User Manual - Mettler Toledo
Contents
1. PT ACC 17012588 PuresSpeed Accessory Kit Contents Base SD card 96 deepwell plate ColorTrak Guides Adapters 200 uL Adapters 1000 uL PT PT96 17012584 PureSpeed Base in PT ACC PT E4 SD 17012587 E4 XLS Micro SD Card with PureSpeed protocol in PT ACC LR P2 96P 5 17012623 96 deepwell plate 5 pack one supplied in PT ACC PT 2 H 17012582 Pack of 10 adapters for 200 uL multichannel cin PT ACC PT 10 H 17012583 Pack of 10 adapters for 1000 uL multichannel cin PT ACC PT 2 S 17013242 Adapter for 200 uL single channel cin Starter Kit PT 10 S 17013226 Adapter for 1000 uL single channel cin Starter Kit 1 3 PureSpeed Accessories PureSpeed Accessories can optimize the purification process allowing you to purify proteins in as little as 15 minutes 2 In Accessory Kit 1 Platform 2 96 deepwell plate 3 Micro SD card 4 2 Mylar ColorTrak Guides for ProA ProG and IMAC 2 sided ee 5 10 each multichannel adapters 200 uL green amp 1000 uL blue of In Starter Kit ma 6 One single channel adapter 200 uL or 1000 uL ee 5 Figure 1 PureSpeed Accessories 2 0 Set Up for Protein Purification with PureSpeed Protein Tips and E4 XLS Pipette 2 1 Buffer Preparation ProA ProG resins IMAC resin Prepare a 5X buffer 50 mM sodium dihydrogen phosphate 700 mM sodium chloride pH 7 4 Then dilute to 1X for the Equilibration and Wash 1 buffers Equilibration buffer See above Wash 1 buffer See above 1X Wash 2 b2. Once a parameter is set to the desired value press or the DONE soft key to save the change Pressing the CANCEL soft key will leave the field unchanged Hint The total time required to complete the protocol is displayed in the lower right above the right soft key in HH MM SS format the example indicates 9 minutes and 32 seconds This value is updated after each parameter change Hint It is possible to bypass a step by entering a Zero in the Aliquots field Hint The NEXT soft key allows you to quickly index through all the step settings cr A Tip Typ IMAC i SuL Saved DEFAULT DEFAULT Protocols ABC PureSpeed SELECT 3 CANCEL IEE AI T DEFAULT Volume Speed Aliquots Cycles DONE 200 0uL Aliquots d 1 Volume Speed 12 CANCEL 3 3 Protocol Save Once all steps are configured properly the DONE key returns you to the main protocol setup screen Then the NEXT soft key advances to the Protocol Options screen Here the option to SAVE or RUN the protocol is given If changes have been made and will be needed it the future it is advisable to save the protocol at this time Deleting a previously saved protocol is also an option See section 3 5 Pressing or gt activates the Protocol Save selection screen By default the active protocol is highlighted Press SELECT to save the settings to the current protocol definition The s
3. resin Chart showing various standardized protocol buffer volumes oY o y o ema s o wo 5 6 a o Ewon o0 100 800 soo 100 vo soo soo 100 100 soo sw 20 500 Feuer 6 o o oo s o o m0 oo 00 For larger samples use multiple aliquots in the Capture step e g Capture 2x Capture 3x etc Take the deepwell plate and slide it into the grooves on the base as shown below The figure shows a 12 channel pipette in use note the adapter positions on the 2nd and 11th tips Depending upon the E4 XLS model you are using your set up may be different 1E Note for 1000 pL PureSpeed Tips using a 1200 uL multichannel E4 XLS pipette Adapter 200 uL shown y When placing the multichannel adapters on t 1000 uL PureSpeed tips they will reach a m certain point then stop See diagram Push the adapters firmly onto the tips until they reach the tip shoulders Deepwell plate Platform groove Figure 3 PureSpeed Accessory Kit Assembled 2 3 Installing the micro SD card into the E4 XLS The PureSpeed mode will only operate with E4 XLS LTS 200 uL and 1000 uL single channel and 200 uL and 1200 uL multichannel models Open the battery compartment on the E4 XLS remove the battery and locate the micro SD slot referring to the diagram on the inside of the battery compartment See Figure 4 Take the micro SD card from the kit and slide it into the slot in the orientation sho
4. LETE is pressed a pop up message will confirm the delete operation Press the DELETE softkey to verify that the protocol is to be deleted Once the delete is complete the E4 will return the Protocol Options page Repeat this operation to delete multiple protocols PureSpeed Protein Tip Data S ul CR PureSpeed Protein Tips provide the highest concen tration of pure functional protein when compared with conventional spin column methods A large E coli lysate sample was spiked with an un known amount of His tagged rubredoxin MW 7500 100 uL of lysate was purified by either spin column or Purespeed IMAC tips packed with 5 uL resin 10 uL of the resulting eluted samples were loaded onto SDS PAGE and the gel was stained by coomassie blue As shown in the gel PureSpeed tips outperformed spin column both in terms of purity and concentration mi TNT Rubredoxin Rainin Instrument LLC www imt com rainin 7500 Edgewater Drive For more information Oakland CA 94621 Sales 800 472 4646 or 1 510 564 1600 Service 800 543 4030 or 1 510 564 1600 Subject fo technical changes 2011 Rainin Instrument LLC Printed in USA Part Number 9920 411 Rev A a METTLER TOLEDO Company
5. MAC SuL 200 0yL Volume ELUTE Start Place tip s in Elution solution WAT es Cycles DEF AULT CONT After the final step is complete normally the Elute step the pipette will prepare for reset by prompting the user to remove the tips Once the CONT soft key is pressed the pipette will reset and return to the Ih Spl options screen 15 0uL Yolume protocol Complete Remove tip s and press CONT to reset Cycles To exit the run mode press the BACK soft key This will return to the protocol steps screen Pressing BACK at this level will return to the Protocol Select screen If protocol edits have been made that were not saved the unit will prompt the user to save them A Protocol Modified Press SAVE to save changes or CONT to select new protocol 3 5 Deleting Saved Protocols saved protocols that are no longer needed can be deleted by activating the delete button on the Protocol Options screen Pressing or the DELETE soft key will bring up the Protocol Delete page NOTE If no protocols are currently saved the Delete option will not appear The protocol delete page displays saved protocols that can be deleted Press s or w io select the protocol to delete and then press DELETE IMi e EE Saved ABC ABC Protocols ABCA Protocols Delete Confirm Press DELETE to remove or CANCEL to keep protocol BACK s gt DELETE sel ones be p Once DE
6. RAININ Pipetting 360 PureSpeed Tips Protein Tips with Affinity Resins i Lue p F d a y A O O D I OLEDO ly Generic Guide for PureSpeed Set up l 10 11 Depending on the type of protein to be purified His tagged recombinant or full length IgG choose the appropriate resin IMAC ProA or ProG to optimize the purification process Determine the resin volume that will be required higher resin volume will enable higher mass of protein fo be captured and subsequently more available for analysis Smaller resin volumes accomplish higher end product concentration Prepare the necessary buffers for the key steps in the process Equilibration Capture Wash 1 Wash 2 and Elution When using either ProA or ProG resins the protein being purified may require a neutralization step after the high acid elution step be sure to have this prepared and ready to use Using the default program within the E4 XLS choose the appropriate resin type and volume This program can be stored with an individual user name if needed See page 8 Using the Rainin ColorTrak Guide as a template place the recommended 96 well deep well plate on top of the guide and add the appropriate volumes of buffers to each marked row Note even if only purifying 2 samples the number of channels on the pipette will determine which guide to use For single channel and 8 channel pipettes use the 8 channel side fo
7. ction in four distinct steps Equilibration Capture Wash and Elute The Protocol Setup screen enables you to view and modify the parameters in each step In the Protocol Setup screen press se or Ww to select the step to view Press or gt to view or modify the step parameters Hint The selected tip type appears in the upper right portion of the screen under the battery icon The selected saved protocol appears in the lower left above the BACK soft key The time required to run the complete protocol appears in the lower right in HH MM SS format This value is updated each time settings are changed Hint Each step is assigned a color that appears here and in the step prompt icons These colors can be used in conjunction with the ColorTrak Guides to facilitate steps and reduce errors Once a step is selected a pop up box presents the parameters and settings Press amp or w io select a particular parameter Each tip type establishes parameter boundaries limiting the range of settings To activate a field press or gt An active field is highlighted white NOTE The defined protocol may limit some parameters to a fixed setting In these cases the values cannot be selected Hint Pressing the jy NEXT soft key in the setup screen advances to the next step This feature also provides a quick way to review all steps You can change an active field s value by pressing s or Ww lo change larger increments use 4 or gt
8. econd save screen presents the user with the selected save name and a SAVE button Hint PureSpeed mode provides space for up to 20 saved protocols per tip type Once this allocation is full use the delete function to make space for new protocols To save the protocol fo a different name press s to select the Save Name field and press amp or gt to activate the name field Once activated you can edit the name Use se or W to select the letter or number Then gt for next symbol You can clear the current name with CLEAR After the name is completed press DONE or to save Pressing the BACK soft key will return to the Protocol Options screen 13PhM i MAC SuL ions DE Name ABCH SAVE PureSpeed i PE Name Ae SAVE DEF AULT DEF AULT DONE DONE 12 CANCEL 12 CANCEL PureSpeed 4 17PM Li Saf Ba TAC SuL RUN DELETE rn 4 13PM i IMAC Spl PureSpeed Save Name see PureSpeed 41 IMAC Syl Save ene ABC1 To perform the save operation select the SAVE field and press or p to activate it Once the save is completed the E4 XLS will return to the protocol options screen Hint The active protocol name displayed in the lower left of the screen is always the last protocol saved If the default settings are active the name will appear as the tip identifier resin type bed size 3 4 Protocol Execution To run the protocol select the run scr
9. een button on the Protocol Options screen and activate it by pressing or b or press the RUN key Activating the run option will bring up the run screen which is similar to the other E4 XLS pipette screens The step name is displayed in yellow in the sub mode area of the screen The familiar green boxes on the right portion of the screen display the step setup data Cycle and aliquot numbers will update as the protocol progresses The E4 XLS will instruct the user to install the proper tips Press the CONT soft key when the appropriate tips have been mounted See page 4 for preparing the 96 deepwell plate and loading tips When the unit is ready to begin operation another pop up message will instruct the user to place the pipette into the Equilibration solution Again press the CONT soft key to start this cycle verify the process is operating DELETE DEFAULT PureSpeed Mode Ready Install IMAC Sul Tip s and Press CONT or CANCEL to IMAC Spl A 200o EQUILIBRATION Start Place tip s in Equilibration solution 0 0L MECCA T DEFAULT CONT During protocol execution the process can be paused at any time by pressing the PAUSE soft key any motion or timeouts are paused The soft keys present an option to RESET or to RESUME operation If RESET is pressed a confirmation message pops up asking to confirm the reset or to CANCEL the request A reset terminates
10. h each adapter fully onto the tip Add the remaining tips to the pipette and press the CONT softkey on the E4 XLS Follow the screen instructions and place the assembly into the Equilibrate row The adapters will maintain the correct height for the tips in each stage of the process In particular the elution step with the lowest volume of buffer requires the fips fo fouch the elution buffer and the adapters are manufactured to accommodate this With the assembly in place in the Equilibration buffer press the CONT softkey to continue the purification process and follow the instructions given on the E4 XLS screen 1 0 Introduction Rainin PureSpeed Protein Tips radically simplify purification of native or recombinant proteins This innovative approach results in highly pure and concentrated purified protein allowing many options for downstream functional assays Up to 12 samples can be processed in parallel using Rainin s state of the art E4 XLS multichannel electronic pipette in PureSpeed mode 1 1 Storage Store the tips at 4 C after receipt all other PureSpeed components can be stored at room temperature Glycerol that is added to the tips to protect the resin can form droplets on the tip walls during shipment but this is normal and will not affect a tip s performance 1 2 PureSpeed Tips and Accessories PureSpeed Protein tips are offered with 3 different resins ProA for purifying IgG antibodies ProG for purifying IgG a
11. ication process Cycle One complete aspiration and dispense 3 1 Protocol Selection Tip Type With the PureSpeed icon visible press or W to launch PureSpeed mode The next screen is the Protocol Select screen MEL DEFAULT where protocols are defined by the type of PureSpeed tip to be used The top field allows selection by resin type IMAC in the AG example and resin bed size 20 uL in the example Under the Tip Type field is a list of up to 20 user defined saved protocols If no protocols have been saved only the DEFAULT indicator is displayed To change tip type press or b to activate the tip type field Once activated the value can be changed by pressing s VW m or gt Each time the tip type is changed the Saved Protocol box is updated with the list of previously saved protocols SELECT s CANCEL When the tip type field is active pressing or the SELECT soft key selects the tip type shown and activates the Saved Protocols scrollable box Select the desired protocol name by pressing or W Press the SELECT soft key or press to load the selected protocol If DEFAULT is selected the default values for the selected tip type will be loaded Pressing CANCEL will restore the original selection and return focus back to tip type selection 3 2 Protocol Setup After selecting the correct protocol the protocol setup screen appears The PureSpeed system is designed to fun
12. ntibodies IMAC for purifying His tagged recombinant proteins These resins are offered in 2 different tip volumes 200 uL and 1000 UL each with 2 volumes of resins to accommodate a variety of sample volume needs PureSpeed tips are supplied in clamshells to protect the resins 12 tips to a clamshell PureSpeed products are supplied in Starter Kits or as individual items Cat No MT Order No Description Cat No MT Order No Description PT 2 A5 17012561 5 uL ProA resin 12 x 200 uL tips PT 10 A20 17012568 20 uL ProA resin 12 x 1000 uL tips PT 2 A20 17012562 20 uL ProA resin 12 x 200 uL tips PT 10 A80 17012569 80 uL ProA resin 12 x 1000 uL tips PT 2 G5 17012563 5 uL ProG resin 12 x 200 uL tips PT 10 G20 17012570 20 uL ProG resin 12 x 1000 uL tips PT 2 G20 17012564 20 uL ProG resin 12 x 200 uL tips PT 10 G80 17012571 80 uL ProG resin 12 x 1000 uL tips PT 2 N5 17012566 5 uL IMAC resin 12 x 200 uL tips PT 10 N20 17012572 20 uL IMAC resin 12 x 1000 uL tips PT 2 N20 17012567 20 uL IMAC resin 12 x 200 uL tips PT 10 N80 17012573 80 uL IMAC resin 12 x 1000 uL tips PureSpeed Starter Kits contain PT ACC E4 XLS and PureSpeed tips PT S2 A20 17012577 Starter Kit E4 200XLS ProA PT S10 A20 17012579 Starter Kit E4 1OOOXLS ProA PT S2 G20 17012578 Starter Kit E4 200XLS ProG PT S10 G20 17012580 Starter Kit E4 1000XLS ProG PT S2 N20 17012576 Starter Kit E4 200XLS IMAC PT SIO N20 17012581 Starter Kit E4 1000XLS IMAC PureSpeed Accessories
13. r 12 channel pipettes use the 12 channel side This preparation step should be checked before starting the purification process make sure reagents are in each of the pre marked rows for equilibration through to elution Finally add the sample to the row marked Capture Remove the buffer filled deep well plate and place the platform over the ColorTrak Guide note the cut outs in the guide should align with the platform s rubber feet Place the deep well plate on the platform and slide into position along the rails of the platform The appropriate rows containing buffer should be aligned over the ColorTrak Guide With the sample and buffers now prepared remove the appropriate clamshell with PureSpeed Tips containing the specific resin type and volume from the refrigerator ready for loading onto the E4 XLS pipette that has been prepared in step 4 With the E4 XLS program for the appropriate PureSpeed function ready press RUN on the E4 XLS softkey The E4 XLS will instruct the user to install the appropriate tips Depending on the number of samples to be purified prepare the appropriate pipette holding system For single channel pipette use the Single Pipette Adapter It should be placed over the well containing the Equilibration buffer For multi channel use place two adapters green adapters for 200 uL tips and blue adapters for 1000 uL tips over the next to outer tips to secure the pipette tip assembly in place in each of the rows Pus
14. the active protocol by dispensing the liquid in the tip A second prompt alerts to remove the tip which can then be reused The E4 XLS then completes the reset and performs a blowout operation 200 0yL Volume MEDIUM Speed 1 of 1 Aliquots U 1 of 1 ran ee Cycles 39 6uL Cycles JEF AULT 02 13 DEF AULT PECE ee LIL E Li Fae a PECE Ma LPL i RESET RESUME PureSpeed 4 20PM Gl MAL Spl A Reset Confirm Press RESET to expel liquid from the tip and end process Press CANCEL to resume current process Hint In RUN mode the time value that appears in the lower right is the active number of minutes and seconds until the next intervention prompt When the Equilibration step is complete the unit prompts the user to move the tip to the Capture solution and then to the Wash and Elution solutions The prompts for each step are color coded to match the rows in the ColorTrak Guides The intervention prompts will generate a warning beep and screen flash along with the pop up message The message will re activate lighting the screen and generating additional beeps at 60 second intervals until the prompt is answered PureSpeed 422 6 PureSpeed 42276 IM AC Spb IMAC SuL 200 0uL 200 0uL Volume Volume CAPTURE Start WASH Start Place tip s in Capture Place tip s in Wash solution solution Cycles Next Step Press CONT When Cycles PureSpeed 4 37PM Gl I
15. uffer Saline solution 140 mM NaCl 1X Elution buffer 200 mM sodium dihydrogen phosphate 140 mM sodium chloride pH 2 5 1X Neutralization buffer 1 M Tris buffer pH 9 0 Note With PureSpeed ProA and ProG we recommend using neutralization buffer after elution of the IgG Affer the elution step add neutralization buffer at 25 v v of elution buffer volume eg if protein eluted in 20 uL elution volume add 5 uL neutralization buffer Prepare a 5X buffer 50 mM sodium dihydrogen phosphate 1 5 M sodium chloride 25 mM imidazole pH 7 4 Then dilute to 1X for the Equilibration and Wash 1 buffers Equilibration buffer See above Wash 1 buffer See above 5X Wash 2 buffer 50 mM sodium dihydrogen phosphate 1 5 M sodium chloride 0 1M imidazole pH 7 4 Note Dilute the wash 2 buffer 5 fold to obtain 20 mM imidazole in the wash 1X Elution buffer 200 mM sodium dihydrogen phosphate 250 mM imidazole pH 7 4 Note For high protein yield use up to 0 5 M imidazole and 0 5 M NaCl However this may lead to poor protein purity 2 2 Purification Set up To remove sample particles spin sample and remove supernatant for processing in PureSpeed tips As a guide set up samples and buffers in a deepwell plate as follows Row Equilibration buffer Row 2 Samples including negative and positive controls if any Row 3 Wash 1 Row 4 Wash 2 Row 5 Elution buffer Figure 2 Deepwell Plate Set up for antibody purification using ProA
16. wn on the diagram inside the battery compartment Cord Orientation When the micro SD card is properly aligned in the slot push the card until it locks in place you may need fo use a flat bladed screwdriver to gently but firmly push it in Replace the battery The E4 XLS will detect the micro SD card and activate the PureSpeed mode Replace the battery cover Figure 4A Micro SD slot Figure 4B Micro SD card in slot 3 0 Using E4 XLS in PureSpeed Mode The PureSpeed mode is activated in the E4 XLS pipette when a PureSpeed enabled SD card is inserted into the card slot and E4 XLS is restarted The PureSpeed icon will appear in the carousel This manual assumes that you have a basic understanding of protein purification methods and that you are familiar with the E4 XLS electronic pipette see also E4 XLS manual Part No 9920 396 When describing E4 XLS the movements and actions of the joystick control are represented herein by the following icons PURESPEED press joystick move joystick up W move joystick down 4 move joystick left gt move joystick right The terms Speed Aliquot Step and Cycle have special meanings in this manual Speed LOW MEDIUM and HIGH result in the following flow rates Saved Protocols Se 200 uL 50 uL min 250 uL min 500 uL min 1000 uL 250 uL min 500 uL min 1000 uL min Aliquot Number of discrete liquid samples per purification step Wash Step Number of discrete wash steps in the purif
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