Hz 1 9A 47 63Hz 0 5A Output SVDC 3 0A Chiller Power Supply Output Chiller Power Supply Input DC Voltage and Current AC Voltage and Current 100 240V 47 63Hz 1 9A WARNING Pollution Degree 1 Version 1 7 9 Mar 7 2013 73 Copyright 2013 PhyNexus Inc All Rights Reserved MEA Personal Purification System Warranty The MEA Personal Purification System has a warranty period of one 1 year from the date of purchase If the system should fail during the warranty period please contact your local PhyNexus office for further instructions and information Limited Warranty and Limitation of Liability Seller PhyNexus warrants the merchandise to conform to specificatio
18. CAPTURE 1 3 0 1108 A1302530U PURIFY Auto Controller X Axis steps Z Axis kasen Rotts ENRICH Retum to L1 COMB 3 2 Execute method Before executing the method make sure of the following see section 2 2 above 1 The Controller is correctly mounted on the Auto Controller 2 The App s serial parameters are set to match those of auto controller 3 The App s capacity setting matches the Controller capacity Version 1 7 9 Mar 7 2013 49 Copyright 2013 PhyNexus Inc All Rights Reserved To execute the currently open method select the Method gt Execute menu option The app will then go online with the controller much as it would while executing a protocol When a Pause method command is executed the app displays the message specified in the command and stops to wait for operator intervention as shown below A PhyNexus Inc Capture Purify Enrich Protocol Method Options Help Method name Method Volume mL Program Files VPhyNesusVE 4 AutoController SWAT est Method pba Elapsedtime 000000 Ph A 0000 d yNexus p o Total time Stop 00 00 Capacity mL 0 2000 Controller version 1 3 0 1109 41302530U PURIFY Auto Controller ENRICH X Axis steps Z Axis steps CARTURE 1 66 Home Retumto L1 COMB MEA Software 1 4 Test Pause Command The operator can press
19. OK 0 proceed with method or Cancel to abort the method The appearance of the app while ezecuting a protocol within a method is very similar to that when simply executing a protocol standalone the only difference is that the method name is shown instead of the protocol name as shown here Version 1 7 9 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 50 Method name C Program Files PhyNexus E 4 AutoController Sw Test Method pba r Manual Override SU Pause Volume mL Rate mL min o Stop Method Volume mL Capacity mL Elapsed time 00 00 Total time 0000 0 2000 Intake Expel gt Controller version CARTURE 1 3 0 1109 A1302530U PURIFY r Auto Controller ENRICH K Ane steps ey ZAS ers Version 0 Retumto L1 6 w Method Progress _ 4 AutoControl In progress Expel step 2 of 2 Similarly Home command cause the status bar to be updated and Volume to be zeroed See section 2 2 3 above for information on stopping execution and possible errors that may occur since this information applies equally to methods as well as protocols Version 1 7 9 Mar 7 2013 51 Copyright O 2013 PhyNexus Inc All Rights Reserved 4 Manual Commands The controller be instructed to perform individual intake a
20. Repeat step b using a towel dampened with clean water Repeat step c with a second water wash step Dry the instrument exterior using a dry towel Warning do not allow any liquid to enter the instrument or come in contact with any electrical components The instrument must be thoroughly dry before you reconnect the power or turn the instrument on Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 70 Safety Notices Warning When cleaning instrument do not allow any liguid to enter the instrument come in contact with any electrical components The instrument must be thoroughly dry before you reconnect the power or turn the instrument on Caution Power Cord Set Requirements The power cord set supplied with your instrument meets the requirements of the country where you purchased the instrument If you use the instrument in another country you must use a power cord set that meets the requirements of that country To reduce the risk of fire hazard and electrical shock do not expose the unit to rain or humidity To reduce the risk of electrical shock do not open or remove the instrument casing All maintenance is to be performed by an authorized PhyNexus service provider Warning This equipment is designed for connection to a grounded earthed outlet The grounded type plug is an important safety feature To reduce the risk of electrical shock or damage to the instrument do not disable
21. The volume of air will vary depending on the specific set of protocols being used Position 1 Row 1 moves the robotics tower over position number 1 and row 1 6 tray of PhyTip columns 2 Moving the PhyTip Columns and Capturing the Sample Load tips Position 7 1 Down 4225 Execute Protocol IMAC Capture Load tips moves the 12 channel system down towards the tips in row 1 then positions the 12 channel system at an angle to load and then secure all 12 PhyTip columns Position 7 Row 1 automatically moves the robotics tower to the full extent Position 0 and then moves it over to position 7 row 1 the plate holding the sample to be purified Down moves the robotics head with PhyTip columns down into the sample solution in row 1 of position 7 This is a predefined position of 4225 assumes use of PhyNexus recommended plates with known dimensions in readiness for the Capture step Execute IMAC Capture executes the stored protocol that implements the Capture process with a given number of cycles flow rates volumes and pauses e g in the standard method this will be a volume of 170 uL for 2 cycles at a flow rate of 250 uL min with 10 second pauses at the end of each step Also note that at the end of the capture step the last cycle expels 172 UL to make sure all of the sample solution is removed 3 Wash and Elution Steps Position 4 1 Down 4280 Execute Protocol IMAC
22. Wash1 Position 5 1 Down 4280 Execute Protocol IMAC Wash2 Position 8 1 Down 4225 Execute Protocol IMAC Elution Position 1 1 Down 4100 Eject Home Auto Controller Position 4 Row 1 moves the robotics tower to the full extend position Position 0 and then moves it over to position 4 row 1 ready for the first wash step Version 1 7 9 Mar 7 2013 23 Copyright 2013 PhyNexus Inc All Rights Reserved Down 4280 moves the robotics tower down to predefined position of 4280 assumes use of PhyNexus recommended plates with known dimensions for the wash step Execute IMAC Wash 1 implements the stored protocol for the first wash step This protocol like Capture will control the flow rate volume pause and number of cycles involved in this step Position 5 Row 1 moves the robotic tower up to the fully extended zero position and then moves it to position 5 and row 1 in readiness for the next wash step Down 4280 moves the robotics tower down to predefined position of 4280 assumes use of PhyNexus recommended plates with known dimensions for the next wash step Execute IMAC Wash 2 implements the stored protocol for the second wash step This protocol like Wash 1 will control the flow rate volume pause and number of cycles involved in this step Position 8 Row 1 moves the robotics tower to the full extend position Position 0 and then moves it over to position 8 row 1 ready for the elution step Down moves the robotics t
23. ate Rate units Delay seconds O mmn 0 OK Cancel Note If no protocol step was selected in the editor dialog then the new step is placed at the end of the protocol If any protocol steps were selected then the new step is placed at the end of the selected steps The Move Up or Move Down button may be used to move the new step if desired Make sure to enter the correct values and units for the volume and rate select the direction intake expel the number of times to execute the step and the post step delay Delay times are rounded to the nearest millisecond If a step is to be repeated then the post step delay is also repeated when executed The Volume prior to this step indicator shows the calculated volume prior to the step being added This dialog will not allow the volume to exceed the capacity and also checks the rate against the capacity see Appendix C Version 1 7 9 Mar 7 2013 55 Copyright O 2013 PhyNexus Inc All Rights Reserved 5 4 Edit protocol step To edit an ezisting protocol step select the step in the editor window and either double click on the step or press the Edit button The protocol step dialog described above is then shown 5 5 Save protocol to file After a user presses OK in the editor window the protocol is assumed to have been changed and the app s caption indicates this via an asterisk next to the title as shown below PhyNexus Inc Capture Purify Enric
24. ber e g 2500 However if the 12 channel system is not at the zero position e g 2500 and you want to move the system down a further 100 stepper motor points type in Down 100 and this moves the system to position 2600 Up moves the 12 channel system back to zero position fully up X pos where any number between and 39 100 moves the robotics tower and 12 channel system to a specific point on the x axis along the deck It is recommended to place two Tips that will be used in the process on positions 1 and 12 of the 12 channel system in order to position the tip correctly Version 1 7 9 7 2013 40 Copyright 2013 PhyNezus Inc Rights Reserved Section B 1 Introduction PhyNexus auto controller software is a graphical user interface application which is intended to allow the user to perform the following tasks 1 Open verify and execute files which perform intake and expel operations Edit and save protocol files 3 Open verify and execute method files which execute protocol files and other commands such as instructions to the auto controller 4 Edit and save method files 5 Perform manual intake expel and blowout home operations The auto controller software is intended for use on Microsoft Windows platforms Windows XP 7 and 8 1 1 Conventions The auto controller software will afterwards be referred to as the app Menu and submenu options will be referred to using the gt nota
25. c Row 1 8 This command assumes the use of plates that have 8 rows e g standard 96 well plates certain 48 well plates etc loads a row of 12 PhyTip columns onto the 12 channel system This also incorporates the function of going down to the tips loading the tips and moving them out from the tip box This is set for the default PhyTip columns and standard Rainin LTS tips for neutralization ejects all 12 PhyTip columns from the 12 channel system Auto movement to specify position along x axis CAUTION This action does NOT move tower to zero position 15 Copyright O 2013 PhyNexus Inc All Rights Reserved 3 Easy to Use Ouick Guide Operating Instructions A Calibrating the Instrument Once installed in the correct position the instrument should be recalibrated in order to correctly zero the position of the robotic tower It is also recommended that for each pipettor 200 or 1200 calibration should be checked and stored To access the calibration software go to the general toolbar on the operating software and click on Options this will display Communications Syringe Calibration Manual Positioning and SubMethods Click on Syringe and select either 200 or 1200 for the 12 channel system that uses 200 tips choose 200 for 12 channel system that uses 1000 tips choose 1200 Next click on Calibration this will now display a screen with Check Alignment and three dialog boxes for x position rotation and z axis Auto C
26. city and verification If the capacity setting is changed while a protocol is open the user is prompted as follows MEA Software 1 Capacity has changed protocol verification could Fail when executed This message indicates that the protocol may not execute properly with the new capacity setting 2 2 2 2 Capacity and protocols If a protocol specifies a different capacity than the current app settings the user is prompted as follows when the protocol file is opened Version 1 7 9 Mar 7 2013 45 Copyright O 2013 PhyNexus Inc All Rights Reserved MEA Software Capacity used to create protocol differs From current capacity Protocol capacity 0 2 Current capacity 1 2 mL Change current capacity to match protocol now 7 Yes Mo Answer yes to set the app s capacity setting to match the protocol or no to do so later Note Answering no may prevent the protocol from executing properly 2 2 3 Start protocol execution Once the protocol has been opened and the controller has been prepared for protocol execution select the Protocol gt Execute option The app will establish communication with the controller go online get the controller s software version and capacity disable the controller s keyboard and speaker and get the current volume The protocol will then be automatically verified as noted in section 2 3 below using the controller s current volume Then the app will instruct th
27. d only be PhyTip columns with the appropriate part numbers for the 12 channel system that is being used e g 200 or 1000 and the affinity resin that is required e g PTR 92 05 01 Pack of 96 PhyTip columns for the 200 system with 5 UL Protein A as affinity resin Position 2 Empty box for used PhyTip columns or standard Rainin LTS pipette tips Used PhyTip columns can be replaced in their original box or if required in a separate empty box Position 3 Extra plate position or Equilibration Plate This extra position can be used for a number of different applications e g extra wash step equilibration etc Position 4 Wash Plate 1 Depending on the specific application a number of different types of wash plates may be placed in this position e g deep well 96 well plates Care must be taken when choosing the type of deep well 96 well plate When using the 1000 system PhyNexus recommendis the use of 2 mL square well plates with a pyramid bottom PhyNexus Part Number PCP 96 20 10 Position 5 Wash Plate 2 Depending on the specific application a number of different types of wash plates may be placed in this position e g deep well 96 well plates Care must be taken when choosing the type of deep well 96 well plate When using the 1000 system PhyNexus recommends the use of 2 mL square well plates with a pyramid bottom PhyNexus Part Number PCP 96 20 10 Position 6 Reservoir Position Depending on the application and spec
28. dicator will show how much volume is in the syringe at the end of the protocol The protocol is automatically terminated if an error occurs during execution and the error is shown in the status bar See Possible errors and corrective action below section 2 2 3 2 2 2 3 1 Stopping protocol ezecution If the protocol needs to be stopped before it s complete then press the Stop button Pressing stop terminates the app s execution of the protocol and will stop the protocol right away before the last command step is completed 2 2 3 2 Possible errors and corrective action If a Port in use error results when the protocol is executed then some other application is using the Com port selected See section 2 2 1 above Possibly a modem is plugged in and is being used or the HyperTerminal app which is found on most versions of Windows To fix this select a different port from the Options gt Communications dialog and try again or find the application which is using the port and shut it down Version 1 7 9 Mar 7 2013 47 Copyright O 2013 PhyNexus Inc All Rights Reserved The error No response from controller offline indicates that the port selected is not actually connected to the controller Select a different port and try again Controller error command not echoed bad checksum invalid parameter or command indicates a problem communicating with the controller over the USB cable or an incompatible version of controller f
29. ds The following standard methods have been built into the standard operating software For 2004 PhyTip columns and 200 4 12 channel system Position 7 is for the sample and position 8 is for elution 1 Protein with included neutralization step 2 Protein G with included neutralization step 3 IMAC 4 Glutathione For 1000 PhyTip columns and 1000 12 channel system Position 7 is for the sample and position 8 is for elution 1 Protein A with included neutralization step 2 Protein G with included neutralization step 3 IMAC 4 Glutathione Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 32 Full x axis and z axis positions For the 200 12 channel system PhyTip columns z axis Rainin LTS Tips z axis Load 4270 Eject 4100 Load 4270 Eject 4100 PhyTip columns z axis PhyTip columns z axis Sample Plate 4280 Wash Plate 4280 Position 6 x axis Neutralization Reservoir 27674 Rainin LTS Tips z axis Neutralization 4070 PhyTip columns z axis Wash Plate 4280 With Cooling System z axis With Cooling System z axis PhyTip Elution 4225 PhyTip Elution 4225 Rainin LTS 4140 Rainin LTS 4140 For the 1000 12 channel system Version 1 7 9 Mar 7 2013 Copyright 2013 PhyNexus Inc All Rights Reserved 33 Position 1 PhyTip columns PhyTip columns z axis Load 3600 Eject 3500 Position 3 Sample Plate PhyTip Deep Well PhyTip colu
30. e comm port can only be changed when the app is not communicating with the auto controller not executing a protocol method or manual override command Pressing Stop terminates communication and allows these settings to be changed see below 2 2 2 Check capacity setting The capacity setting may be adjusted via the Options gt Syringe menu which pops up the following dialog Version 1 7 9 7 2013 44 Copyright O 2013 PhyNexus Inc All Rights Reserved WA PhyNexus Inc Capture Purify Enrich Method ptions He Method name Method Volume mL Elapsed time 0 00000 NI Manual Overrides 00 00 HEN lig V Volume mL Rate mL min o Total tine d 1 Stop 00 00 Capacity D Intake lt Expel gt Controller version CAPTURE PURIFY Auto Controller X Axis steps Z Axis steps 0 Capacity mL Syringe Options Pot ENRICH COME IET The setting shown above is for the 1200 uL capacity The app is initially set up for 200 uL but remembers the last settings used See Registry Settings below Also note that the current capacity is displayed on the main window of the app The capacity setting can only be changed when the app is not communicating with the auto controller not executing a protocol method or manual override command Pressing Stop also stops communication so that these settings can be changed 2 2 2 1 Capa
31. e controller to execute the intake or expel steps contained in the protocol The app displays the following during protocol execution 1 Status bar displays the protocol step and step type intake expel 2 Progress bar to the right of the status bar text shows progress in total execution time as calculated by the volume and rate of each step 3 Volume displays the current volume in the syringe as reported by the controller after the most recently executed step 4 Controller version shows the firmware version of the controller 5 Version shows the firmware version of the auto controller 6 Port shows the Com port used by the Auto Controller An example is as follows Version 1 7 9 Mar 7 2013 46 Copyright O 2013 PhyNexus Inc All Rights Reserved Ii PhyNexus Inc Capture Purify Enrich Eile Options Help Protocol name Method Volume mL Program Files PhyNexus AutoController SW 200 AminoLink Wash 200uL ps Elapsed time 0 17400 Ph N 5 yNexus a o gt Total time ze Sk 00 00 Capacity x 02000 Controller version CAPTURE 1 3 0 1109 41302530U PURIFY Auto Controller ENRICH Version X Axis steps Z Axis steps Port 0 0 Retumto 1 1 Expel step 2012 When the protocol is completed the status bar will display Protocol execution complete and the Volume in
32. e that is attached to the 12 channel system and route it through the cable management channel on the robot track down to the computer USB port This is achieved by just pressing the cable into the track and snapping it in place Take the MEA power supply and attach to the supply port on the rear of the MEA labeled Power Connect the power supply to the rear of the instrument in the port labeled Chiller Plate Take the MEA USB communications cable and connect between the MEA USB port and another USB port on the computer Version 1 7 9 Mar 7 2013 6 Copyright 2013 PhyNexus Inc All Rights Reserved Front view of MEA system uid Rack Bars PhyNexus Inc Capture Purity Enneh Side view of MEA system Version 1 7 9 7 2013 7 Copyright 2013 PhyNexus Inc All Rights Reserved Rear view of MEA system showing power supply inlets and cable attachment points Power inlet and On Off Switch for cooling accessory a Power inlet and On Off x Switch for MEA system e Rui TO PIPETTOR 2 3 8 USB 9 SUNT pes COMI TO COMPUTER Version 1 7 9 7 2013 Copyright 2013 PhyNexus Inc All Rights Reserved 2 Install the software on the PC optional The MEA Personal Purification System comes eguipped with a computer The user may choose to install the software on additional computers The PhyNezus Operating Software is designed to operate with PCs r
33. ect the MEA USB communications cable between MEA USB port and the computer USB port Connect the power supply to the MEA and separate power to the chiller The PhyNexus software is preloaded on the supplied computer Locate the Software CD and install on any additional computer if desired Run software and attach MEA head to the MEA tower Detailed description Instrument placement Remove the MEA system from its packing box and place on the specified lab bench This must be close enough for three power supplies that are required to operate the MEA system Enough vertical clearance must be given for the MEA tower to move along the x axis maximum height above the bench 24 and there must be enough space on the right hand side of the instrument to reach press the red Emergency Stop button and the control switch for the chiller The surface for the MEA should be smooth flat and stable The full instrument dimensions are Width 32 Depth 14 plus 6 to accommodate all necessary power supplies and cables Height 24 Weight 55 1 5 Installing the instrument Each MEA is shipped with the ordered 12 channel MEA head s either ME200 for 200 PhyTip columns that operate with volumes up to 200 uL or ME1000 for 1000 PhyTip columns that operate with volumes up to 1000 uL with the alignment clamp Version 1 7 9 7 2013 5 Copyright 2013 PhyNezus Inc Rights Reserved attached Remove the MEA system from it
34. ed to comply so that protocol edits to not affect other methods Version 1 7 9 7 2013 61 Copyright 2013 PhyNexus Inc All Rights Reserved Edit method step r Type Protocol Pause for operator DK Audible alert Comment hidden Home Controller Blowout Home Auto Controller C Down n Edit method step Save as different File C Up Load Cancel External pump Botate Submethod Home Rotate Axis 9 9 9 9 9 r Protocol filename 000 40uL Pra amp 1000 Capture 1mL psc m 6 6 Save method to file After a user presses OK in the editor window the method is assumed to have been changed and app s caption indicates this via an asterisk next to the title as shown below PhyNexus Inc Capture Purify Enrich Elle Protocol Method Options Help Method name Method 1 Volume 4 C Program Files PhyNexus E4 AutoController S w 200 200 20uL Elapsed time 0 00000 7 Manual Overrides 00 00 1 yN Volume mL Rate mL min Total time 0 0 002 Capacity mL 1 20 Intake lt Expel gt Controller version CAPTURE 1 301 109 41302530U PURIFY m Auto Controller X Ais steps Z Aris steps keson orte ENRICH mj Retumto 1 1 The asterisk means that there changes to the method that not been saved S
35. eeded add 40 to the final volume number to obtain the programmed elution volume e g if 30 uL is the final elution volume required program 30 40 70 uL for the final elution volume This guideline is valid for elution volumes less than 75 ul For elution volumes greater or equal to 75 ul simply program that same volume Again note that the final step expels 56 UL to maximize the volume of Elution Buffer returned Note Initial Conditioning step has Intake volume of 10 uL Capture has excess expel of 2 uL Wash 2 has excess expel of 2 uL and the final Elution step has an excess expel of 6 uL thus balancing the Intake and Expel for the entire process 200 ProA Condition 2 Volume mL Rate mL min Delay sec aa MEE ae 0 02 Version 1 7 9 7 2013 37 Copyright 2013 PhyNezus Inc Rights Reserved Conditioning 2 this step conditions the 12 channel system prior to adding the Rainin LTS Tips for the Neutralization Step Air 20 uL is taken in at a flow rate of 1 mL min 200 ProA Intake 5 uL Rate mL min 0 2 Intake 5 uL This step pulls 5 uL of neutralization buffer into Rainin LTS Tips at a flow rate of 6 mL min 200 ProA Expel 5 uL Volume mL Rate mL min Delay sec 0 025 Expel 5 uL This step expels the 5 uL of Neutralization Buffer from the Rainin LTS Tips into the specified row at a flow rate of 2 mL min The step requires 25 uL of expel the first 20 uL is air and the remaining 5 i
36. elect File gt Save to save the changes back to the previous filename or File gt Save As to save changes to a new filename Version 1 7 9 7 2013 62 Copyright 2013 PhyNexus Inc All Rights Reserved Appendix A Protocol File Format Protocols are stored as simple text files and may be edited with Notepad if desired however this may result in a protocol which cannot be opened and or verified by the app The app registers the file extension psc for PhyNexus protocol files so that clicking on a psc file opens the protocol in the app if the app isn t already running Each line of the file represents one step of the protocol and is a comma delimited list of the following fields Volume in mL positive for intake negative for expel Rate in mL per minute Number of times to do the step optional Post step delay in seconds app rounds this to nearest millisecond optional Toup For example the following line indicates an expel step of 20 uL with a rate of 5 uL per minute done once 0 02 0 005 The following line indicates an intake step of 1 5 mL with a rate of 10 mL per minute done three times 1 5 10 3 This line indicates an intake step of 0 5 mL with a rate of 2 mL per minute done once with a post step delay of 2 seconds 0 5 2 1 2 The line below indicates an intake step of 1 5 mL with a rate of 10 mL per minute done three times each with a post step delay of 1 5 seconds if a s
37. em down towards the tips in row 1 then moves the 12 channel system at an angle to load and secure all 12 PhyTip columns Position 7 Row 1 automatically moves the robotics tower to the full extent Position 0 and then moves it over to position 7 row 1 the plate holding the sample to be purified Down moves the robotics head with PhyTip columns down into the sample solution in row 1 of position 7 This is a predefined position of 4225 assumes use of PhyNexus recommended plates with known dimensions in readiness for the Capture step Execute ProA Capture executes the stored protocol that implements the Capture process with a given number of cycles flow rates volumes and pauses e g in the standard method this will be a volume of 170 uL for 2 cycles at a flow rate of 250 uL min with 10 second pauses at the end of each step Also note that at the end of the capture step the last cycle expels 172 uL to make sure all of the sample solution is removed Version 1 7 9 Mar 7 2013 27 Copyright 2013 PhyNexus Inc All Rights Reserved 3 Wash and Elution Steps Position 4 1 Down 4280 Execute Protocol Pro A Wash1 Position 5 1 Down 4280 Execute Protocol Pro A Wash2 Position 8 1 Down 4225 Execute Protocol Pro A Elution Position 1 1 Down 4100 Eject Eject Position 4 Row 1 moves the robotics tower to the full extend position Position 0 and then moves it over to position 4 row 1 ready
38. eps Therefore four cycles of Capture and that at the end of the fourth cycle 172 uL is expelled from the PhyTip column This extra volume is part of the original 10 uL Condition step and helps to remove as much sample solution as possible prior to the Wash steps The Flow Rate is 250 uL min expressed as 0 25 mL min with a delay of 10 seconds at the end of each step This delay step of 10 seconds is built into the system to allow for the full equilibration of all PhyTip columns in all positions To modify any of the parameters double click on the parameter line and an Edit Protocol Step screen will display This has several boxes that can be modified see Section B Detailed software Information Volume sets the desired volume Volume Units references the volume with mL or uL available Direction Expel or Intake Volume Prior to this Step indicates last volume input How Many Times number of times to repeat sequentially this specific step Rate the flow rate Rate units shows mL hr mL min uL hr uL min and Delay in seconds 200 Pro A Wash 1 Volume mL Rate mL min Times to do Delay sec os ol 0 17 Wash 1 This is the first of two washing Purification steps here two cycles of moving 170 uL of Wash Buffer 1 is accomplished at a flow rate of 500 uL min expressed as 0 5 mL min with a delay of 10 seconds at the end of each step Note that on the final expel step the volume is the sa
39. eserved 13 14 15 16 Move tower down so that PhyTip columns enter Elution buffer Begin preprogrammed Enrichment step Upon completion of this step bring tower up and move back to row 1 of the original tip box in position 1 Move tower down and discard tips in row 1 Repeat the process for row 2 etc The robotics tower is stepper motor driven and as such can be controlled by discrete steps in both the X and Z axis In the Z axis there are 4 725 steps and in the X axis there are 39 100 steps The robotics head can be driven to any of these steps by entering position numbers in the appropriate part of the software Standard methods built into the software already have these positions preprogrammed To simplify this process the following standard and guidelines will help to speed up the process of programming Version 1 7 9 7 2013 18 Copyright 2013 PhyNezus Inc Rights Reserved When using the 200 and 1200 1 channel systems The simple programming of the software allows the user to move to any position and any row However to set the height of the tips use these guidelines For z axis heights in positions 3 4 5 7 amp 8 it is assumed a PhyTip plate is used For z axis heights 2 mL Deep Well Plate PCP 96 20 10 Column Resin Bed Volume Volume Position Position Position Position Position Position uL pL 3 4 5 6 7 8 1000 transfer tip 1000W transfer tip 1200 transfer tip 2160 2160
40. ettings to be changed Version 1 7 9 7 2013 52 Copyright 2013 PhyNezus Inc Rights Reserved 5 Protocol editing 5 1 How to create a new protocol To create a new protocol select File gt New and select Protocol as shown below NI PhyNexus Inc Capture Purify Enrich File Protocol Method Options Help Method Volume mL Elapsed time 0 00000 Reset Method name Manual Overrides 1 PEST 00 00 Volume Rate mL min Total time 1 D 0000 Capacity mL 1 20 Intake Expel gt Controller version Method or Protocol Auto Controller r Open Port X Axis steps Z Axis steps 0 Method COME Then press OK which will pop up the editor dialog Edit protocol Volume after selected Capacity mL step mL 02000 Delay sec 9 PhyNexus PURIFY ENRICH Press the Add button to insert new steps New steps may be edited after they have been added see below Version 1 7 9 Mar 7 2013 Copyright 2013 PhyNexus Inc Rights Reserved 53 Steps may be re ordered either individually or in groups by selecting the steps to be re ordered and using the Move Up and Move Down buttons A single step or a group of steps may be cut copied or re inserted using the Cut Copy and Paste buttons these work as most Windows users would expect When a single step is selected hig
41. h File Protocol Method Options Help Protocol name Method Volume mL Elapsed time 0 00000 3 m Manual Overrides gt 00 00 B Pause eset Volume mL Rate mL min Pause Total time Bes d otal time 0 00 00 Capacity mL 1 20 Intake lt Expel gt Controller version CARTURE 301108 13025 PURIFY ENRICH Version Port X Axis steps Z Axis steps C Home Retum to 1 1 COME Auto Controller The asterisk means that there are changes to the protocol that have not been saved Select File gt Save to save the changes back to the previous filename or File gt Save As to save changes to a new filename Note that the app s current capacity setting is also saved to the protocol file as a comment see Appendix B Version 1 7 9 Mar 7 2013 56 Copyright 2013 PhyNexus Inc All Rights Reserved 6 Method editing The steps involved in creating and editing a method are similar to that for a protocol 6 1 How to create a new method To create a new method select File gt New and select Method as shown below 22 PhyNexus Inc Capture Purify Enrich File Protocol Method Options Help Protocol name Method Volume mL C Program Files PhyNexus AutoController SW 200 AminoLink Wash 200uL ps Elapsed time 0 16200 5 A 7 gt Manual verrides 00 00 1 yN Volume mL Rate mL min o Total time
42. have v bottoms which maximize the process of elution It is also recommended to cover the plates once the elution buffer has been added with a PhyTip Plate Cover Part number PCP 96 10 20 and PhyTip Plate Holder Part number PCP 96 10 50 With the chiller activated both positions 7 and 8 can be maintained at sub ambient temperatures Version 1 7 9 Mar 7 2013 12 Copyright O 2013 PhyNexus Inc All Rights Reserved 2 Easy to use Ouick Guide Software Instructions The Auto controller Operating Software has been designed for simple to use automated methods that have been preprogrammed into the system However any combination of movements and positions can be built into a user defined method To begin PhyNexus recommends that the user start with the built in methods and subsequently modify these to match the requirements of the application Methods manage the entire process of moving the robotics tower with 12 channel system attached to perform the Capture Purify and Enrich process for all of the defined samples Protocols manage the individual steps of the Capture Purify and Enrich process A method is a series of protocols and robotic movement position instructions that fully automate the entire process of purifying a full plate of 96 samples To load the software onto a PC simply insert the CD into the CD or DVD drive of the PC and follow the installation instructions on the screen For simplest use of the system run the preprogra
43. hlighted using the mouse or keyboard the Volume after selected step indicator shows the volume in the syringe after the current step has been executed The volume at the beginning of the protocol is assumed to be the value in the Initial volume edit box The user may change the Initial volume as desired 5 2 How to edit an existing protocol To edit a protocol select Protocol gt Edit to pop up the editor dialog as described above An example is shown below Edit protocol Volume after selected Capacity step mL 01 Volume mL Rate mL min Times to do 1 See below for a description of creating and editing steps Version 1 7 9 7 2013 54 Copyright 2013 PhyNexus Inc Rights Reserved 5 3 Create protocol step To create a new protocol step press the Add button this pops up the following dialog PhyNexus Inc Capture Purify Enrich 5 53 Protocol name Method Volume mL Elapsed time 0 00000 Manual Overrides 00 00 Volume mL Rate mL min Edit protocol 0 0 Volume after selected Capacity mL step Intake Expel gt 1 20 0 Volume Rate mL min Times to do Delay sec Auto Controller 01 0 5 1 0 X Axis steps Z Axis steps Enter protocol step Volume Volume units Direction D mi C Expel zl Intake Volume prior to this step 81 How many times 0 1 B
44. ied step i Position Row moves Controller to specified position based upon 96 well SBS plates j Eject tips from controller k Move controller down towards tray by specified number of steps l Load tips onto controller 1 3 Compatibility The auto controller MEA system is assumed to be connected in between the host computer and the controller the 12 channel system No other system or options are compatible with the MEA system 2 How to open and execute a protocol The following window appears when the app first runs LITER Inc Capture Purify Enrich File t M Options Help Method name Method Volume mL Elapsed time 0 00000 A f Manual Overrides 00 00 i E eset p lt lt Volume mL Rate mL min asa 0 00 00 Capacity mL 120 Intake lt Expel gt Controller version CAPTURE PURIFY qe ENRICH X Axis steps Z Axis steps Bole 0 Home Retumto L1 COME Auto Controller The status bar lower portion of the screen shows the app status at any time Initially the app is not communicating with the controller so the status bar shows the message Offline when it starts Version 1 7 9 Mar 7 2013 42 Copyright 2013 PhyNexus Inc All Rights Reserved 2 1 Open protocol file Use the File gt Open menu option and select Protocol to browse for a protocol file as shown below NI PhyNexus Inc Capture Pu
45. ific affinity resin used the Reservoir Position can be used as a holder for neutralization buffer When used in this mode the reservoir is filled with sufficient volume of neutralization buffer Also Tip box position 2 should be used to hold standard Rainin LTS Pipette Tips The system should then include the incorporation of a neutralization step using the Reservoir Position use PhyNexus reservoir inserts PhyNexus Part Number 96 60 00 The maximum total volume per well for each of the reservoir wells is approximately 300 uL therefore maximum recommended volume to be transferred assuming 8 rows in a full 96 well plate is 35 uL Version 1 7 9 Mar 7 2013 11 Copyright O 2013 PhyNexus Inc All Rights Reserved Position 7 Sample Plate samples to be purified Depending on the specific application a number of different types of sample plates may be placed in this position e g deep well 96 well plates Care must be taken when choosing the type of deep well 96 well plate When using the 1000 system PhyNexus recommendis the use of 2 mL square well plates with a pyramid bottom PhyNexus Part Number PCP 96 20 10 Position 8 Elution Plate When using the 1000 system PhyNexus recommends the use of 2 mL square well plates with a pyramid bottom PhyNexus Part Number PCP 96 20 10 When using the 200 system PhyNexus recommends the use of the PhyTip Elution Plates Part number PCP 96 00 10 for the final step of elution These plates
46. irmware Check that the USB cable is in good condition and that the controller firmware version is supported by the app see section 1 above 2 3 Verify protocol Verification is performed automatically by the app prior to protocol but may also be invoked manually In this process the app checks that the following rules are followed during the protocol 1 Volume never falls below zero 2 Volume never rises above the capacity see also section 2 2 2 1 3 Flow rates within limits for the capacity see Appendix D When verification is performed automatically just prior to protocol execution the initial volume is assumed to be that reported by the controller A protocol may be manually verified using the Protocol Verify menu option In this case whatever current volume is presently reported by the controller is assumed to be the initial volume for verification purposes If the app has not yet gone online then the initial volume is assumed to be zero If protocol verification fails then an error message appears showing the line in the protocol at which the error occurred 3 How to open and execute a method Handling of methods is similar to that of protocols 3 1 Open Method Use the File gt Open menu option and select Method to browse for a method file as shown below Version 1 7 9 7 2013 48 Copyright 2013 PhyNezus Inc All Rights Reserved Inc Capture Purify E
47. lable to run Select the required file and press Open The Method will now be available for the system to run To verify the method click on Method Edit this displays the robotic steps and all of the linked protocols that constitute the given method To run this method press Method Execute The PC is in constant communication with the MEA Never run other programs while running the PhyNexus Operating Software The PC is a part of the MEA system and as a dedicated system should never be used on a network 5 Store analyze samples Once the final enrichment step has been completed samples will be ready for storage analysis Version 1 7 9 7 2013 20 Copyright 2013 PhyNezus Inc Rights Reserved Standard Methods Example 1 IMAC Figure 1 below is an ezample of a standard method of Capture Purify Enrich using Ni IMAC PhyTip columns for one row of 12 x 200 uL samples Deck configuration for this example is Position 1 Row 1 IMAC 200 PhyTip columns Position 2 Row 1 Empty Position 3 Row 1 Empty Position 4 Row 1 Recommended volume 200 uL of Wash 1 Position 5 Row 1 Recommended volume 200 uL of Wash 2 Position 6 Row 1 Empty Position 7 Row 1 Recommended volume 200 uL of Sample Position 8 Row 1 20 uL Elution Front Rear 5 PhyTip Columns Sample plate Elution Figure 1 Standard Method for Capture Purify amp Enrich Version 1 7
48. ller of Capture Purify amp Enrich for Protein A for one row of 12 x 200 uL samples For information on configuration of the instrument with and without chiller see page 29 Deck configuration for this example is as below Position 1 Row 1 Protein A 200 PhyTip columns Position 2 Row 1 Rainin LTS Tips Position 3 Row 1 Empty Position 4 Row 1 200 uL of Wash 1 Position 5 Row 1 200 uL of Wash 2 Position 6 Reservoir with Neutralization Buffer Position 7 Row 1 200 uL of Sample Position 8 Row 1 20 uL Elution Front Rear PhyTip Columns Standard Tips Wash 1 Wash 2 Neutralization Reservoir Sample 8 Elution Version 1 7 9 Mar 7 2013 25 Copyright 2013 PhyNezus Inc Rights Reserved Figure 2 Standard method including addition of Neutralization step Command Parameters if any Home Auto Controller Home Controller Execute Protocol ProA Air Gap Position 1 1 Load tips Position 7 1 Down 4225 Execute Protocol ProA Capture Position 4 1 Down 4280 Execute Protocol ProA Wash1 Position 5 1 Down 4280 Execute Protocol ProA Wash2 Position 8 1 Down 4225 Execute Protocol ProA Elution Position 1 1 Down 4100 Eject Eject Position 2 1 Load tips Execute Protocol ProA Intake 20uL Position 6 1 Down 4070 Exec
49. m out from the tip box This is set for the default PhyTip columns and standard Rainin LTS tips for neutralization ejects all 12 PhyTip columns from the 12 channel system Auto movement to specify position along x axis CAUTION This action does NOT move tower to zero position loads a row of 12 PhyTip columns onto the 12 channel system 60 Copyright O 2013 PhyNexus Inc All Rights Reserved 6 4 Edit method step To edit an ezisting method step select the step in the editor window and either double click on the step or press the Edit button The method step dialog described above is then shown 6 5 Edit protocol within a method To edit a protocol within a method select the protocol in the editor window and either double click on the protocol or press the Edit button The method step dialog described above is then shown Click on the Edit button and proceed with editing the protocol as described in section 3 4 Edit method step Type Protocol Pause for operator C Audible alert Comment hidden Home Controller Blowout Home Auto Controller Position C Down Position row C Up Load Tips External pump C Eject Rotate C XM V Submethod Load Cancel C Rotate Axis Protocol filename 1000 40uL Pra amp N1000 Capture 1mL psc Edit Click OK to accept the changes to the protocol The user will be prompted to save the protocol as a different file It is advis
50. matically return to a safe height above the deck but instead moves the 12 channel system along the specific z axis height that is seen on the screen Manual Positioning X Position Rotation EXEXENS MERERI EBENEN Pes fo C Instrument programming Before programming a specific method note all of the specific steps that are likely to be carried out in the process that needs to be accomplished Below is an example of some of the steps involved in a basic Method Prime the 12 channel system and home 2 Move to Row 1 of Box 1 of PhyTip columns move robot tower down and pick up row 1 of PhyTip columns 3 Move robot tower up and the across to row 1 of sample plate at position 7 4 Move tower down so that PhyTip columns enter the sample 5 Begin preprogrammed Capture Step 6 Upon completion of Capture step bring tower up and move across to row 1 of wash plate 1 in plate position 4 7 Move tower down so that PhyTip columns enter wash buffer 1 Begin preprogrammed Wash 1 Step 9 Upon completion of Wash step 1 bring tower up and move across to row 1 of wash plate 2 in plate position 5 10 Move tower down so that PhyTip columns enter wash buffer 2 11 Begin preprogrammed Wash 2 Step 12 Upon completion of wash step 2 bring tower up and move across to row 1 of Enrichment Step in plate position 8 90 Version 1 7 9 Mar 7 2013 17 Copyright 2013 PhyNezus Inc All Rights R
51. me as all the intake steps At this first step there is no need to expel any further volume since the next step is another wash step Version 1 7 9 7 2013 36 Copyright 2013 PhyNezus Inc Rights Reserved 200 Pro A Wash 2 Volume mL Rate mL min Times to do Delay sec 018 1105 1 o 0 18 0 182 Wash 2 This is the second wash step again two cycles at the same flow rate as Wash Step 1 Now however there is a larger volume of wash buffer 180 uL and at the final expel step there is an extra 2 uL of volume expelled from the PhyTip column 182 uL 200 Pro A Elute Elute The final step in the purification process uses 5 cycles of rapid flow elution over the resin bed In this case although only 10 uL is being used as the Elution volume the protocol is stored as 50 uL This difference between actual physical volume and programmed volume allows for all of the Elution buffer to pass over the resin bed thus maximizing the total yield of purified protein that can be obtained from the column It is essential to overdrive this step 50 uL to pick up 10 uL of liquid to overcome the surface tensions and pressures that are caused by the initial insertion of the tip and bed resistance in the tip The required balance is to pull up enough to get the fluid to move but not too much such that the volume of fluid pulls an air bubble As a programming guideline if a higher volume for elution is n
52. midazole in the wash buffer rows CRD have 5 mM E amp F have 10 mM and G amp H have 20 mM Imidazole Version 1 7 9 7 2013 30 Copyright 2013 PhyNezus Inc Rights Reserved Column 1 2131415 6171891101112 01010101010 10 10 10 0 01010101010 0 10 10 0 BSS 86 2 5 5 5 5 5 5 5 5 5 5 5 5 20 0 Purification Step 2 This can be a repeat of Wash 1 using the exact same methodology Enrich The final elution step can now be compared using two concentrations of Imidazole to remove the his tagged protein e g columns 1 6 are eluted with a 150 mM Imidazole buffer and columns 7 12 are eluted with a 250 mM Imidazole buffer Row 1121314115 9 1j m m A 50 150 150 150 160 150 20 250 250 250 250 250 8 450 150 150 150 150 150 250 250 250 250 260 21 86 585 18 580 80 8 28 280 29 250 80 881191 19119 80 8 28 29 280 8 20 0 881191 19119 80 280 0 20 80 6 19 86 38 o 26 280 8 20 Ew iso 91 so so 80 8 58 2 280 250 This one experiment which can be run in less than 2 hours allows for a statistically significant number of comparisons of capture conditions wash conditions and elution conditions Version 1 7 9 var 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved Standard Metho
53. mmed methods for any of the given affinity resins To do this go to File then Open This will bring down a screen that shows either Method or Protocol click on Method to show all of the stored Methods Open the standard method IMAC this will bring down the operational screen Now go to Method and click on Edit this will show all of the individual steps of the method Click on any one of the individual steps of the methods to show the options screen this will be split into two sections The top section deals with the operation of the 12 channel system controller while the bottom section deals with robotics movement of the autocontroller Actions that are available are Top Section Execute executes a stored protocol e g Capture Audible Alert introduces an audible alert at the end of an action Home controller homes the 12 channel system to a zero volume Pause for Operator builds in an automatic pause in the method Comment allows operator to build in an automatic comment or prompt between actions Version 1 7 9 7 2013 13 Copyright 2013 PhyNezus Inc Rights Reserved Bottom Section Home Autocontroller returns the robotics tower and 12 channel system to registered Home position Down moves the robotics tower with 12 channel system down to designated step position 0 is the highest position above the deck and 4725 is the position closest to the deck A dialog box will appear where the number of steps ca
54. mns z axis Sample Plate 2310 PhyTip Deep Well PhyTip columns z axis Wash Plate 2310 PhyTip Plate With Cooling System z axis PhyTip Elution 2310 Rainin LTS 2120 Protocols Version 1 7 9 Mar 7 2013 Position 2 PhyTip columns Rainin LTS Tips z axis Load 3600 Eject 3500 PhyTip Deep Well PhyTip columns z axis Wash Plate 2310 Position 6 x axis Neutralization Reservoir Rainin LTS Tips z axis Neutralization 2050 With Cooling System z axis PhyTip Elution 2310 Rainin LTS 2120 Copyright 2013 PhyNexus Inc All Rights Reserved 34 Protocols define the flow rates volume and pauses associated with the process of Capture Purify Enrich There are a series of standard protocols that have been stored in the software e g 200 ProA Condition 200 ProA Capture 200 ProA Wash 1 200 ProA Wash 2 200 ProA Elute 200 ProA Condition 2 200 ProA Intake 5 uL 200 ProA Expel 5 uL These protocols represent a series of steps that would be used for the 2004 PhyTip columns containing 5 UL Protein A affinity resin there are others for 2004 Protein G IMAC and Glutathione plus 1000 Protein A Protein G IMAC and Glutathione The standard methods have been written to incorporate a series of the standard protocols so that the process of Capture Purify Enrich can simply be carried out without the need for further programming 200 Protocol As an example of these p
55. n be entered Up moves the robotics tower with 12 channel system up to the designated step position 0 is the highest position above the deck and 4725 is the position closest to the deck A dialog box will appear where the data can be entered if left unchecked the default is all the way up to the zero position If data is entered this information is only a delta meaning that if 20 is entered the tower will move up 20 positions and NOT to location number 20 External Pump operates an external pump when fitted via the rear serial port allowing for the automated addition of reagents or activation of vacuum system Rotate rotates the vertical 12 channel system clockwise 0 to 335 and Counterclockwise 0 to 25 Sub method allows user to define a specific action and store this in memory e g Costar 200 V bottom plate could reference the specific height of a novel plate once stored in memory z numbers would not need to be recalled Submethods Command Costar 200 V bottom Elution Plates Down 4225 Remove Version 1 7 9 Mar 7 2013 14 Copyright 2013 PhyNexus Inc All Rights Reserved Position moves the robotics tower with 12 channel system to the desired x axis location on the deck There are a total of 39 100 positions along the x axis Position Row Load Tips Eject XMOV Version 1 7 9 Mar 7 2013 moves the robotic tower with 12 channel system to the desired Position 1 8 and specifi
56. nd expel steps when the app isn t running a protocol The following procedure should be used Verify that the Controller is mounted on the Auto Controller correctly as above Verify that the app s serial parameters match those of the controller as above Verify that the app s capacity setting matches that of the syringe as above Enter the volume and rate in the text boxes under the Manual Overrides frame and then press the Intake or Expel button 35 qr This will cause the app to go online with the controller and execute the intake or expel step Status will be shown in the status bar and the Volume indicator will be updated when the operation is completed if successful See section 2 2 3 2 above for troubleshooting 4 1 Reset volume The Reset button may be used to manually blow out the syringe contents and reset its volume to the home position zero 4 2 Stopping manual commands Manual intake expel operations may be aborted using the Stop button As with aborting protocol or method execution pressing stop will terminate the app and will stop the protocol right away before the last command step is completed The app will display the current volume as last reported by the controller Note Communications and Syringe settings can only be changed when the app is not communicating with the controller not executing a protocol method or manual override command Pressing Stop terminates communication and allows these s
57. nrich Method Options Help Protocol name Method Volume MER C Program Files PhyN exus AutoController SW 200 AminoLink Wash 200uL ps Elapsed time 1620 18200 a PhyNexus Volume mL Rate mL min o Pu 7 otal time Stop Capacity mL 0 0 0 2000 Intake lt Expel gt Controller version CARTURE 11 3 0 1109 413025300 PURIFY Auto Controller Method or Protocol E Pan ENRICH xis steps xis steps 0 COME Protocol Method files are assumed to have a pba file extension If the method file was opened successfully then it may be executed and or edited If the app displays an error message when you try to open the method file then it may have been corrupted e g via disk error or hand editing or the file selected is simply not a method The MRU most recently used list in the file menu also handles method files After opening the method file the display will indicate the currently open method as shown below Bi PhyNexus Inc Capture Purify Enrich Elle Protocol Method Options Help Method name Method Volume mL C Program Files PhyNexus E4 AutoController S w T est Method pba Elapsed time 0 00000 Manual Overrides 00 00 yNexus Volume mL Rate mL min E o Ez L d m otal time Stop 00 00 Capacity mL 02000 Intake lt Expel gt Controller version
58. ns Under no circumstances shall Seller be responsible for alleged nonconformities with respect to any merchandise which has been used for purposes or in any manner for which it was not intended or any merchandise which has been customized or modified without Seller s prior written consent or damaged or misused As Buyer s exclusive remedy in the event of breach of warranty Seller shall repair or replace as its option any nonconforming merchandise or parts thereof for a period of one 1 year after delivery All claims must be made in writing to the Seller Any claims not made within the period specified above shall be deemed waived and released THE PROVISIONS OF THE FOREGOING WARRANTIES ARE IN LIEU OF ANY OTHER WARRANTY WHETHER EXPRESS OR IMPLIED WRITTEN OR ORAL INCLUDING ANY WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE TITLE OR NON INFRINGEMENT SELLER S LIABILITY ARISING OUT OF THE MANUFACTURE SALE OR SUPPLYING OF A PRODUCT OR ITS USE OR ITS DISPOSITION WHETHER BASED UPON WARRANTY CONTRACT TORT OR OTHERWISE SHALL NOT FOR ANY REASONS EXCEED THE AGGREGATE PURCHASE PRICE PAID BY BUYER FOR SUCH PRODUCT IN NO EVENT SHALL SELLER BE LIABLE TO BUYER OR ANY OTHER PERSON OR ENTITY FOR SPECIAL INCIDENTAL CONSEQUENTIAL OR EXEMPLARY DAMAGES INCLUDING BUT NOT LIMITED TO DAMAGES FOR LOSS OF PROFITS LOSS OF DATA OR LOSS OF USE ARISING OUT OF THE MANUFACTURE SALE SUPPLY USE MARKETING RESALE OR OPERATION OF THE MERCHANDISE
59. ollows Version 1 7 9 Mar 7 2013 43 Copyright 2013 PhyNexus Inc All Rights Reserved PhyNexus Inc Capture Purify Enrich a aaa File Protocol Method Options Help Method name Method Volume mL Elapsed time 000000 Manual Overrides 0000 Volume mL Rate mL min o Lgs ZU Besa 2 p 22 1 20 L Intake Expel gt Controller version CAPTURE PURIFY r Auto Controller X Axis steps Z Axis steps COM 6 0 Controller Device Communication Options m Pot ENRICH L Auto Controller Comm Port 6 Make sure that the Controller Device setting matches the serial number of the Controller To select the proper AutoController Comm Port use the Window s Control Panel Control Panel gt System gt Hardware gt Device Manager Expand the Ports file and note the USB COM Port in use Device Manager File Action View Help Batteries H Computer Disk drives 58 Display adapters B DVD CD ROM drives 028 Human Interface Devices 9 e IDE ATA ATAPI controllers 9 399 Imaging devices Keyboards 9 7 Mice and other pointing devices Monitors iei MB Network adapters E Ports COM amp LPT USB Serial Port COM6 Sf Processors amp 89 Sound video and game controllers System devices 8 Universal Serial Bus controllers e Settings for th
60. ontroller Calibration EZ Position a 55 Rotation fs Z Axis ft 1 Save Click Check Alignment and the 12 channel system will move to the zero position azis and then down to the normal tip loading height for the specific system that is connected e g 200 or 1200 Adjustments can be made for x axis z axis and rotate as follows For the x axis adjust the 1 and 1 settings to align the nozzles on the 12 channel system in the center of the tips For the z axis adjust the 1 and 1 settings to bring the base of the nozzles on the 12 channel system to approximately 0 5 mm or 1 5 mm above the top of the tips for the 200 and 12004 systems respectively For the rotation adjust the 1 and 1 settings to bring the plane of the base of the nozzles on the 12 channel system parallel with the plane of the top of the tips Once complete click Save This will save the calibration settings for the specific 12 channel system The software recognizes the difference between a 2004 system and 1200 system and applies the saved settings accordingly Version 1 7 9 7 2013 16 Copyright 2013 PhyNezus Inc Rights Reserved B Manual Positioning This allows the user to position the 12 channel system anywhere on the deck This 15 particularly useful when used in conjunction with Sub method Care must be taken when using this part of the software as the z azis does not auto
61. other expel steps in order to maximize the total volume of elution buffer returned to the elution plate Position 1 Row 1 moves the robotics tower to the full extend position Position 0 and then moves it over to position 1 row 1 ready for the ejection of the PhyTip columns Down 4100 moves the robotics tower down to predefined position of 4100 ready to eject the PhyTip columns back into their original position in row 1 Eject is a command to remove all of the now used PhyTip columns Version 1 7 9 Mar 7 2013 28 Copyright 2013 PhyNexus Inc All Rights Reserved 4 Neutralization Position 2 1 Load tips Execute Protocol ProA Intake 20uL Position 6 1 Down 4070 Execute Protocol ProA Intake 5uL Position 8 1 Down 4140 Execute Protocol ProA Expel 25uL Position 2 1 Down 4100 Eject Home Auto Controller Position 2 Row 1 moves the robotics tower over position number 2 and row 1 The tray of Rainin LTS Tips Load tips moves the 12 channel head down to the pre assigned position to automatically load the tips onto the 12 channel system Execute ProA Intake 20uL executes a stored protocol which in this case the 12 channel system withdraws air into the syringes before the LTS tips are lowered into the neutralization buffer It does this to create an air buffer in the system that will later be used to push liquid out of the LTS tips The volume of air will vary depending on the
62. ower down to predefined position of this assumes use of PhyNexus recommended labware with known dimensions for the elution step Execute IMAC Elution implements the stored protocol for the elution step This protocol like Capture will control the flow rate volume pause and number of cycles involved in this step Note at the end of this step the last expel step is greater volume than the other expel steps in order to maximize the total volume of elution buffer returned to the elution plate Position 1 Row 1 moves the robotics tower to the full extend position Position 0 and then moves it over to position 1 row 1 ready for the ejection of the PhyTip columns Down 4100 moves the robotics tower down to predefined position of 4100 ready to eject the PhyTip columns back into their original position in row 1 Eject is a command to remove all of the now used PhyTip columns Home Auto Controller takes the robotics tower back to its safe starting position and completes the purification of 1 row of samples If using this program as a template just cut and paste the entire page and replace row 1 with row 2 etc When a method is running each step of the process will display on the screen Completed steps will display Green while steps In Progress will display Blue Version 1 7 9 Mar 7 2013 24 Copyright O 2013 PhyNexus Inc All Rights Reserved Example 2 Protein Figure 2 below is an example of a modified method without chi
63. ptions field Simulation Mode allows the user to run a method on the software only disconnecting the functions of the MEA This can be used to verify check that all of the software programming is compatible without running the MEA Terminal allows the user to move the 12 channel system via a series of typed commands to any position on the deck This function allows the user to verify specific positions on the MEA and to design new methods that are different to the stored methods Specific position commands can be entered in the window and once the send command is hit the robotics tower with 12 channel system will move to that specific position Commands are entered in the window to the right of the send command button Once the command has been entered press send This will initiate the command and in the screen box above this window there will be a confirmation that the command has been implemented e g Response OK Commands Home moves the robotics tower and 12 channel system back to starting home position 0 and 2 0 Down where any number between and 4275 moves the 12 channel system down to a specific point It is recommended to place two Tips that will be used in the process on positions 1 and 12 of the 12 channel system in order to position the base of the tip correctly Note that when the 12 channel system is in the full up position 0 then to move the system down just type in the desired position num
64. rify Enrich e Protocol Method Options Help Method name Method Volume mL 5 Elapsed time 0 00000 Manual Overrides 00 00 lt Volume Rate mL min M d Total time 0 Stop 00 00 Capacity 1 20 Intake lt Expel gt Controller version i CAPTURE Aree Method or Protocol PURIFY uto Controller 1 X Axis steps Z Axis steps Botts ENRICH d COMB 0 mmn Home Protocol files are assumed to have a psc file extension If the protocol file was opened successfully then it may be verified executed and or edited If the app displays an error message when you try to open the protocol file then it may have been corrupted e g via disk error or hand editing or the file selected is simply not a protocol 2 2 Execute Before executing the protocol make sure of the following 1 All PhyTip columns sample plates wash buffers etc are in place App s serial parameters are set to match those of the controller 3 The capacity of the PhyTip columns matches the volume required to be processed After these items have been checked the protocol can be executed 2 2 1 Check Controller Communication The app communicates with the AutoController and the controller The app s Communication setting verifies communication to the AutoController and the Controller Check this by selecting the Options gt Communications menu which pops up a dialog as f
65. rotocols when using the standard 200 Protein A Method there are several Execute steps that reference the following steps 200 Pro A Condition Volume mL Rate mL min Times to do Delay sec 0 01 Air Gap This conditions the 12 channel system to prepare for the complete process of moving liquids over the resin bed in the PhyTip columns The final process of elution will often require only 10 uL of elution buffer to be moved over the resin bed and then expelled In order to be sure that all of this liquid can be blown out a sufficient reservoir of air must be added to the 12 channel system before the PhyTip columns are attached In this case 10 uL of air is moved into the 12 channel system at a rate of 1 mL minute Version 1 7 9 7 2013 35 Copyright 2013 PhyNezus Inc Rights Reserved 200 Pro Capture Volume mL Rate mL min Times to do Delay sec 0 17 0 25 1 10 0 17 0 25 1 10 0 17 0 25 1 10 0 17 0 25 1 10 0 17 0 25 1 10 0 17 0 25 1 10 0 17 0 25 1 10 0 172 0 25 1 10 Capture Passes the sample over the resin bed enabling the Protein A affinity resin to bind the protein of interest In this case 170 uL of sample is moved into the PhyTip column Intake and 170 uL is moved out Expelled Intake can be seen as the positive number and expel the negative number also note that all expel steps are colored red Note that there are four expel st
66. s packing and install this unit onto the 1 on the robot tower as follows 1 The black alignment clamp has been factory installed on the 12 channel system The LCD on the 12 channel system points to the right and the clamp has an alignment slot at the rear which will interface with an alignment bar on the moving Z head on the MEA tower The moving Z head is a round bar with a single flat surface that extends forward of the robotic tower and the alignment bar that is parallel to the x axis of the system 2 Locate the single attachment hole at the rear of the clamp and push the clamp onto Z head bar 3 With the alignment bar and alignment slot parallel to each other push the clamp mechanism in fully until it snaps in place 4 Turn the knurled screw on the side of the clamp to finally tighten the clamping mechanism in place To exchange the 12channel head simply loosen the knurled screw ease the clamp from the alignment slot and pull away from the z head bar Repeat the first four steps with the alternate 12 channel head When replacing a 200 channel system with a 1000 12 channel system remove the front and rear rack bars that are at positions 1 and 2 There are three Allen key screws two at the front and 1 at the rear Unscrew the three screws and remove the rack bars The new height of positions 1 and 2 will be ready for the deep tip boxes used with the 1000 12 channel head Take the MEA 12 channel head USB cabl
67. s the neutralization buffer Note the negative number indicating an expel step 1000 Protocol Below is an example of the protocols for a 10004 PhyTip column system using IMAC as the affinity resin 1000 IMAC Air Gap Volume mL Rate mL min Times to do Delay sec 0 09 3 1 1000 IMAC Capture Volume mL Rate mL min Times to do Delay sec 0 45 0 25 1 20 0 45 0 25 1 20 0 45 0 25 1 20 0 45 0 25 1 20 0 45 0 25 1 20 0 45 0 25 1 20 0 45 0 25 1 20 0 47 0 25 1 20 1000 Wash 1 Volume mL Rate mL min Times to do pov 9 t XE 0 09 31 o 09 05 1 o 09 05 0 Version 1 7 9 Mar 7 2013 38 Copyright 2013 PhyNezus Inc Rights Reserved 1000 MAC Wash 2 EE 1000 YO Elute a 0010 1 2 Note The initial conditioning step of 90 uL and the subsequent combination of excess expels total 90 uL Also the final elution volume of 20 uL requires an intake expel of 250 uL As a programming guideline if a higher volume for elution is needed add 230 to the final volume number to obtain the programmed elution volume e g if 30 uL is the final elution volume required program 30 230 260 uL for the final elution volume This guideline is valid for elution volumes less than 250 uL For elution volumes greater or equal to 250 uL simply program that same
68. stt eh Section A 1 Unpacking Each MEA system contains the following 1 MEA Personal Purification System 2 Two power supplies one for the MEA one for the chiller Use only the power supplies provided see page 73 for ratings MEA 12 channel head s The 12 channel heads s will be provided with attachment clamp already installed ready to attach to the MEA tower MEA 12 channel head USB Cable Software CD MEA USB communications cable Accessory pack including rack bars and evaporation hold down cover Consumables pack including PhyTip Evaporation Covers Holders reservoirs and 96 well plates Literature documents and Operating Instructions pack O If these separate items are not in the original outer packing please call PhyNexus Inc at 408 267 7214 Remove all contents from their individual packs and prepare to install the system Version 1 7 9 Mar 7 2013 4 Copyright 2013 PhyNexus Inc All Rights Reserved 2 Setting up the system This section is divided into Initial location and set up of system parts Installing the software Powering up the system Cleaning the system 1 Initial location and set of system parts Ouick guide Once all contents have been removed from their individual packs locate the MEA instrument and place it close to the computer to be used for operation Using needle nose pliers unhook the springs holding the Tower Support Blocks together Conn
69. t actually implement continuous flow rates but operates at ten discrete motor speeds each of which is computed as follows flow rate mL min 60 x syringe capacity mL full scale duration sec where the motor speed determines full scale duration as per the following table only the 200 and 1000 uL capacities are shown Full scale duration Flow rate for 200 Flow rate for 1000 sec uL capacity uL capacity mL min mL min 1 2 3 4 5 6 7 8 9 The motor speed used to implement 8 particular flow rate is usually arrived at by interpolating between the motor speeds For example a flow rate of 13 mL min for the 200 uL capacity would use motor speed 9 because the midpoint between speeds 9 and 10 is 14 6 mL min For flow rates below motor speed 1 the controller is commanded to perform move wait cycles in which small intake or expel volume increments are made interspersed by delays of several hundred to several thousand milliseconds These cycles are used when the flow rate falls below 2 10 or 12 mL min for the 200 and 1000 uL capacities respectively Motor speed 1 is used to perform the cycles Version 1 7 9 7 2013 67 Copyright 2013 PhyNezus Inc Rights Reserved The volume increments for move wait cycles are as follows 20 100 0 5 for 200 uL capacity 1 otherwise 100 200 1 200 400 2 400 800 5 800 1500 10 1500 2000 15 2000 4000 2
70. tep is to be repeated then the post step delay is also repeated when executed 1 5 10 3 1 5 Any line which begins with is treated as 8 comment and ignored The app stores only one comment line to a file to indicate the capacity milliliters An example is as follows vol 21 This indicates that when the file was saved the app s capacity setting was 1 mL Version 1 7 9 7 2013 63 Copyright 2013 PhyNezus Inc Rights Reserved Appendix B Method File Format Methods are also stored as text files The app registers the file extension pba for PhyNexus method files Each line of a method file represents a step of the method and may take the following forms Execute specified protocol file execute protocol file path For example the following line indicates that the protocol file C PhyNexusHome AutoController SW test1 psc should be executed test1 psc Note that file paths may be absolute as above or relative Relative paths are relative to the app executable file For example if the app resides at C PhyNexusHome AutoController SW AutoController exe and the path Xestl psc is specified then the protocol file C PhyNexusHome AutoController SW test1 psc will be executed 2 Audible alert alert 3 Pause show message and wait for operator intervention pause message The message is shown with OK and Cancel buttons OK continues the method and Cancel terminates it New lines may be embedded in
71. this feature Environmental Operating Temperature 4 to 30 Relative Humidity 096 to 9596 Altitude 0 to 10000 Ft AWARNING INSTRUMENT CONTAINS CTRICAL CIRCUITS Devic ES S VOLTAGES col WITH THESE CIRCUITS DEVICES AND COMPONENTS CAN CAUSE DEATH SERIOUS INJURY OR PAINFUL ELECTRICAL SHOCK OPERATORS UNAUTHORIZED m LN ya ST NEVER OPEN THE OR APPROVED SERVICE ENGINEERS Version 1 7 9 Mar 7 2013 71 Copyright 2013 PhyNexus Inc All Rights Reserved A AVERTISSEMENT TOUT CONTACT AVEC LES HAUTES TENSIONS PEUT ENTRAINER LA MORT OU DES BLESSURES SEVERES CE PANNEAU NE DOIT ETRE ENLEVE QUE PAR UN REPARATEUR QUALIFI WARNING THIS INSTRUMENT CONTAINS ELECTRICAL CIRCUITS DEVICES AND COMPONENTS OPERATING AT DANGEROUS VOLTAGES CONTACT WITH THESE CIRCUITS DEVICES AND COMPONENTS CAN CAUSE DEATH SERIOUS INJURY OR PAINFUL ELECTRICAL SHOCK OPERATORS AND OTHER A AVERTISSEMENT TOUT CONTACT AVEC LES HAUTES TENSIONS PEUT ENTRAINER LA MORT OU DES BLESSURES SEVERES CE PANNEAU NE DOIT ETRE ENLEVE QUE PAR UN R PARATEUR QUALIFI ANWARNINC CONTACT WITH DANGEROUS VOLTAGES CAN CAUSE DEATH OR INJURY COVER TO BE REMOVED ONLY BY TRAINED SERVICE PERSONNEL A AVERTISSEMENT TOUT CONTACT AVEC LES HAUTES TENSIONS PEUT ENTRAINER LA MORT OU DES BLESSURES SEVERES CE PANNEAU NE DOIT ETRE ENLEVE QUE PAR UN REPARATEUR QUALIFIE Version 1 7 9
72. tion For example the Open option under the File menu is referred to as File gt Open 1 2 Definitions The Controller is the 12 channel pipette A protocol is a series of steps which command the controller to intake or expel liquid Each step of a protocol specifies a volume positive to intake negative to expel a rate and a number of times to repeat The post step delays may also be specified A method is a series of steps which group protocol s together and also provide some user interface functionality The following actions can be steps of a method 1 Execute specified by filename 2 Audible alert beep 3 Pause for operator display message and wait for user input to either continue method or abort 4 Home Controller moves Controller pistons to the home position 5 Comment solely for documentation of the method file not seen by the operator 6 Auto controller instructions a Home AutoController reset all axes to home position Version 1 7 9 Mar 7 2013 41 Copyright 2013 PhyNezus Inc Rights Reserved b Down moves Controller down to specified position c Up moves Controller up to specified position performed automatically by tray command i d External Pump turns external pump on off e Rotate rotates Controller to specified location f Submethod utilizes heights of stored plate definitions g Home Rotate Axis reset the rotation axis h Position above tray at specif
73. unning Windows 8 With the computer turned on insert the software disc into the CD or DVD drive of the system double click on the setup icon and follow the install instructions on the screen 3 Powering up the system With all power supplies connected and the software installed in the computer turn the system on by pressing the power supply switch at the rear of the MEA A green Power On light will illuminate at the front of the instrument If the green light does not illuminate first check that all connections have been made and secondly make sure that the red emergency stop button has not been depressed To do this turn the emergency button in the direction for Reset clockwise Turn on the chiller by pressing the power switch at the rear of the instrument and then turning the setting switch on the right of the instrument to the appropriate setting 4 Cleaning the system The MEA Personal Purification System can be cleaned by wiping with a damp cloth Do not use solutions containing high concentrations of bleach or other ozidizing agents Version 1 7 9 Mar 7 2013 Copyright O 2013 PhyNexus Inc All Rights Reserved 3 Using the PhyTip MEA Purification System This portion of the manual is divided into the following sections Setting the instrument deck Easy to Use Ouick Guide Software Instructions Easy to Use Ouick Guide Operating Instructions Detailed Software Information and Instructions TEU O 1 Setting the
74. ute Protocol ProA Intake 5uL Position 8 1 Down 4140 Execute Protocol ProA Expel 25uL Position 2 1 Down 4100 Eject Home Auto Controller This method shows the automated movement of the 12 channel system for 1 row of samples In detail this can be broken down as follows 1 Homing the system Version 1 7 9 Mar 7 2013 Copyright 2013 PhyNexus Inc All Rights Reserved Command Parameters if any Home Auto Controller Home Controller Execute Protocol ProA Air Gap Position 1 1 Home Auto Controller moves the robotics tower to the Home position full left position Blowout performs a complete blowout of the 12 channel system Home Controller performs a zeroing of the 12 channel system followed by a resetting of the system to zero Execute ProA Air Gap executes a stored protocol which in this case is setting the 12 channel system prior to attachment of PhyTip columns The 12 channel system withdraws air into the syringes to create an air buffer in the system that will later be used to push liquid out of the PhyTip columns The volume of air will vary depending on the specific set of protocols being used Position 1 Row 1 moves the robotics tower over position number 1 and row 1 The tray of PhyTip columns 2 Moving the PhyTip Columns and Capturing the Sample Load tips Position 7 1 Down 4225 Execute Protocol Pro A Capture Load tips moves the 12 channel syst

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