GPR®-800
Contents
1. 8 1 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 For run to run reproducibility in both the protein gel separation and gel protein recovery the SDSPAGE gels should be run until the dye front reaches the bottom of the gel NOTE 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 of which may be recovered during gel protein recovery NOTE 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 8 3 Staining and Visualization Supported stains e Zinc Sypro Ruby Coomassie R 250 non colloidal G 250 colloidal Non supported stains Silver Imperial blue Colloidal gold Gel Fixation 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 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 4 e 877 776 8321 Supported fixing solutions e 102. e SIOPISET button If the system is delivering electroelution voltage as indicated by illuminated Channel LEDs pressing the STOP SET LEDs 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 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 to the timer relay interface 25 e 877 776 832 5 5 22 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 NOT Figure 5 2 Timer Relay E The timer re
3. Supported gel characteristics Percent acrylamide e 5 10 12 and 15 polyacrylamide e 10 and 12 gels work best for most proteins 15 gels should not be used for proteins over 30 kDa Crosslinker ratio Higher polyacrylamide monomer crosslinker ratios produce larger average pore sizes that improve gel protein recovery e Recovery trends 37 5 gt 29 1 gt 19 1 Non supported gel characteristics 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 proteins by the GPR 800 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 2 acrylamide which increases the resistance of electrophoretic movement within the gel and decreases the efficiency of recovery 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 Gradient gels should only be used for advanced GPR 800 users who are performing optimized methods development for gel protein recovery Consequently Protea does not support the use of gradient gels for routine gel protein recovery www proteabio com 40
4. GPR 800 An appropriate computer will meet the following requirements at a minimum Windows 2000 XP or later version 64 MB RAM for Pentium 200MHz processor or equivalent 256 MB RAM for Pentium lll Celeron 600 MHz processor or equivalent 800x600 pixels for Pentium 200 MHz processor or equivalent 1024x768 pixels for Pentium Ill Celeron 600 MHz processor or equivalent and USB interfaceport 42 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 WARNING Read the Safety information starting on page prior to setting up or operating the GPR 800 4 Unpacking and Setup of the GPR 800 Step 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 WARNING 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 www proteabio com 20 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
5. Methanol 7 Acetic Acid SYPRO Ruby protein gel stain e 50 Methanol 1076 Acetic Acid Coomassie Blue R250 stain 0 1 Methanol 5 Phosphoric Acid Coomassie Blue G250 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 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 NOTE The use of acetonitrile is not recommended for protein fixation as it is very effective at precipitating proteins gt 50 kDa at concentrations 75096 Gel Destaining Zinc and SYPRO Ruby stained protein gels do not require destaining and can be used directly for gel protein recovery 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 Protocols Zinc stain Remove the gel from the cassette once gel el
6. spike in current 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 with the he platinum electrodes are fragile and can be bent by rough handling When working electrode assembly or electrodes please handle gently NOTE he 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 28 Protein GPRchip in center chip position 3 Power up the GPR 800 system by turning on the switch at back of instrument The green POWER STATUS LE GPR 800 Chip Holder Figure 5 4 Protein GPRchip in the chip holder s center position T U will light up to indicate power to the GPR 800 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 controls he left timer relay controls and displays the run time for channels 4 The right timer relay and displays the run time for channels 5 8 NOTE that will must be he bottom number on the timer relay is the target time the top number is the run time be applied to each bank of channels When the target time is changed the STOP SET button pressed to appl
7. 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 45 877 776 8321 8 14 Gel Sample Handling and Storage 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 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 crystals within and around the gel can damage the gel matrix leading to sample loss and reduced efficiency of gel protein recovery 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 www proteabio com 46 8 1 5 Optional Buffer Formulations In order to facilitate better protein extraction for the multitude of protein properties several different buffer formulations have been developed Each of the buffers contain a Protea Anionic Acid Labile Surfactant which provides a safe alternative to SDS based electroelution buffers These surfactants ar
8. GPR 060 GPR 065 GPR 070 A l 2 10 I Sample collection and detergent degradation Collect sample from protein GPRchip Reservoir C into microcentrifuge tube using a micropipette Add Surfactant Degradation Reagent 10X to the sample to achieve a 0 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 or 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 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 HL aliquot cycles Wash the sample to elute salts and other non retained components by ad
9. 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 A 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 computers CD ROM drive Software installation should start 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 Welcome to the Compressed zipped Folders Extraction Wizard The extraction wizard helps you copy files ftom inside a ZIP archive Figure 4 GPR Software Installation Parameters 2 e 877 776 832 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
10. 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 Stop Datalogging Figure 4 2 Figure 4 3 GPR 800 Calibration Value Entry Software Process Screen Step 6 Remove electrode alignment tool Step 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 relays should turn on www proteabio com 22 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 materials involved Protea cannot be held responsible for any injuries to persons or propert
11. If the channel LEDs are blinking the chip holder may not be installed www proteabio com 14 22 E ECTRICAL SAFETY 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 WARNIING Turn off electrical power to the system and unplug the power supply cord before performing any cleaning service or maintenance on the GPR 800 WARNIING Remove power from the system immediately upon fluid spills on or near the system Do not operate the unit if fluid spills have occurred 5 877 776 832 AcWTPEIFASZEN 3 Component Locations TAEVA This chapter provides the locations and descriptions of the components that make up the GPR 800 3 EXTERNAL AND INTERNAL PARTS Indicate if the corresponding channels are powered and if they are receiving over 50 HA 5 uA LED states Off No electroelution voltage provided to channel Green Electroelution voltage provided Channel is drawing at least 50 yA of cur rent Channel EDs Red Electroelution voltage provided Channel is drawing less than 50 uA of cur rent 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 Timer Relay Left Controls
12. Incomplete electroelution Increase electroelution time Coomassie stained gels still Electroelution from an old gel Electroelute proteins run on fresh gels colored at the end of the run with proteins that are strongly fixed in the gel Difficulty in recovering high Slow movement of proteins out Electroelute proteins run on a lower percent molecular weight proteins of the polyacrylamide gel acrylamide gel www proteabio com 38 Table 7 2 Error Message Guide Device Not Found USB connection is not responding Calibration file not found Communication with the GPR 800 data acquisition device has been lost Communication with the GPR 800 data acquisition device has been lost Device specific calibration file has been deleted relocated or renamed Ensure that the GPR 800 is powered on Power Status LED is lit and that the USB cable is properly connected to the GPR 800 and the computer running the GPR 800 software See resolution to Device Not Found error warning Input device specific calibration values when prompted by the software 39 e 877 6 832 8 Gel Guidelines for Optimal GPR Performance AcWTPEIFAZEN TAXE UTC Had 8 1 SDS PAGE GEL RECOMMENDATIONS FOR GPR 800 SAMPLES 8 1 1 SDS PAGE Gel Recommended gel vendors Protea Biosciences ProteaGels BioRad Mini Protean and Criterion gels Life Technologies formerly Invitrogen Novex NuPAGE gels
13. Volumes of Reagents 200 uL 400 uL 600 HL Reagent Methanol Water 3parts X 150gL X 300pgL 450hL Methanol 1pat X 50gL 100pgL 150gL 35 8 7 776 832 1 5 54 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 NOT E Toggling the main power switch on the rear of the unit removes all power from the GPR 800 NOT E 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 22 for further descriptions of the timer relay set and process times www proteabio com 36 Lia Pat Peace JAE Pia eal 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 electrodes are delicate and sharp Use care when working around the electrodes 5 2 MAINTENANCE No aspect of the GPR 800 is serviceable by th
14. also provides information about this User s Manual 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 sample recovery process that minimizes set up operation time and user interaction The operator begins the process by excising poylacrylamide gel plugs and placing them into the designated GPRchip 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 detergent degradation and desalting using SPE or other methods prior to mass spectrometry For instructions on general use of the GPR 800 see the General Operation section starting on page 23 of this manual l2 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 prior to setting up or operating the GPR 800 as this section contains information necessary for the safe operati
15. be stopped before the run time reaches 00 00 by pressing the STOP SET button at any time e If it is 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 www proteabio com 30 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 f the blue safety lid is opened to pause the instrument the voltage will be restored when the lid is closed After the sample has been electroeluted with the GPR 800 a decision must be made to determine how to analyze the sample Depending on the specific downstream application the chart below diagrams some possible post electroelution procedures Recommended Post electroelution Procedure Extracted Sample LC ESI MS Desalt on LC Column Analyze Data Sample Prep Application Kits Desalt with Spintips MALDI TOF MS LC MALDI MS LG ESIEMS ESI MS Direct Infusion Analyze Data Desalt on LC Column Analyze Data 31 e 877 7 6 832 5 5 3 4 LC MS analysis of intact proteins Sample collection and clean up Collect sample from protein GPRchip Reservoir C into microcentrifuge tube using a micropipette 2 Dry down sample in a lyophilizer
16. container containing 50 mL X 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 e Dilute the fixation solution by adding 100 mL of ddH O and add a folded paper towel to absorb additional Coomassie from the solution www proteabio com 44 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 overnight because it will de stain your protein bands Reagent recipes X Coomassie R 250 e 400 mL ddH O 500 mL methanol 00 mL acetic acid 110 mL of 10X Coomassie R 250 stock solution Fixation solution e 400 mL ddH O 500 mL methanol 00 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 e 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 e Add
17. or SpeedVac 3 Reconstitute the sample with at least 20 uL of Surfactant Degradation Reagent and incubate at room temperature for at least 10 min Dilute either ESI or MALDI surfactant degradation reagents to 10x and use the solution for sample reconstitution NOTE The ESI and MALDI Surfactant Degradation reagents are supplied in as a 10x solution Dilute to x with deionized water ddH O and use the solution for sample reconstitution 4 Inject the sample onto the C Lith Column Part no LC131 1 and rinse the sample to remove unbound salt and surfactant breakdown products A LC MALDI e Rinse the sample bound on the column using 97 Buffer A 3 Buffer B for 15 min at 2 uL min e Buffer A 0 1 TFA in 2 acetonitrile Buffer B 0 1 TFA in 98 acetonitrile Separation conditions e 80 3 Buffer A for 15 min e 3 Buffer A for 10 min Equilibrate column at initial run conditions for 5 min e Separation Flowrate 2 HUmin B LC ESI MS e Rinse offline to waste the sample bound on the column using 97 Buffer A 3 Buffer B for 15 min Buffer A 0 Formic acid in 2 acetonitrile Buffer B 0 1 Formic acid in 98 acetonitrile Separation conditions e 80 3 Buffer A for 15 min 3 Buffer A for a further 10 min www proteabio com 32 Equilibrate column at initial run conditions for a further 5min e Separation Flowrate 2 uL min 5 3 5 GPR 800 Application Kits Sample desalting using C and C SpinTips GPR 055
18. 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 43 877 776 8321 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 Alternatively 10 ethanol can be used to replace the 10 methanol Wash gel with deionized water prior to imaging Add 50 mL deionized water and shake gently for 30 seconds Discard rinse water Add fresh 50 mL deionized water Gel imaging 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
19. the GPR Electroelution Buffer Catalog Number Size GPR 055 Kit GPR 800 Reagent Bundle MALDI Low MW Contains Protein GPRchips 10 C LithTips with reagents 96 pk GPR Electroelution buffer 2 x 25 mL and surfactant degradation reagent for MALDI 10 mL Catalog Number Size GPR 060 Kit GPR MALDI Bottom Up Prep Kit Low MW GPR Application Kits are used for post GPR processing of recovered proteins from gel protein recovery experiments prior to analysis All Application Kits contain a surfactant degradation reagent for breakdown of the Progenta Anionic Acid Labile Surfactant that is contained in the GPR Electroelution Buffer Catalog Number Size GPR 065 Kit www proteabio com 48 AiRWFEIFAZEN DAE eel GPR Centrifugal UF Sample Prep Kit 3 kDa MWCO Catalog Number Size GPR 067 Kit GPR Centrifugal UF Sample Prep Kit 3 kDa MWCO Catalog Number Size GPR 067 Kit GPR Centrifugal UF Sample Prep Kit 10 kDa MWCO Catalog Number Size GPR 068 Kit GPR 800 Accessory Kit Contains one gel spot picker 2 6 mm diameter and forceps pair Catalog Number Size GPR 075 Kit 9 Consumables GPR 800 Accessories Protein GPRchip Contains 8 channels for protein electroelution from polyacrylamide gel samples PMMA pack of 10 Catalog Number Size GPR 200 10 10 pk Protein GPRchip Contains 8 channels for protein electroelution from polyacrylamide gel samples PMMA pack of 25 Catalog Number Size G
20. APRPOAF PERRA TAEVAS proted GPR 800 ADVANCED PROTEIN RECOVERY FROM GEL SAMPLES USER S MANUAL www proteabio com 2 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 2010 Protea Biosciences Inc All Rights Reserved Revision 1 0 04 2010 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 professional
21. PR 200 25 25 pk GPR Electroelution Buffer Catalog Number Size GPR 020 25mL 25 mL GPR 020 4x25mL 4X25 mL GPR Electroelution Buffer Il Acetate Catalog Number Size GPR 021 25mL 25 mL GPR 021 4x25mL 4x25mL GPR Electroelution Buffer Ill CAPS Catalog Number Size GPR 022 25mL 25 mL GPR 022 4x25mL 4x25mL GPR Electroelution Buffer IV Tris Glycine Plus Catalog Number Size GPR 023 25mL 25 mL GPR 023 4x25mL 4x25mL GPR Electroelution Buffer V CAPS Plus Catalog Number Size GPR 024 25mL 25 mL GPR 024 4x25mL 4x25mL Surfactant Degradation Reagent for MALDI 10 mL Catalog Number Size GPR 025 10mL 10 mL Surfactant Degradation Reagent for ESI 10 mL Catalog Number Size GPR 026 10mL 10 mL Gel Spot Picker Catalog Number Size GPR 045 ea Protein GPRchip Catalog Number Size GPR 200 1 chip GPR 200 10 10 pk GPR 200 25 25 pk C LithColumns 250 pm I D 20 cm Catalog Number Size LC 131 1 column LC 131 3 3 columns 49 e 877 776 832 www proteabio com 50 51 877 776 8321 lt e a ss SSS TSS APRPORS PEREA JAE THT Annee 2010 Protea Biosciences Inc 877 776 8321 e www proteabio com LN0222101 e C Se
22. bes As an alternative below is the protocol for preparing GPR 800 samples using Methanol Chlorofom Water and Acetonitrile precipitation www proteabio com 34 100 uL protein sample solution NOTE ADD 400 uL methanol Vortex for 10 seconds Centrifuge at 9000g for 30 seconds Add 100 uL chloroform Vortex for 10 seconds Centrifuge at 9000g for 30 seconds ADD 300 uL deionized water for phase separation Vortex vigorously 30 to 60 seconds Centrifuge at 9000g for minute 0 Carefully remove upper phase with a gel loading pipette tip and discard NOTE the protein IS present prior to pellet formation at the interface Do NOT disturb this interface when discarding the supernatant I ADD 100 uL methanol 2 Vortex for 10 seconds 13 Centrifuge at 9000s for 2 minutes to pellet the protein 14 Remove supernatant carefully with a gel loading pipette tip 15 ADD 200 uL of acetonitrile to the protein pellet 16 Vortex for 10 seconds 7 Centrifuge at 9000g for 2 minutes to pellet the protein 18 Remove supernatant carefully with a gel loading pipette tip and allow the pellet to air dry 19 Reconstitute in appropriate buffer for analysis a LC aqueous buffer for LC MS and LC MALDI analysis b Infusion buffer for direct infusion or static spray ESI MS analysis Digestion buffer digestion M C W A Reagent Ratio Guide LONDAN Volume of Protein Sample and Corresponding
23. cetal 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 hazardous liquids CAUTION The total volume of flammable liquids used in the GPR 800 per chip run should not exceed 4 mL during proper operation of the device www proteabio com lO ee eT 2 Safe ty AE JEU 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 wanna Only operate the system when the system is fully assembled WARNIING Do not operate the system if unit safety lid and elec
24. ding 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 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 SpeedVac Reconstitute in LCA buffer or MALDI reconstitution buffer for ESI or MALDI mass spec analysis respectively NOTE for proteins gt 75 kDa a C SpinTip should be used 33 8 7 776 832 1 5 GPR 800 Sample Clean up using an Ultrafiltration Device GPR 067 A Sample collection and detergent degradation Collect sample from protein GPRchip Reservoir C into microcentrifuge tube using a micropipette 2 Add Surfactant Degradation Reagent 10X GPR 026 to the sample to achieve a 0 fold dilution e g add 15 uL of Surfactant Degradation Reagent to a 150 HL GPR processed sample and incubate at room temperature for 15 30 minutes 15 minutes for MALDI analysis 30 minutes for ESI analysis B Desalt protein sample using a 10 kDa Ultrafiltration Device Prior to Mass Spec analysis interfering components can be removed from t
25. e compatible with mass spectrometry due to the degradation of the detergent under acidic conditions Each buffer has been formulated for the optimum electroelution of proteins with varying hydrophobic properties and isoelectric points The table below details the recommended buffer to use for each specific protein property GPR 800 Buffer Selection Guide High and low pl hydrophilic proteins GPR Electroelution Buffer GPR 020 Intermediate and high pl hydrophilic proteins GPR Electroelution Buffer Il GPR 021 Acetate High and low pl hydrophobic proteins and antibodies GPR Electroelution Buffer IV GPR 023 Tris Glycine Plus Low and intermediate pl hydrophilic proteins GPR Electroelution Buffer Ill GPR 022 CAPS Low and intermediate pl hydrophobic proteins and GPR Electroelution Buffer V GPR 024 antibodies GAPS Plus 47 877 7 6 832 9 Consumables GPR 800 Reagent Bundles and Kits GPR 800 Reagent Bundle ESI Low MW Contains Protein GPRchips 10 C LithTips with reagents 96 pk GPR Electroelution buffer 2 x 25 mL and surfactant degradation reagent for ESI 10 mL Catalog Number Size GPR 050 Kit GPR MALDI Intact Mass Prep Kit Low MW GPR Application Kits are used for post GPR processing of recovered proteins from gel protein recovery experiments prior to analysis All Application Kits contain a surfactant degradation reagent for breakdown of the Progenta Anionic Acid Labile Surfactant that is contained in
26. e 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 serviceable only by Protea trained service technicians 877 776 8321 37 877 7 6 832 AcWTPEIFPBZEN 74 Troubleshooting abseil Table 7 Troubleshooting Guide System is paused Press start to begin the run Blinking red LEDs Blue safety lid is open Lower lid run will start automatically Chip holder not properly placed Make sure the chip holder is in it proper place Insufficient current is flowing Aspirate the buffer from the collection reservoir through that channel due to a to remove the bubble Add the aspirated buffer bubble in the back to the electrode reservoirs One or more channels shows red micro channel LED at the start of the run Insufficient volume in the buffer Check to ensure buffer was placed in all 4 or 8 reservoir channels Top up buffer in buffer reservoirs In most cases the bubble will move through the One or more channels showsa A bubble has formed or entered channel and the current will return to normal If red LED during the run the micro channel during the run the LED is red for a prolonged period of time several minutes re run that channel One or more channels LEDs do Contact Protea for replacement of the circut not light up at the start of the run board fuse by a trained service technician
27. ectrophoresis is completed and place in an appropriate size staining container www proteabio com 42 Add 50 mL of 0 2 M imidazole solution NOTE Use sufficient volume to immerse gel Discard Add 50 Equilibrate on shaker for 10 minutes imidazole solution 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 NO gel s E The color transition time is sample dependent Larger gel protein loads can develop the pots in as little as 10 seconds Smaller gel protein loads may take 60 seconds or longer NO E 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 NO stop E Place the receptacle over a dark background to observe the staining reaction and it at the correct time point before over development e Rinse the gel in 100 mL ddH O for 3 minutes 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 Discard Add 50 mL deionized water and shake gently for 30 seconds rinse water mL of SYPRO Ruby protein gel stain to cover the gel NOTE Use
28. er 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 WARNIING Do not attempt to run the system with the safety cover open as doing so may cause physical injury NOTE 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 e 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 all 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
29. g Channel LEDs will illuminate indicating that current is flowing 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 If no safety interlocks 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 flowing through the channel If safety interlocks are activated pressing the PAUS E 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
30. he GPR samples by filtering with an ultrafiltration tube 10 kDa MWCO SP 022 2 Place the 10 kDa reservoir into a microcentrifuge tube 3 Load all of sample post surfactant degradation into the MWCO reservoir and top the reservoir with the cap 4 With the cap s hinge facing the rotor place the reservoir and tube into the centrifuge 5 Centrifuge at 14 000 rpm for 30 min or until the sample has moved through the reservoir the filter is built to retain 20 uL 6 Rinse the reservoir by adding 100 uL Wash Solution A and centrifuging at 14 000 rpm for 30 min or until the sample has moved through the reservoir 7 Rinse the reservoir by adding 100 uL Wash Solution B and centrifuging at 4 000 rpm for 30 min or until the sample has moved through the reservoir 8 Ina clean microcentrifuge tube place the reservoir upside down and centrifuge at 2 000 rpm for minute to collect the retentate which contains the cleaned up sample 9 Discard the filter reservoir 10 Freeze and lyophilize the retentate Additional ddH O may need to be added to dilute the sample in order for sample to freeze NOTE At least twice as much ddH O as the amount of retentate collected needs to be added because of the percentage of organics in Wash Solutions A and B which don t freeze Reconstitute in LCA buffer or MALDI reconstitution buffer for ESI or MALDI mass spec analysis respectively GPR 800 Sample Prep M C W A Protocol for 1 5 mL centrifuge tu
31. lay 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 NOT E 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 www proteabio com 26 53 GEL PROTEIN RECOV ERY The gel protein recovery process steps are described below 5 3 1 Priming the Protein GPRchip for Gel Protein Recovery 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 NOT E Protea recommends 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 NOT E Protea recommends the use of its gel spot cutters to produce 2 6 mm circular gel spots for optimum efficiency in the GPR 800 However the GPR 800 chips are compatible with gel spots cut o ut with razor blades and other coring devices provided that the gel spots are placed down in the bottom of Reser
32. nd 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 USB Connector Communication link for the GPR 800 software Calibration Values Precalibrated values used in GPR 800 software www proteabio com 18 32 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 normal use are described in Section 9 US 5 47 9 cousct Figure 3 4 The GPR 800 accessories for loading the consumables and for powering and communicating with the GPR 800 1 Chip Holder olds the Protein GPRchip Vial Rack olds the sample and waste collection vials Electrode Alignment Tool Used to align electrodes USB Cable Connects the GPR 800 to a computer computer not provided Power Cable Connects the GPR 800 to an appropriate AC power source 19 e 877 776 8321 AcHTPEZFAZEN 4 Installation and Preparation AE JE CH an 4 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
33. on of the unit Important information contained in this manual is marked as follows WARNING Impending Danger May cause death or physical injury A CAUTION Dangerous Situation May cause equipment material damage or data loss NOTE Helpful additional information and tips for use www proteabio com 8 l3 TECHNICAL SPECIFICATIONS The GPR 800 has the specifications listed below 1 3 Physical Dimensions Physical U S Units SlUnits Dimensions 13 inx9inx 14 in 33 cm x 23 cm x 36 cm 20 Ib 9 0 kg Dimensions and RUNS 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 85 non condensing 35 to 85 non condensing Exposing the system to direct sunlight is not recommended 1 3 3 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 30A 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 877 776 832 1 35 Chemical Compatibility Exterior surfaces of the device enclosure are ABS a
34. s with adequate training and experience in handling of the 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 CE s 3 877 776 832 1 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 4 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 5 e 877 776 832 Contents Introduction 8 Introducing the GPR 800 8 2 About the User s Manual 8 3 Technical Specifications 9 1 3 Physical Dimensions 2 1 3 2 Environmental Ranges 9 1 3 3 Con
35. sumables Storage Specifications 9 1 34 Electrical Specifications 9 1 32 Chemical Compatibility 10 1 3 6 Flammable Liquids 10 2 Safety 2 1 Safety Stops 13 2 Unit Safety Cover 13 2 1 2 Chip Holder 14 2 2 Electrical Safety 5 3 Component Locations l6 3 l External and Internal Parts 16 3 2 System Accessories 9 4 Installation and Preparation 20 4 Accessory Equipment 20 42 Initial Setup 20 42 Unpacking and Setup of the GPR 800 20 www proteabio com 6 Instructions for Use 5 General Operation 5 2 Operating Controls Keypads and Timer Relays 5 2 1 Keypads 52 2 Timer Relays 5 3 Gel Protein Recovery 52 3 Priming the Protein GPRchip for Gel Protein Recovery 5 3 2 Sample Extraction and Collection 5 33 Stopping Electroelution 5 3 4 LC MS Analysis of Intact Proteins 5 35 GPR 800 Application Kits 54 Power Off Cleaning and Maintenance 6 1 Cleaning 6 2 Maintenance Troubleshooting Gel Guidelines for Optimal GPR Performances 8 1 SDS Page Gel Reccomendations for GPR 800 Samples 8 1 1 SDS Page Gel 8 1 2 Electrophoresis 8 1 3 Staining and Visualization 8 1 4 Gel Sample Handling and Storage 8 1 5 Optional Buffer Formulations GPR Consumables Notes 29 23 23 24 26 27 27 28 30 32 33 36 3T 37 37 38 40 40 40 40 4 46 47 48 50 7 877 776 832 Introduction This chapter contains introductory information on the Gel Protein Recovery System GPR 800 including specifications It
36. the time voltage is applied for electrode channels 4 Timer Relay Right Controls the time voltage is applied for electrode channels 5 8 Controls the timer relays by starting pausing or stopping the process and by allowing the electroelution time setpoint to be modified www proteabio com l6 Figure 3 2 External and internal parts located on GPR 800 Ga Vial Lift Knob Raises and lowers the vial holder for positioning of sample vials Positions the vial holder and vials below the outlet channel bridges 8 Safety Cover Protects user from high voltages EH Electrode Hinge oo the positioning of the electrodes into either the OPEN RESTING or SEALED posi Leveling Mounts Interfaces with the E position Interfaces with the E Provide electroelution voltage 16 electrodes supply 8 channels B EH Knob 14 Catch Plate Catch Plate to position the EH in either the RESTING or SEALED Knob to position the EH in either the RESTING or SEALED position Allow the GPR 800 to sit level on a work surface These mounts can be rotated to adjust the level of the GPR 800 7 877 7 6 832 CONTROL BOARD 1156 0015 001 CALIBRATION VALUES S N QOH5 REV 2 cH 506b cus 50d cH2 595 cua SoS CH 502 cuz be CH 4 5bo7 cug 222 Figure 3 3 External parts located on the rear of the GPR 800 Power Entry Module AC power entry port a
37. trode hinge are not properly installed or if the unit safety lid or electrode hinge are damaged or broken WARNIING 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 AN emi 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 malfunction WARNIING Use caution when working near the operating system as the system contains high voltages ANUNG Remove power from the system immediately upon fluid spills on or near the system Do not operate the system after fluid spills have occurred Awessins No modification of this equipment is permitted 877 776 832 1 WARNING Do not operate the system if damage or abnormal erratic operation has been detected A CAUTION Keep open containers of fluid away from the system A CAUTION Keep areas around the system clean at all times A CAUTION Maintain at least 6 inches of unobstructed space around the unit to ensure proper use of the system www proteabio com 2 2 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 and the user is prevented from applying voltage to the electrodes 2 l Unit Safety Cov
38. voir A NO prote E Multiple gel spot cores can be loaded into a single Reservoir to increase the amount of in available for recovery but a longer electroelution time may be required NO E 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 7 6 832 Figure 5 3 Protein GPRchip reservoirs for a single processing channel 5 3 2 Sample Extraction and Collection Place the microfluidic chip in the GPR 800 chip holder center chip 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 the resivors should not be overfilled
39. y 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 WARNING Read the Safety information starting on page 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 52 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 relays as this may damage the component surfaces Use your finger for all keypad and timer relay selections 23 e 877 776 832 Figure 5 1 Shows the GPR 800 user interface 5 2 Keypads Each keypad has three control buttons START PAUSE and STOP SET Each set of buttons controls bank CH 1 4 or CH 5 8 of 4 electrodes 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 correspondin
40. y this new value as the run time 29 e 877 776 832 Table 5 1 Electroelution Time Guidelines for the GPR 800 Size of Protein Number of Gel Plugs per channel 2 mm circular Gel Plug 2 Gel Plugs 0 Ea x gt 2575 IDs 2030 75 kDa and up 20 30 20 30 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 uA 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 lt 50 uA to indicate the electrical current error The channel LED will be green if the channel current is gt 50 pA 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 3 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 33 X Stopping Electroelution Electroelution will stop when the timer run time reaches 00 00 Electroelution can
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