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GPRTM-800 - Protea Biosciences

1. US Unis St Units Dimensions I3inx9 inx 14 in 33 cm x 23 cm x 36 cm 20 Ib 9 0 kg Dimensions and weight are approximate 1 3 2 Environmental Ranges 10 to 50 C 50 to 122 F 10 to 50 C 50 to 122 F Relative Humidity 35 to 8576 non condensing 35 to 8576 non condensing Exposing the system to direct sunlight is not recommended 1 3 8 Consumables Storage Specifications CAUTION Consumable materials to be used with the GPR 800 must be stored in accordance with the requirements set forth on their labels and packaging 1 3 4 Electrical Specifications Input Voltage 100 240 V 0 304 maximum 50 60 Hz Fast acting 250 V 5x20 mm Mains Fusing 1A Bell Fuse P N 5SF 1 R qty 2 High Voltage DC Supply Fusing 200 mA Bell Fuse P N 5SF 200 R qty 2 A CAUTION Fuses are not serviceable by the end user Please call Protea Technical Support 9 e 877 776 8321 1 3 5 Chemical Compatibility Exterior surfaces of the device enclosure are ABS acetal aluminum EPDM neoprene polycarbonate and polyester Care should be taken to ensure chemicals used in operating cleaning and maintaining the system do not remain in contact with materials with which they are incompatible as system damage may occur 1 3 6 Flammable Liquids Flammable liquids may be used during operation of the GPR 800 Follow your institutional requirements for the handling and disposal of the flammable or otherwise hazardou
2. NOTE The use of acetonitrile is not recommended for protein fixation as it is very effective at precipitating proteins gt 50 kDa at concentrations gt 50 Gel Destaining e Zinc and SYPRO Ruby stained protein gels do not require destaining and can be used directly for gel protein recovery e Coomassie stained protein gels that are used for gel protein recovery will recover the Coomassie blue dye along with the protein This dye will produce an intense chromatographic peak and it may also lead to the production of Coomassie adducts in the mass spec NOTE At this time there is not a recommended destain protocol for use with Coomassie stained protein gels NOTE The use of acetonitrile based destain protocols can lead to the precipitation of the proteins in the gel matrix and or non reproducible gel protein recovery PROTEABIOSCIENCES www proteabio com ATO Protocols Zinc stain Remove the gel from the cassette once gel electrophoresis is completed and place in an appropriate size staining container e Add 50 mL of 0 2 M imidazole solution NOTE Use sufficient volume to immerse gel e Equilibrate on shaker for 10 minutes e Discard imidazole solution e Add 50 mL 0 2 M zinc sulfate solution and shake tray rapidly for 10 60 seconds The gel becomes opaque white and the protein spots remain transparent NOTE The color transition time is sample dependent Larger gel protein loads can develop the gel spots in as little as 1
3. spectrometry then the Elution Solution for ESI should be used formic acid based NOTE Monitor the level of liquid in the waste centrifuge tube being careful the flow through volume does not cover the bottom point of the SpinTip Dry down sample in a lyophilizer or SoeedVac NOTE for proteins 75 kDa a C SpinTip should be used www proteabio com e 34 3 Solution phase digestion e Dissolve protein pellet in 100 uL of Digestion Buffer 50 mM ammonium bicarbonate in 20 acetonitrile e Vortex or sonicate the tube to aid in solubilization Using a centrifuge spin the tube at 5 000g for 1 minute to make sure that the sample solution is collected in the bottom of the tube e Reduction DTT Add 5 uL of 250 mM DIT in 50 MM ammonium bicarbonate in 20 acetonitrile to the sample solution This dilution will give a final concentration of 10 mM DTT NOTE Add 0 386 g DTT to 10 mL of 50 MM ammonium bicarbonate in 2076 acetonitrile to make the 250 mM solution e Incubate at 40 C for 1 hour on heat plate e Alkylation lodoacetamide e Add Sul of 625 mM iodoacetamide in 50 mM ammonium bicarbonate in 20 acetonitrile to the sample solution This dilution results in a final concentration of 25 mM iodoacetamide NOTE Add 1 156 g iodoacetamide to 10 mL of 50 mM ammonium bicarbonate in 2076 acetonitrile to make the 625 mM solution Incubate at room temperature in the dark for 1 hour Add 2 0 uL of trypsin solution 1 0 ug
4. NOTE Electroelution can be stopped before the electroelution timer reaches 00 00 by pressing the PAUSE button pressing the STOP SET button or by opening the Safety Cover Refer to Section 5 3 4 for additional details 6 When the electroelution time has expired the GPR 800 automatically turns off the electroelution voltage 7 Sample Collection Pipette out the sample that collects in Reservoir C into a clean 0 5 mL vial 5 3 8 Stopping Electroelution Electroelution will stop when the timer run time reaches 00 00 Electroelution can be stopped before the run time reaches 00 00 by pressing the STOP SET button at any time e fitis necessary to shut off the electroelution voltage during the gel protein recovery process either push the PAUSE button or open the blue safety lid to automatically place the GPR 800 in a pause mode AT 0 L01010 0K00 aa C0 PROTEABIOSCIENCES e When the pause feature is engaged the LED s will blink red If the PAUSE button is depressed to pause the instrument then press START to re start the sample processing at the time point where the pause was initiated e If the blue safety lid is opened to pause the instrument the voltage will be restored when the lid is closed 5 3 4 LC MS Top Down Analysis 1 Sample collection and detergent degradation Collect sample from protein GPRchip into microcentrifuge tube using a micropipette Add Surfactant Degradation Reagent 10X to the sample to achieve a 10
5. fold dilution e g add 15 uL of Surfactant Degradation Reagent to a 150 uL GPR sample and incubate at room temperature for 15 30 minutes 15 minutes for MALDI analysis 30 minutes for ESI analysis 2 Desalt protein sample using a C SpinTip e Ensure that the packing material is at the bottom of the tip by gently tapping the tip to displace any packing material sticking to the top cap e Place a centrifuge adaptor onto a 2 mL centrifuge tube Remove the cap from the SpinTip and place into the centrifuge adaptor e Wash the SpinTip to wet the packing material by adding 50 uL of Equilibration Solution to the top of the SpinTip using a micropipette Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip wash e Rinse the SpinTip by adding 50 uL of Sample Reconstitution and Rinse Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip rinse Load 10 to 200 uL of the GPR Sample Solution by adding it to the top of the SpinTip and centrifuging the system at 4000 x g for 3 min Additional sample volumes can be added in 200 uL aliquot cycles e Wash the sample to elute salts and other non retained components by adding 100 uL of the GPR Rinse Buffer for ESI to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip sample wash Transfer the SpinTip and adapter to a new clean centrifuge tube to collect the sample during elution 31 877 776 8321 Elute
6. overnight because it will de stain your protein bands www proteabio com m 48 Reagent recipes e 1X Coomassie R 250 400 mL ddH O 500 mL methanol 100 mL acetic acid 110 mL of 10X Coomassie R 250 stock solution Fixation solution e 400 mL ddH O e 500 mL methanol e 100 mL acetic acid Coomassie G 250 stain SB G250 Remove the gel from the cassette once gel electrophoresis is completed and place in an appropriate size staining container containing 100 ml ddH O Gently shake for 5 minutes Replace with fresh ddH O and wash an additional 2 times for 5 minutes each Remove the ddH O from the gel and discard Add 50 mL Coomassie G 250 to the gel and continue shaking for an additional 45 60 minutes Remove the Coomassie solution from the gel and dispose in an appropriate waste container Add 50 mL ddH O to the gel and continue shaking for additional 30 minutes Gently shake until the required intensity of protein staining is accomplished and background staining is reduced May be left overnight A Gel sample handling and storage e Gel protein recovery experiments produce the highest recoveries for freshly run gels When possible it is always recommended that the SDS PAGE gel separation and gel protein recovery be performed on the same day e Gel storage It is recommend to store whole gels at 4 C in a minimal amount to water to reduce diffusional protein losses NOTE Do not freeze the gels as the formation of ice cry
7. page 11 prior to setting up or operating the GPR 800 4 2 1 Unpacking and Setup of the GPR 800 Step 1 Unpack System Unpack the system from its shipping container and place the GPR 800 on a sturdy level flat surface Ensure that the AC power disconnect is accessible when the system is positioned for use Nome Do not position the system in a location where liquids can spill onto electrical components NOTE Be sure to place the GPR 800 unit on a flat level stable surface that can easily support 30 pounds Adjust the leveling mounts GPR 800 feet so that the instrument is secure and level in position VANAN 0 L01010 0K00 00a PROTEABIOSCIENCES Step 2 Install AC Power Cord Ensure that the main power switch on the rear of the unit is in the OFF position Unpack the AC power cord from the shipping container and connect the GPR 800 via the power entry module to an electrical power source with earth ground using the supplied three prong grounded power cord Ensure that the supply outlet is grounded and that the voltage of your main power supply matches the voltage of the unit CAUTION Make sure the outlet is grounded and that the voltage of your main power supply matches the voltage of the unit Step 3 Install GPR Works Software If using the GPR Works software install the software on an appropriate computer Install the software by placing the installation CD into the computer s CD ROM drive Software installation should sta
8. relays as this may damage the component surfaces Use your finger for all keypad and timer relay selections 23 877 776 8321 Figure 5 1 Shows the GPR 800 user interface 5 2 1 Keypads Each keypad has three control buttons START PAUSE and STOP SET Each set of buttons controls 1 bank CH 1 4 or CH 5 8 of 4 electrodes e START button If no safety interlocks are activated i e the Safety Lid is closed and the Chip Holder is in place pressing the START button begins the timer countdown and applies a voltage to the corresponding electroelution channels Additionally the corresponding Channel LEDs will illuminate indicating that current is lowing through the channel If safety interlocks are activated pressing the START button transfers the corresponding set of electrodes into a PAUSED state All channels are identified as paused by blinking Channel LEDs When the safety interlocks are disengaged e g the safety lid is closed the system begins the timer countdown and applies a voltage to the corresponding electroelution channels e PAUSE button If the system is delivering electroelution voltage as indicated by illuminated Channel LEDs pressing the PAUSE button will pause the timer countdown and remove the applied voltage from the corresponding electroelution channels Channels which are in a paused state are identified by blinking Channel LEDs www proteabio com 24 PROTEABIOSCIENCES If no safety inter
9. sample e Wash and equilibrate a C LithTip using the Equilibration Buffer Add 25 UL of buffer to a clean vial and then perform 10 cycles of aspiration and expulsion of the buffer using the micropipette set at 10 UL e Rinse the C LithTip using Reconstitution and Rinse Solution Add 25 uL of buffer to a clean vial and then perform 10 cycles of aspiration and expulsion of the buffer using the micropipette set at 10 uL e Load the sample into the C8 LithTip by aspirating and expelling the sample 5 10 times within the sample vial Wash the sample on the C LithTip with GPR Rinse Buffer for MALDI e Add 25 uL of buffer to a clean vial and then perform 10 cycles of aspiration of the buffer using the micropipette set at 10 uL e lute the sample with 2 uL of Elution Solution by spotting the 2 uL directly onto the MALDI target pre spotted with 0 5 uL MALDI matrix e g CHCA and allowing to air dry Load the MALDI target into the mass spectrometer and begin mass spec analysis 33 877 776 8321 B 5 3 6 Bottom Up Analysis by ESI Post GPR Digestion 1 Sample collection and detergent degradation Collect sample from protein GPRchip into microcentrifuge tube using a micropipette Add Surfactant Degradation Reagent 10X to the sample to achieve a 10 fold dilution e g add 15 uL of Surfactant Degradation Reagent to a 150 uL GPR sample and incubate at room temperature for 15 30 minutes 15 minutes for MALDI analysis 30 minutes for
10. to the timer relay interface 25 877 75 892 B 5 2 2 Timer Relays The GPR 800 has two timer relays see Figure 5 1 Each relay is dedicated to the control of one bank CH 1 4 or CH 5 8 of 4 electrodes Each up down button of the timer relay see Figure 5 2 controls one digit of the process time mmiss Pressing the up or down arrows changes the corresponding value of the set time When the appropriate set time is displayed in the timer pressing the SET key on the keypad sets the process time to equal the set time Figure 5 2 Timer Relay NOTE The timer relay set time is displayed in orange below the process time The timer relay process time is displayed in red above the set time The process time controls the electroelution time of the GPR 800 NOTE The yellow buttons to the left of the timer relay should not be pressed at any time They are not part of the GPR 800 user interface PROTEABIOSCIENCES www proteabio com 26 5 3 GEL PROTEIN RECOVERY The gel protein recovery process steps are described below 5 3 Priming the Protein GPRchip for Gel Protein Recovery 1 Place the chip against a dark background and pipette 200 uL of GPR Electroelution Buffer GPR 020 into Reservoirs A and B for every channel that will be run Watch as the buffer completely fills the micro channel and ensure that there are no bubbles present The hydrodynamic flow will begin as soon as the Reservoirs are filled NOTE Protea recommend
11. uL stock solution to the reduced and alkylated sample solution Incubate overnight or for 12 hours at 37 C on heat plate Stop the digestion by adding 10 uL of glacial acetic acid The digestion solution can be transferred to a labeled 1 5 mL centrifuge tube and dried down in a lyophilizer or SoeedVac to produce the crystallized peptides Freeze the peptides at 80 C until ready for use e For MALDI analysis the protein digest can be spotted directly on the plate or with a C LithTip using CHCA matrix e For LC MS or LC MALDI analysis the protein digest can be reconstituted in LC aqueous buffer and then loaded onto the chromatographic column for separation and analysis 35 e 877 776 8321 5 5 3 7 Bottom Up Analysis by MALDI Post GPR Digestion 1 Sample collection and detergent degradation Collect sample from protein GPRchip into microcentrifuge tube using a micropipette Add Surfactant Degradation Reagent 10X to the sample to achieve a 10 fold dilution e g add 15 uL of Surfactant Degradation Reagent to a 150 uL GPR sample and incubate at room temperature for 15 30 minutes 15 minutes for MALDI analysis 30 minutes for ESI analysis 2 Desalt protein sample using a C SpinTip PROTEABIOSCIENCES Ensure that the packing material is at the bottom of the tip by gently tapping the tip to displace any packing material sticking to the top cap Place a centrifuge adaptor onto a 2 mL centrifuge tube Remove the cap f
12. 0 seconds Smaller gel protein loads may take 60 seconds or longer NOTE If the gel is incubated for too long in the zinc sulfate solution it will over develop and the protein spots will develop the same opaque white color as the gel background NOTE Place the receptacle over a dark background to observe the staining reaction and stop it at the correct time point before over development e Rinse the gel in 100 mL ddH O for 3 minutes e Replace with fresh ddH O for storage Sypro Ruby fluorescent stain Remove the gel from the cassette once gel electrophoresis is completed and place in an appropriate size staining container Wash gel with deionized water to remove residual TGS running buffer Add 50 mL deionized water and shake gently for 30 seconds Discard rinse water Add 50 mL of SYPRO Ruby protein gel stain to cover the gel NOTE Use sufficient SYPRO Ruby protein gel stain to sufficiently cover the gel In general use ten times the volume of the gel Using too little stain will reduce sensitivity 47 877 776 832 Stain the gel with continuous gentle agitation for at least 3 hours for maximal sensitivity NOTE Specific staining can be seen within 30 90 minutes NOTE For convenience gels may be left in the stain overnight 16 18 hours without overstaining Rinse the gel in 50 100 mL Rinse Solution 10 methanol 7 acetic acid for 30 60 minutes NOTE This rinse step decreases background fluorescence NOTE Alternati
13. 4 Set the timers to the required electroelution time Each timer controls a bank of four electrodes 1 4 and 5 8 See Table 5 1 for eletroelution time guidelines NOTE The left timer relay controls and displays the run time for channels 1 4 The right timer relay controls and displays the run time for channels 5 8 NOTE The bottom number on the timer relay is the target time the top number is the run time that will be applied to each bank of channels When the target time is changed the STOP SET button must be pressed to apply this new value as the run time 29 e 877 776 8321 5 Table 5 1 Electroelution Time Guidelines for the GPR 800 Time minutes Size of Protein Number of Gel Plugs per channel 2 mm circular 1 Gel Plug 2 Gel Plugs I25bkba 17 1 2 25 75 kDa 30 40 75 kDa and up 20 30 40 60 5 Press the START button s to apply the electric field and initiate the gel protein recovery process When operating the system will apply a constant voltage of 500 V across Reservoirs A and B of each channel The LED indicators for each channel will change from unlit OFF to green when both the high voltage is ON and the current through the channel is above 50 pA NOTE If the electroelution voltage is ON the eight channel LEDS will be illuminated The channel LED will be red if the channel current is 50 pA to indicate the electrical current error The channel LED will be green if the channel current is gt 50 pA
14. ESI analysis 2 Desalt protein sample using a C SpinTip PROTEABIOSCIENCES Ensure that the packing material is at the bottom of the tip by gently tapping the tip to displace any packing material sticking to the top cap Place a centrifuge adaptor onto a 2 mL centrifuge tube Remove the cap from the SpinTip and place into the centrifuge adaptor Wash the SpinTip to wet the packing material by adding 50 uL of Equilibration Solution to the top of the SpinTip using a micropipette Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip wash Rinse the SpinTip by adding 50 uL of Sample Reconstitution and Rinse Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip rinse Load 10 to 200 uL of the GPR Sample Solution by adding it to the top of the SpinTip and centrifuging the system at 4000 x g for 3 min Additional sample volumes can be added in 200 uL aliquot cycles Wash the sample to elute salts and other non retained components by adding 100 uL of the GPR Rinse Buffer for ESI to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip sample wash Transfer the SpinTip and adapter to a new clean centrifuge tube to collect the sample during elution Elute the sample by adding 100 uL of Elution Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min NOTE If the sample is targeted for LC MS analysis by electrospray ionization ESI mass
15. Electrical Specifications 9 1 3 5 Chemical Compatibility 10 1 3 6 Flammable Liquids 10 2 Safety 11 2 1 Safety Stops 13 2 1 1 Unit Safety Cover 13 2 1 2 Chip Holder 14 2 2 Electrical Safety 15 3 Component Locations 16 3 External and Internal Parts 16 3 2 System Accessories 19 4 Installation and Preparation 20 4 Accessory Equipment 20 4 2 Initial Setup 20 4 2 1 Unpacking and Setup of the GPR 800 20 PROTEABIOSCIENCES www proteabio com 4 5 Instructions for Use 23 5 General Operation 23 5 2 Operating Controls Keypads and Timer Relays 23 5 2 4 Keypads 24 5 2 2 Timer Relays 26 5 3 Gel Protein Recovery 27 5 3 1 Priming the Protein GPRchip for Gel Protein Recovery 27 5 39 2 Sample Extraction and Collection 28 5 3 3 Stopping Electroelution 30 5 3 4 GPR 800 LC MS Top Down Kit 31 5 3 5 GPR 800 MALDI Intact Mass Measurement Prep Kit 32 5 3 6 GPR 800 Bottom Up Kit for ESI Post GPR Digestion 34 5 3 7 GPR 800 Bottom Up Kit for MALDI Post GPR Digestion 36 5 4 Power Off 38 6 Cleaning and Maintenance 39 6 Cleaning 39 6 2 Maintenance 39 7 Troubleshooting 40 8 GPR Consumables 42 9 Gel Guidelines for Optimal GPR Performance Appendix 44 5 e 877 776 8321 Tables Table 5 1 Electroelution Time Guidelines for the GPR 800 30 Table 7 1 Troubleshooting Guide 36 Table 7 2 Error Message Guide 37 www proteabio com Mo Figures Figure 2 1 GPR 800 with Safety Cover Closed 13 Figure 2 2 GPR 800 with Safet
16. Electroelution voltages will not be applied to reservoir samples if the safety cover is not closed If the channel LEDs are blinking the safety cover may be open 13 877 776 8321 2 2 1 2 Chip Holder The chip holder protects the user from touching the high voltage electrodes inside the GPR 800 when it is in place and the safety cover is closed The system is designed to automatically remove voltage from alll electrodes when the chip holder is removed from or improperly installed in the system The chip holder contains a magnetic safety lock that must be engaged by properly placing the chip holder into position for the GPR 800 to operate under high voltage Figure 2 3 GPR 800 with Chip Holder installed Figure 2 4 GPR 800 with Chip Holder removed WARNING Do not attempt to run the system without the chip holder in place as doing so may cause physical injury NOTE Electroelution voltages will not be applied to reservoir samples if the chip holder is not installed on the chip platform If the channel LEDs are blinking the chip holder may not be installed PROTEABIOSCIENCES www proteabio com m 14 2 2 ELECTRICAL SAFETY A WARNING Connect the system to an electrical power source with earth ground using the three prong grounded power cord Ensure that the supply outlet is grounded and that the voltage of your main power supply matches the voltage of the unit WARNING Turn off electrical power to the system and unplug the powe
17. PROTEABIOSCIENCES Gl i 800 ADVANCED PROTEIN RECOVERY FROM GEL SAMPLES E USERS MANUAL Copyright This manual is copyrighted with all rights reserved No portion of this manual may be copied or reproduced by any means without the prior written consent of Protea Biosciences Inc Protea While every precaution has been taken in the preparation of this document Protea assumes no liability to any party for any loss or damage caused by errors or omissions or by statements resulting from negligence accident or any other cause Protea further assumes no liability arising out of the application or use of any product or system described herein nor any liability for incidental or consequential damages arising from the use of this document Trademarks The Protea Gel Protein Recovery System hereafter GPR 800 is a trademark of Protea Biosciences Inc Protea reserves the right to make changes without further notice to any product or system described herein to improve reliability function or design 2008 Protea Biosciences Inc All Rights Reserved Revision 1 0 11 2008 Protea Biosciences Inc 955 Hartman Run Rd Morgantown WV 26507 Main Switchboard 877 776 8321 Fax 304 292 7101 Safety Warning and Limitation of Liability Use of the GPR 800 involves the use of and potential exposure to hazardous materials and therefore the system is intended for use only by professionals with adequate training and experie
18. SoeedVac to produce the crystallized peptides Freeze the peptides at 80 C until ready for use e For MALDI analysis the protein digest can be spotted directly on the plate or with a C LithTip using CHCA matrix e For LC MS or LC MALDI analysis the protein digest can be reconstituted in LC aqueous buffer and then loaded onto the chromatographic column for separation and analysis 37 e 877 776 8321 B 5 4 POWER OFF At any time the user may remove power from the GPR 800 by toggling the main power switch on the rear of the unit NOTE Toggling the main power switch on the rear of the unit removes all power from the GPR 800 NOTE The timer relay set time displayed in orange below the process time at the time the unit is powered off will become the process time when the unit is powered on See Section 5 2 2 for further descriptions of the timer relay set and process times www proteabio com 38 6 Cleaning and Maintenance 6 1 CLEANING After each use of the GPR 800 clean the electrodes electrode hinge vial lift assembly chip holder vial holder and exterior surfaces of the GPR 800 Surfaces should be cleaned with a lint free wipe dampened with isopropyl alcohol Use care not to wet any internal electronic components or exposed wiring Also take care not to bend the electrodes WARNING Turn off electrical power to the system and unplug the power supply cord before cleaning the GPR 800 A CAUTION The system el
19. al use are described in Section 8 Figure 3 4 Shows the GPR 800 accessories for loading the consumables and for powering and communicating with the GPR 800 1 Chip Holder Holds the Protein GPRchip I2 Vial Rack Holds the sample and waste collection vials USB Cable Connects the GPR 800 to a computer computer not provided nmi Power Cable Connects the GPR 800 to an appropriate AC power source 19 e 877 776 8321 4 Installation and Preparation 4 1 ACCESSORY EQUIPMENT The following equipment is not supplied with the GPR 800 but is recommended for use with the system e Computer A computer running the GPR Works software shows the current for each channel in real time This data can be logged and saved for record keeping and data evaluation purposes The computer software feature is not required for operation of the GPR 800 An appropriate computer will meet the following requirements at a minimum e Windows 2000 XP or later version 64 MB RAM for Pentium 200MHz processor or equivalent 256 MB RAM for Pentium Ill Celeron 600 MHz processor or equivalent e 800x600 pixels for Pentium 200 MHz processor or equivalent 1024x768 pixels for Pentium Ill Celeron 600 MHz processor or equivalent and USB interfaceport 4 2 INITIAL SETUP This chapter provides information on installing and preparing the GPR 800 for initial use Follow these steps prior to using the GPR 800 Nie Read the Safety information starting on
20. dation Reagent 25 mL and C GPR SpinTips Sample Prep Kit for ESI 24 pack Catalog Number Size Price GPR 050 ea 145 GPR MALDI Intact Mass Prep Kit Contains Surfactant Degradation Reagent 25 mL C GPR SpinTip Sample Prep Kit for MALDI 24 pack C LithTip Sample Prep Kit 24 pack and Ultrapure CHCA MALDI Matrix 5 x 10mg vials Catalog Number Size Price GPR 055 ea 275 GPR LC MS Bottom Up Prep Kit Contains Surfactant Degradation Reagent 25 mL and C GPR SpinTips Sample Prep Kit for ESI and Trypsin Digestion Kit Catalog Number Size Price GPR 060 ea 245 GPR MALDI Bottom Up Prep Kit Contains Surfactant Degradation Reagent 25 mL C GPR SpinTip Kit for MALDI 24 pack C LithTip Sample Prep Kit 24 pack Ultrapure CHCA MALDI Matrix 5 x 10mg vials and Trypsin Digestion Kit Catalog Number Size Price GPR 065 ea 375 GPR 800 Reagent Bundle Protein Recovery Contains Protein GPRchips 10 and GPR Electroelution buffer 2 x 25 mL Catalog Number Size Price GPR 070 ea 550 GPR 800 Accessory Kit Contains spot cutter holder metal spot cutter cores with 0 rings 50 and forceps 1 pair Catalog Number Size Price GPR 075 ea 145 www proteabio com M 42 Protein GPRchip Contains 8 channels for protein electroelution from polyacrylamide gel samples PMMA pack of 10 Catalog Number Size Price GPR 200 10 10 pk 450 Protein GPRchip Contains 8 channels for protein electroelution from polyacrylamide g
21. ectrodes are delicate and sharp Use care when working around the electrodes 6 2 MAINTENANCE No aspect of the GPR 800 is serviceable by the end user Only a Protea trained service technician should perform maintenance on the GPR 800 CAUTION Users should not attempt to repair the GPR 800 The GPR 800 is designed to be A serviceable only by Protea trained service technicians 877 776 8321 39 e 877 776 8321 Troubleshooting Table 7 1 Troubleshooting Guide Problem Cause Resolution System is paused Press start to begin the run Blue safety lid is open Lower lid run will start automatically Blinking red LEDs Chip holder not properly Make sure the chip holder is in it proper placed place Insufficient current is flowing Aspirate the buffer from the collection through that channel due reservoir to remove the bubble Add to a bubble in the the aspirated buffer back to the micro channel electrode reservoirs One or more channels shows red LED at the start Insufficient volume in the of the run buffer reservoir Check to ensure buffer was placed in all 4 or 8 channels Top up buffer in buffer reservoirs In most cases the bubble will move One or more channels A bubble has formed or through the channel and the current shows a red LED during the entered the micro channel will return to normal If the LED is red run during the run for a prolonged period of time several minutes re run that channel One or more channels LED
22. el samples PMMA pack of 25 Catalog Number Size Price GPR 200 25 25 pk 1000 GPR Electroelution Buffer Catalog Number Size Price GPR 020 25mL 25mL 95 GPR 020 4x25mL 4X25 mL 295 Surfactant Degradation Reagent for MALDI 10 mL Catalog Number Size Price GPR 025 10mL lOmL 45 Surfactant Degradation Reagent for ESI 10 mL Catalog Number Size Price GPR 026 10mL lOmL 45 Spot Cutter Holder Catalog Number Size Price GPR 030 ea 50 Metal spot cutter cores with o rings 50 pk Catalog Number Size Price GPR 035 50 pk 100 8 GPR 800 Accessories Forceps 1 pair Catalog Number Size Price GPR 040 ea 12 LithTins Sample Prep Kit 1 10 pL 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution Equilibration Solution and Elution Solution Catalog Number Size Price SP 420 24 24 tip kit 95 LithTins Sample Prep Kit 1 10 pL 96 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution Equilibration Solution and Elution Solution Catalog Number Size Price SP 420 96 96 tip kit 345 C GPR SpinTips Sample Prep Kit for ESI 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for ESI Equilibration Solution and Elution Solution Catalog Number Size Price SP 161 24 24 tip kit 125 C GPR SpinTips Sample Prep Kit for ESI 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for ESI Equil
23. eraction The operator begins the process by excising poylacrylamide gel plugs and placing them into the designated receptacles The GPR 800 is primed by the addition of GPR Electroelution Buffer into the electrode reservoirs After completing the system setup the operator starts the sample recovery process using the front panel interface on the GPR 800 After electroelution the recovered protein samples are transferred to vials for subsequent desalting and detergent degradation using reversed phase functionalized pipette tips e g LithTips For instructions on general use of the GPR 800 see the General Operation section starting on page 23 of this manual 1 2 ABOUT THE USER S MANUAL This manual contains information on the features and functions of the GPR 800 It provides instructions on operating the unit Read the Safety Information starting on page 11 prior to setting up or operating the GPR 800 as this section contains information necessary for the safe operation of the unit Important information contained in this manual is marked as follows A WARNING Impending Danger May cause death or physical injury CAUTION Dangerous Situation May cause equipment material damage or data loss NOTE Helpful additional information and tips for use patent pending www proteabio com 8 PROTEABIOSCIENCES 1 3 TECHNICAL SPECIFICATIONS The GPR 800 has the specifications listed below 1 3 1 Physical Dimensions Physical
24. ibration Solution and Elution Solution Catalog Number Size Price SP 161 96 96 tip kit 375 GPR SpinTips Sample Prep Kit for MALDI 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for MALDI Equilibration Solution and Elution Solution Catalog Number Size Price SP 171 24 24 tip kit 125 GPR SpinTips Sample Prep Kit for MALDI 96 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for MALDI Equilibration Solution and Elution Solution Catalog Number Size Price SP 171 96 96 tip kit 375 LithTips Sample Prep Kit 1 10 pL 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution Equilibration Solution and Elution Solution Catalog Number Size Price SP 410 24 24 tip kit 95 LithTips Sample Prep Kit 1 10 pil 96 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution Equilibration Solution and Elution Solution Catalog Number Size Price SP 410 96 96 tip kit 345 C GPR SpinTins Sample Prep Kit for ESI 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for ESI Equilibration Solution and Elution Solution Catalog Number Size Price SP 162 24 24 tip kit 125 C GPR SpinTips Sample Prep Kit for ESI 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for ESI Equilibration Solution and Elut
25. ion Solution Catalog Number Size Price SP 162 96 96 tip kit 375 GPR SpinTips Sample Prep Kit for MALDI 24 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for MALDI Equilibration Solution and Elution Solution Catalog Number Size Price SP 172 24 24 tip kit 125 GPR SpinTips Sample Prep Kit for MALDI 96 Tip Kit Kit contains C LithTips Sample Reconstitution and Rinse Solution GPR Rinse Buffer for MALDI Equilibration Solution and Elution Solution Catalog Number Size Price SP 172 96 96 tip kit 375 43 877 776 832 9 Gel Guidelines for Optimal GPR Performance 9 1 SDS PAGE Gel Recommendations for GPR 800 Samples SDS PAGE gel Recommended gel vendors e Protea Biosciences ProteaGels e BioRad Mini Protean and Criterion gels e Life Technologies formerly Invitrogen Novex NUPAGE gels Supported gel characteristics Percent acrylamide e 5 10 12 and 15 polyacrylamide e lOand 12 gels work best for most proteins e 15 gels should not be used for proteins over 30 kDa Crosslinker ratio e Higher polyacrylamide monomer crosslinker ratios produce larger average pore sizes that improve gel protein recovery Recovery trends 37 5 1 gt 29 1 gt 19 1 e Non supported gel characteristics e Gradient gels gradient gels have variable pore sizes across the length of the gels This variation in pore size can have an unpredictable effect on the recovery of p
26. locks are activated i e the Safety Lid is closed and the Chip Holder is in place and the system is in a paused state pressing the PAUSE button resumes the timer countdown and resumes application of voltage to the corresponding electroelution channels Additionally the corresponding Channel LEDs will illuminate not blink indicating that current is lowing through the channel If safety interlocks are activated pressing the PAUSE button pauses the corresponding Channels When the safety interlocks are disengaged e g the safety lid is closed the user must press the START button to resume current delivery e STOP SET button If the system is delivering electroelution voltage as indicated by illuminated Channel LEDs pressing the STOP SET button will stop the timer countdown reset the timer value to the initial value and remove the applied voltage from the corresponding electroelution channels Channels which are in a stopped state are identified by unlit Channel LEDs If the system is paused as indicated by blinking Channel LEDs pressing the STOP SET button will reset the timer value to the initial values and remove the applied voltage from the corresponding electroelution channels Channels which are in a stopped state are identified by unlit Channel LEDs If the system is in a stopped state pressing the STOP SET button sets the target time on the timer relays as The relay run time See Section 5 2 2 for information related
27. nce in handling of the dangerous materials involved Protea cannot be held responsible for any injuries to persons or property or for any other damage resulting from the improper use of this equipment or its use in any manner not specifically described in this manual Furthermore Protea disclaims all responsibility and liability in connection with any accidents arising from any use of the GPR 800 in situations where inadequate safety procedures are in place or where such procedures have not been followed e 877 776 8321 Warranty WARRANTY Products are Warranted to be free of defects in materials and workmanship and to perform to the published specifications for a period of one year from the date of the product s shipment see current Protea Biosciences Inc catalog for additional warranty language and Terms of Sale www proteabio com 2 Preface This user s manual is written for use with the Protea Biosciences Inc Protea Gel Protein Recovery System 800 hereafter GPR 800 This device s intended use is to serve as a platform for developing The gel protein recovery process The audience of this device is the intended eventual end users i e practitioners of the device 3 e 877 776 8321 Contents 1 Introduction 8 1 1 Introducing the GPR 800 8 1 2 About the User s Manual 8 1 3 Technical Specifications 9 1 3 1 Physical Dimensions 9 1 3 2 Environmental Ranges 9 1 3 3 Consumables Storage Specifications 9 1 3 4
28. nction gt WARNING Use caution when working near the operating system as the system contains high voltages A WARNING Remove power from the system immediately upon fluid spills on or near the system Do not operate the system after fluid spills have occurred WARNING No modification of this equipment is permitted 11 e 877 776 8321 WARNING Do not operate the system if damage or abnormal erratic operation has been detected CAUTION Keep open containers of fluid away from the system CAUTION Keep areas around the system clean at all times PPP A CAUTION Maintain at least 6 inches of unobstructed space around the unit to ensure proper use of the system www proteabio com A 12 2 1 SAFETY STOPS The GPR 800 employs automatic safety stops as outlined below When an automatic safety stop is required the system enters an abort mode where all voltage is removed from the electrodes or the user is prevented from applying voltage to the electrodes 2 1 1 Unit Safety Cover A safety cover is provided to protect the user from coming into contact with the high voltage elements of the GPR 800 The system is designed to automatically remove voltage from all electrodes when the cover is opened or removed Figure 2 1 GPR 800 with Safety Cover closed Figure 2 2 GPR 800 with Safety Cover open WARNING Do not attempt to run the system without the safety cover closed as doing so may cause physical injury NOTE
29. ns gt 75 kDa a C SpinTip should be used www proteabio com 36 3 Solution phase digestion e Dissolve protein pellet in 100 uL of Digestion Buffer 50 mM ammonium bicarbonate in 20 acetonitrile e Vortex or sonicate the tube to aid in solubilization Using a centrifuge spin the tube at 5 000g for 1 minute to make sure that the sample solution is collected in the bottom of the tube e Reduction DTT Add 5 uL of 250 mM DIT in 50 MM ammonium bicarbonate in 20 acetonitrile to the sample solution This dilution will give a final concentration of 10 mM DTT NOTE Add 0 386 g DTT to 10 mL of 50 MM ammonium bicarbonate in 2076 acetonitrile to make The 250 mM solution e Incubate at 40 C for 1 hour on heat plate e Alkylation lodoacetamide e Add Sul of 625 mM iodoacetamide in 50 mM ammonium bicarbonate in 20 acetonitrile to the sample solution This dilution results in a final concentration of 25 mM iodoacetamide NOTE Add 1 156 g iodoacetamide to 10 mL of 50 mM ammonium bicarbonate in 2076 acetonitrile to make the 625 mM solution Incubate at room temperature in the dark for 1 hour Add 2 0 uL of trypsin solution 1 0 ug uL stock solution to the reduced and alkylated sample solution Incubate overnight or for 12 hours at 37 C on heat plate Stop the digestion by adding 10 uL of glacial acetic acid The digestion solution can be transferred to a labeled 1 5 mL centrifuge tube and dried down in a lyophilizer or
30. of which may be recovered during gel protein recovery NOTES This recommendation is very important for investigators who opt to utilize gradient gels as The pore size of the gel varies along the length of the gel as the percent acrylamide changes 3 Staining and visualization Supported stains e Zinc e Sypro Ruby e Coomassie R 250 non colloidal G 250 colloidal Non supported stains e Silver Imperial blue e Colloidal gold Gel Fixation e Fixation of proteins after SDS PAGE separation is often performed to prevent diffusional protein movement with the gel loss of resolution and migration out of the gel protein sample loss from the gel e Fixation of the proteins is a precipitation event that is typically induced by using a fixing solution containing high percentages or organic solvent and or organic acids 45 877 776 832 y Supported fixing solutions e 10 Methanol 7 Acetic Acid SYPRO Ruby protein gel stain e 50 Methanol 10 Acetic Acid Coomassie Blue R250 stain e 0 1 Methanol 5 Phosphoric Acid Coomassie Blue C250 stain NOTE The staining methods in parentheses above are the methods that typically call for these individual fixing solution recipes Each fixing solution can be used for other staining techniques e Harsher protein fixation protocols can produce non reversible protein precipitations that lead to reduced efficiency and or inhibition of both in gel digestion and gel protein recovery
31. ows the external and internal parts located on GPR 800 6 Vial Lift Knob Raises and lowers the vial holder for positioning of sample vials Vial Lift Positions the vial holder and vials below the outlet channel bridges Safety Cover Protects user from high voltages Controls the positioning of the electrodes into either the OPEN RESTING EH Electrode Hinge or SEALED position Electrodes Provide electroelution voltage 16 electrodes supply 8 channels M Chip Holder See Section 5 12 Vial Holder See Section 5 Interfaces with the EH Catch Plate to position the EH in either the RESTING or SEALED position Interfaces with the EH Knob to position the EH in either the RESTING or EH Catcher Plate SEALED position Leveling Mounts Allow the GPR 800 to sit level on a work surface These mounts can be 9 rotated to adjust the level of the GPR 800 17 e 877 776 8321 Figure 3 3 Shows the external parts located on the rear of the GPR 800 Power Entry Module AC power entry port and hard power switch controls all power to the system Requires an input from an electrical power source with earth ground using the supplied three prong grounded power cord I2 USB Connector Communication link for the GPR 800 software PROTEABIOSCIENCES www proteabio com 18 3 2 SYSTEM ACCESSORIES These accessories organize the GPR 800 consumables and allow them to be loaded onto the unit The consumables needed for the system during norm
32. p position Figure 5 4 Ensure that the chip is pushed down to the bottom left corner in the center position of the holder Load the holder in its slot inside the instrument NOTE Protea recommends returning the buffer that has flowed into Reservoir C in equal amounts to Reservoirs A and B This will minimize the collection volume and maximize the concentration of the recovered protein If you load the buffer and the gel plugs quickly this will not be necessary 2 Lower the electrode assembly onto the chip and secure the knob in the top hole resting position of the metal closure plate Close the blue safety lid to disengage the safety interlock on the power supply NOTE The platinum electrodes are fragile and can be bent by rough handling When working with the electrode assembly or electrodes please handle gently NOTE The top latch position is the resting i e unsealed position The lower latch position seals the reservoirs and should not be used with the Protein GPRchip www proteabio com MPs PROTEABIOSCIENCES L iod 5 Gs Porch nd mi ee Protein GPRchip in center chip position alu E dcos cum Ro aa Li GPR 800 Chip Holder 1 iii 1 f fi Figure 5 4 Protein GPRchip in the chip holder s center position 3 Power up the GPR 800 system by turning on the switch at back of instrument The green POWER STATUS LED will light up to indicate power to the GPR 800
33. r supply cord before performing any cleaning service or maintenance on the GPR 800 A WARNING Remove power from the system immediately upon fluid spills on or near the system Do not operate the unit if fluid spills have occurred 15 e 877 776 8321 PROTEABIOSCIENCES 3 Component Locations This chapter provides the locations and descriptions of the components that make up the GPR 800 3 1 EXTERNAL AND INTERNAL PARTS Figure 3 1 Shows the external parts located on the front of the GPR 800 Indicate if the corresponding channels are powered and if they are uis over 50 uA 5 uA LED states Off No electroelution voltage provided to channel Green Electroelution voltage provided Channel is drawing at least 50 uA of current Channel LEDs Red Electroelution voltage provided Channel is drawing less than 50 pA of current Blinking Electroelution process is PAUSED by user or because safety mechanisms are not in place see section 2 1 No electroelution voltage provided to channel 2 Power Status LED Indicates whether or not the device is powered I3 Timer Relay Left Controls the time voltage is applied for electrode channels 1 4 4 Timer Relay Right Controls the time voltage is applied for electrode channels 5 8 Keven Controls the timer relays by starting pausing or stopping the process and yp by allowing the electroelution time setpoint to be modified www proteabio com 16 Figure 3 2 Sh
34. rom the SpinTip and place into the centrifuge adaptor Wash the SpinTip to wet the packing material by adding 50 uL of Equilibration Solution to the top of the SpinTip using a micropipette Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip wash Rinse the SpinTip by adding 50 uL of Sample Reconstitution and Rinse Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip rinse Load 10 to 200 uL of the GPR Sample Solution by adding it to the top of the SpinTip and centrifuging the system at 4000 x g for 3 min Additional sample volumes can be added in 200 uL aliquot cycles Wash the sample to elute salts and other non retained components by adding 100 uL of the GPR Rinse Buffer for MALDI to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip sample wash Transfer the SpinTip and adapter to a new clean centrifuge tube to collect the sample during elution Elute the sample by adding 100 uL of Elution Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min NOTE If the sample is targeted for LC MS analysis by electrospray ionization ESI mass spectrometry then the Elution Solution for ESI should be used formic acid based NOTE Monitor the level of liquid in the waste centrifuge tube being careful the flow through volume does not cover the bottom point of the SpinTip Dry down sample in a lyophilizer or SoeedVac NOTE for protei
35. roteins by the GPR 800 e While some proteins may be not affected or even positively influenced by the use of a gradient gel with the GPR 800 many proteins can be located in gel regions containing greater than 12 acrylamide which increases the resistance of electrophoretic movement within the gel and decreases the efficiency of recovery e Variations in the gel vendor type of gradient linear vs non linear percent gradient e g 5 20 lot to lot reproducibility and length of electrophoretic run can all significantly influence the reproducibility and efficiency of gel protein recovery for gradient gel samples e Gradient gels should only be used for advanced GPR 800 users who are performing optimized methods development for gel protein recovery Consequently Protea Biosciences does not support the use of gradient gels for routine gel protein recovery PROTEABIOSCIENCES www proteabio com A P 2 Electrophoresis e SDS PAGE gels should be run according to the manufacturer s instructions for voltages currents and run times e In general run voltages of 100 to 180V are recommended e Forrun to run reproducibility in both the protein gel separation and gel protein recovery the SDS PAGE gels should be run until the dye front reaches the bottom of the gel NOTES If the run time is too short for complex samples then the sample proteins may not fully resolve leading to protein bands that may contain multiple protein species all
36. rt automatically When the installation parameters appear on the computer screen Figure 4 1 accept the default values The installation will proceed automatically and may take several minutes When the installation is complete press the Finish button If prompted reboot the computer to complete the installation Figure 4 1 GPR Software Installation Parameters 21 e 877 776 8321 4 Step 4 Unpack USB Cable If using the GPR Works software unpack the USB cable and connect the end of the cable with the mini USB connector to the rear of the GPR 800 Connect the other end of the cable to the computer running the GPR Works software Step 5 Enter Calibration Values Start the GPR Works software on the controlling computer Click the calibration button Enter the 8 calibration values for the device being used Select OK to save the calibration values and dismiss the dialog NOTE Calibration values are specific to each GPR 800 and are supplied by Protea The calibration values are located on the back of each GPR 800 on a label If using more than one GPR 800 with a given computer the calibration values will need to be appropriately set each time a different GPR 800 is running Figure 4 2 Figure 4 3 GPR 800 Calibration Value Entry Software Process Screen Step 6 Power Unit ON Toggle the main power switch on the rear of the unit After the unit is powered ON the GPR 800 Power Status LED should illuminate and the timer relay
37. s Contact Protea technical service for do not light up at the start Blown circuit board fuse guidance in replacing of circuit board of the run fuse Incomplete electroelution Increase electroelution time Coomassie stained gels still colored at the end of the run Electroelution from an old Electroelute proteins run on fresh gels gel with proteins that are strongly fixed in the gel Slow movement of proteins Electroelute proteins run on a lower out of the polyacrylamide percent acrylamide gel gel www proteabio com m 40 Difficulty in recovering high molecular weight proteins PROTEABIOSCIENCES 7 Table 7 2 Error Message Guide Error Message Cause Solution O Ensure that the GPR 800 is powered on Communication with the Power Status LED is lit and that the GPR 800 data acquisition USB cable is properly connected to the device has been lost GPR 800 and the computer running the GPR 800 software Device Not Found See resolution to Device Not Found Communication with the error warning GPR 800 data acquisition device has been lost USB connection is not responding Device specific calibration Calibration file not found file has been deleted relo cated or renamed Input device specific calibration values when prompted by the software Al 877 776 8321 PROTEABIOSCIENCES 8 Consumables GPR 800 Reagent Bundles and Kits GPR LC MS Top Down Prep Kit Contains Surfactant Degra
38. s liquids CAUTION The total volume of flammable liquids used in the GPR 800 per chip run should not exceed 1 mL during proper operation of the device A 0 C0111010 0K00 aa mnie 2 Safety 2 Safety This chapter contains safety information necessary for the safe operation of the GPR 800 Read and understand this chapter prior to setting up or operating the unit Always adhere to the safety standards regulating your operating environment Also adhere to the safety rules outlined below in addition to your specific standards and guidelines WARNING Use of the system involves the potential use of and exposure to potentially harmful materials Only use the system if you have adequate training and experience in handling such materials WARNING Turn off electrical power to and unplug the system prior to any factory or end user maintenance or cleaning WARNING Do not operate the system if unit safety lid and electrode hinge are not properly installed or if the unit safety lid or electrode hinge are damaged or broken A WARNING Only operate the system when the system is fully assembled WARNING Do not attempt to disassemble alter override or otherwise defeat safety switches or features of the system Doing so may result in injury or death WARNING Use caution when coming in close contact with the electrodes and other components within the system as these components could cause physical harm especially in cases of a malfu
39. s should turn on www proteabio com m 22 PROTEABIOSCIENCES 5 Instructions for Use This chapter provides an overview of operation of the GPR 800 See the Installation and Preparation instructions in Section 4 for guidance on installing and preparing the GPR 800 for initial use Safety Warning and Limitation of Liability Use of the GPR 800 involves the use of and potential exposure to hazardous materials and therefore the system is intended for use only by professionals with adequate training and experience in handling of the dangerous materials involved Protea cannot be held responsible for any injuries to persons or property or for any other damage resulting from the improper use of this equipment or its use in any manner not specifically described in this manual Furthermore Protea disclaims all responsibility and liability in connection with any accidents arising from any use of the GPR 800 in situations where inadequate safety procedures are in place or where such procedures have not been followed 5 1 GENERAL OPERATION Zn rens Read the Safety information starting on page 11 prior to setting up or operating the GPR 800 See the component location information in Section 3 for descriptions of GPR 800 components and interfaces 5 2 OPERATING CONTROLS KEYPADS AND TIMER RELAYS The GPR 800 user interface includes two keypads and two timer relays NOTE Do not use a writing pen or other sharp object when operating the keypad or timer
40. s that the GPR buffer be placed in a sonicator for 5 minutes prior to use to minimize the risk of bubbles 2 Add a cored gel plug to Reservoir A for each channel that will be run See figure 5 3 for designation of the Reservoirs NOTE Protea recommends the use of its gel spot cutters to produce 2 mm circular gel spots for optimum efficiency in the GPR 800 However the GPR 800 chips are compatible with gel spots cut out with razor blades and other coring devices provided that the gel spots are placed down in the bottom of Reservoir A NOTE Multiple gel spot cores can be loaded into a single Reservoir to increase the amount of protein available for recovery but a longer electroelution time may be required NOTE It is very important to ensure that air bubbles are not present in the microfluidic channels as air bubbles can cause breakdown of the electrical pathway and current fluctuations during electroelution 3 Ensure that all gel plugs are at the bottom of Reservoir A for each channel containing a gel sample An entire bank of electrodes 1 4 or 5 8 must be run together If you have less than 4 gel pieces the remaining empty Reservoirs from the unused channels must be filled with 200 uL of de ionized water before starting the run 27 e 877 776 8321 Figure 5 3 Protein GPRchip reservoirs for a single processing channel 5 3 2 Sample Extraction and Collection 1 Place the microfluidic chip in the GPR 800 chip holder center chi
41. stals within and around the gel can damage the gel matrix leading to sample loss and reduced efficiency of gel protein recovery e Over time proteins will diffuse out of gel stored at 4 C and leach into the water It is recommended to use gels for gel protein recovery that have been stored properly for less than two weeks Gels older than two weeks are not recommended for typical gel protein recovery experiments 49 877 776 8321 Protea Biosciences Inc 877 776 8321 e www proteabio com copyright 2009 patent pending
42. the SpinTip to wet the packing material by adding 50 uL of Equilibration Solution to the top of the SpinTip using a micropipette Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip wash Rinse the SpinTip by adding 50 uL of Sample Reconstitution and Rinse Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip rinse Load 10 to 200 uL of the GPR Sample Solution by adding it to the top of the SpinTip and centrifuging the system at 4000 x g for 3 min Additional sample volumes can be added in 200 uL aliquot cycles www proteabio com e 32 e Wash the sample to elute salts and other non retained components by adding 100 uL of the GPR Rinse Buffer for MALDI to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min Repeat the SpinTip sample wash Transfer the SpinTip and adapter to a new clean centrifuge tube to collect the sample during elution lute the sample by adding 100 uL of Elution Solution to the top of the SpinTip Centrifuge the system at 4000 x g for 3 min NOTE Monitor the level of liquid in the waste centrifuge tube being careful the flow through volume does not cover the bottom point of the SpinTip e Dry down sample in a lyophilizer or SoeedVac NOTE for proteins 75 kDa a C SpinTip should be used 3 MALDI Analysis of Intact Proteins Add 10 20 uL of the Sample Reconstitution and Rinse Solution to the solid sample and sonicate briefly to dissolve the
43. the sample by adding 100 uL of Elution Solution to the top of the SpinTip Centrifuge the system at 4000 x for 3 min NOTE Monitor the level of liquid in the waste centrifuge tube being careful the flow through volume does not cover the bottom point of the SpinTip Dry down sample in a lyophilizer or SoeedVac NOTE for proteins 75 kDa a C SpinTip should be used LC MS Analysis of Intact Proteins Add 10 20 uL of the LC aqueous sample loading solution to the solid sample and sonicate briefly to dissolve the sample Load sample into sample loop on LC MS system Load sample from sample loop onto C column and begin LC MS analysis 5 3 5 MALDI Intact Mass Measurement Prep Analysis N PROTEABIOSCIENCES Sample collection and detergent degradation Collect sample from protein GPRchip into microcentrifuge tube using a micropipette Add Surfactant Degradation Reagent 10X to the sample to achieve a 10 fold dilution e g add 15 uL of Surfactant Degradation Reagent to a 150 uL GPR sample and incubate at room temperature for 15 30 minutes 15 minutes for MALDI analysis 30 minutes for ESI analysis Desalt protein sample using a C SpinTip Ensure that the packing material is at the bottom of the tip by gently tapping the tip to displace any packing material sticking to the top cap Place a centrifuge adaptor onto a 2 mL centrifuge tube Remove the cap from the SpinTip and place into the centrifuge adaptor Wash
44. vely 10 ethanol can be used to replace the 10 methanol Wash gel with deionized water prior to imaging e Add 50 mL deionized water and shake gently for 30 seconds e Discard rinse water e Add fresh 50 mL deionized water Gel imaging PROTEABIOSCIENCES SYPRO Ruby protein gel stain has two excitation peaks at 280 nm and 450 nm and has an emission maxima near 610 nm Stained proteins can be visualized using a variety of excitation sources including a 300 nm UV or blue light transilluminator or laser based systems SYPRO Ruby protein gel stain also has exceptional photostability and a long emission lifetime allowing for long exposure times while minimizing background fluorescence Coomassie R 250 stain SB R250 Remove the gel from the cassette once gel electrophoresis is completed and place in an appropriate size staining container containing 50 mL 1X Coomassie R 250 solution Gently shake for 45 60 minutes Remove the Coomassie solution from the gel and dispose in an appropriate waste container Add 50 mL of fixation solution to the gel and continue shaking for an additional 30 minutes Dilute the fixation solution by adding 100 mL of ddH O and add a folded paper towel to absorb additional Coomassie from the solution Gently shake until the required intensity of protein staining is accomplished and background staining is reduced This step will take several hours NOTE When using ProteaGels do not leave gels on final step
45. y Cover Open 13 Figure 2 3 GPR 800 with Chip Holder Installed 14 Figure 2 4 GPR 800 with Chip Holder Removed 14 Figure 3 1 Shows the external parts located on the front of the GPR 800 16 Figure 3 2 Shows the external and internal parts located on GPR 800 17 Figure 3 3 Shows the external parts located on the rear of the GPR 800 18 Figure 3 4 Shows the GPR 800 accessories for loading the consumables and for powering and communicating with the GPR 800 19 Figure 4 1 GPR 800 Software Installation Parameters 21 Figure 4 2 GPR 800 Calibration Value Entry 22 Figure 4 3 Software Process Screen 22 Figure 5 1 Shows the GPR 800 user interface 24 Figure 5 2 Timer Relay 26 Figure 5 3 Protein GPRchip reservoirs for a single processing channel 28 Figure 5 4 Protein GPRchip in the chip holder s center position 29 7 e 877 776 8321 Introduction This chapter contains introductory information on the Gel Protein Recovery System GPR 800 including specifications It also provides information about this User s Manual 1 1 INTRODUCING THE GPR 800 The GPR 800 is intended for use in a research laboratory to support extraction of intact proteins peptide mixtures from protein digests and other biomolecules from polyacrylamide gel plugs by electroelution or electroextraction The GPR 800 is designed to efficiently recover biomolecules from acrylamide gel plugs via a decoupled sample recovery process that minimizes set up operation time and user int

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