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Procise® Protein Sequencing System

1. Topic See Page Basic Connections 2 2 Pressures and Temperatures 2 5 Sequencer Reagents and Solvents 2 6 Bottle Change 2 7 PTH Amino Acid Standard 2 9 BioBrene Plus Solution 2 10 B lactoglobulin Solution 2 11 Waste Bottle 2 12 Sequencer Setup 2 1 Basic Connections About Connections Electrical Connections Communication Connections 2 2 Sequencer Setup During installation all the correct physical connections are made by your Applied Biosystems Service Representative If the instrument is moved or shut down for an extended period of time review this section to ensure that all the necessary connections are made before restarting the sequencer A power strip with four power connections is provided with the standard Procise system Additional connections may be needed for optional modules such as a chart recorder It is recommended that the instrument have a dedicated electrical line with a circuit breaker The outlet must be located within 2 5 m 8 ft of the instrument For additional details see the Procise Procise c LC Protein Sequencing Systems Site Preparation and Safety Guide P N 4314377 The Procise system has an automatic line switching power supply that accepts an ac voltage between 90 and 264 Vac ata frequency of 50 or 60 Hz The Microsoft Windows NT based computer for system control and data analysis has an automatic switching power supply and operates between 90 and 264 Vac at a frequenc
2. HPLC Setup 3 1 Preparing Solvents Aged Mobile Phase Sufficient Quantities 3 2 Overview Chemicals used with the Model 140C Microgradient Delivery System include two HPLC Setup solvents Solvent A3 3 5 aqueous tetrahydrofuran and Solvent B2 acetonitrile isopropanol and Premix Buffer Concentrate Use these solvents and the buffer concentrates to prepare the HPLC mobile phases that elute the PTH AAs from the analytical column See Chemical Warnings on page 1 5 Table 3 1 Typical Mobile Phase Composition New Column Bottle Fluid Volume Part Number Solvent A 3 5 THF H O 1000 mL 401464 approximately Solvent A3 100 analyses Premix Buffer 25 mL 401446 Concentrate 1 Acetone 1 mL 1 M NaH PO or KH PO 100 ul Solvent B Acetonitrile isopropanol 1000 mL 401570 approximately Solvent B2 150 analyses a The amount of Premix Buffer Concentrate added must be properly adjusted to achieve optimal separation of PTH glutamic acid from DMPTU and both PTH histidine and PTH arginine from other PTH amino acids Note Column temperature 55 C may vary slightly for optimum separation Note The column insert shipped with every column provides specific optimization guidelines If the system is not used for a week or more prepare new Solvents A3 and B2 and optimize the separation before sequencing a protein or peptide The following conditions may indicate aged mobil
3. Note Adding sodium phosphate or potassium phosphate to Solvent A3 can help prevent the occurrence of a downward sloping baseline associated with metal contamination of the HPLC pumps column or solvents 4 Enter the following information in the Bottle Change window and the sequencer logbook the date the lot numbers of the solvents and buffer concentrate and the amount of the buffer concentrate added to the solvents HPLCSetup 3 3 Replacing Solvents Overview When installing new solvents purge the separation system A purge rapidly expels solvents and trapped gases from the pump syringes Before sequencing or evaluating the separation equilibrate the column with the new solvents until the baseline is stable Use the procedure below to change the HPLC mobile phase For additional information refer to the Model 140C User s Manual Control the pump from the front panel keyboard of the Model 140C pump Changing Solvents To change solvents Step Action 1 Remove the old solvent bottle s Comply with all applicable laws and regulations related to chemical storage handling and disposal 2 Check the solvent lines for obstructions or salt deposits If the lines are not clear clean or replace them Check all fittings for salt deposits or indications of leakage Clean or replace as necessary 3 Empty the pump cylinders and solvent inlet tubing The Model 140C pump is equipped with an automatic purge valv
4. Table 1 1 Warnings on Chemicals Used in the Procise System continued Chemical Chemical Hazard Methanol PUZGNING CHEMICAL HAZARD Methanol is a Also in R2B flammable liquid and vapor Exposure may cause eye skin ANSON and respiratory tract irritation and central nervous system depression and blindness Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves PMTC NUNNE CHEMICAL HAZARD PMTC in acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Premix Buffer Concentrate CHEMICAL HAZARD Premix Buffer Concentrate causes burns to the eyes skin and respiratory tract It is a combustible liquid and vapor Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R1 5 phenylisothiocyanate in n heptane CHEMICAL HAZARD R1 5 phenylisothiocyanate in n heptane causes burns to the eyes skin and respiratory tract It is a flammable liquid and vapor Exposure may cause an allergic skin reaction and central nervous system effects such as drowsiness dizziness headache etc and irregular heartbeats Please read the MSDS and follow the handling instructions W
5. To start a run Step Action 1 Perform the Start run checklist a Check that the quantities of the sequencing chemicals and HPLC solvents are sufficient for the entire run Replace chemicals as necessary See Bottle Change on page 2 7 b Check the sequencer and HPLC waste levels The sequencer waste bottle must be emptied when the liquid level rises to within 2 inches of the bottle top Do not empty the waste bottle when the sequencer is running c Check that the argon tank has enough gas for the entire run Sequencer Operation 4 9 4 10 Sequencer Operation To start a run continued Step Action 2 Enter the sample and method information E PROCISE Procise 1 0 L o xj File Edit Sequencer Help Idle Start Run h Pause Navy Pause later Run E File Name File Name File Name File Name JFsmeie 1 Ena 2 Empe 3 ample 4 Cycles J 10 zl Cycles I 12 Cycles f 2 zi Cycles J 15 zl Method Method Method Method Pulsed liquid hi GP PYDF Peptide v Fast Precycle v PL PYDF Peptide h Status Idle Status Idle Status Idle Status Idle IV Collect Data IV Collect Data IV Collect Data IV Collect Data Sample fag o pmol Sample B y pmol Sample 5 o pmol Sample 4 0 pmol Stdlk n pmol StdlE o pmol Std ll o pmol Std ilo pmol Startup None h Shutdown None hd Start Run ii a Select Start Run view from the View menu b Select the cartridge run order
6. www appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number c Use the index number when requesting documents following the procedures below by phone for fax delivery a From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request through the Internet for fax or e mail delivery a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery Getting Help A 5 Warranty Applied Biosystems Limited Warranty Statement Applied Biosystems warrants to t
7. 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Product or Product Area Telephone Dial Fax Dial Integrated Thermal Cyclers ABI PRISMO 877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 BioInformatics includes BioLIMS BioMerge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 32 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic aci
8. Cartridges can be run in any order Selecting the run order for a cartridge activates the File Name Cycles and Methods fields c Enter a unique file name for each sample d Enter the number of cycles to be run by highlighting each Cycles field and typing the number or by using the scroll up down keys For filter precycling enter 4 When sequencing samples using the standard methods please note that the first three cycles are Prepare Pump a blank and a standard Therefore if 20 residues of sequencing are required enter 23 in the Cycles field e Move the cursor to the Method drop down menu in the lower half of the window Select the appropriate method for each cartridge f Choose the sequencer startup procedure if the sequencer has been idle for more than one week prior to the current run g Be sure the Collect Data box es are checked Type the sample and standard amounts h Click the Start Run button or press the Return key Sequencing parameters are downloaded to the sequencer and the Monitor Run window is displayed Data Collection Overview Figure 4 1 provides a graphic representation of data collection on the Procise system Procise Protein Sequencer Internal Analog Signal HPLC A D Converter Detector When the A D State is set to finished and all data has been Maximum of 90 collected by the Procise Data f dat Analysis software the Virtual A D minutes of data file is deleted by S
9. SequencePro software is data acquisition software for collection storage analysis and reporting of protein peptide sequence data SequencePro software is described in detail in the SequencePro User s Manual P N 905007 Note The Procise Protein Sequencing System is intended for research use only It is not to be used for reporting patient diagnostic or therapeutic results Introduction 1 3 Safety Documentation User Attention Words Chemical Hazard Warning Chemical Waste Hazard Warning 1 4 Introduction Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation VN NU eo Cautions the user that a potentially hazardous situation could occur causing injury to the user or damage to the instrument if this information is ignored NONIN Warns the user that serious physical injury or death to the user or other persons could result if these precautions are not taken L Aa Indicates an imminently hazardous situation that if not avoided will result in death or serious injury WIEGINDE CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safe
10. state provincial or national environmental and health regulations Chemical Warnings Potentially hazardous chemicals used on the Procise system are discussed in Table 1 1 Table 1 1 Warnings on Chemicals Used in the Procise System Chemical Chemical Hazard A3 3 5 tetrahydrofuran in water CHEMICAL HAZARD A3 tetrahydrofuran in water is a flammable liquid and vapor It may be harmful if swallowed Exposure may cause eye and respiratory tract irritation central nervous system depression and liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Acetone CHEMICAL HAZARD Acetone is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Acetonitrile Also in R5 S4B B2 CHEMICAL HAZARD Acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Argon CHEMICAL HAZARD Argon is a nonflammable high
11. 22358 2838 886 2 2358 2839 Thailand Bangkok 66 2719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 3575 0 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420 2 61 222 164 420 2 61 222 168 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 231206 05 4723120575 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 095 935 8888 7 095 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 All other countries not listed 44 0 1925 282481 44 0 1925 282509 Warrington UK Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81
12. 3 5566 6507 Latin America Del A Obregon Mexico 305 670 4350 305 670 4349 To Reach Technical We strongly encourage you to visit our Web site for answers to frequently asked Support Through questions and for more information about our products You can also order technical the Internet documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the product area of interest 3 Enter the requested information and your question in the displayed form then click Ask Us RIGHT NOW blue button with yellow text 4 Enter the required information in the next form if you have not already done so then click Ask Us RIGHT NOW You will receive an e mail reply to your question from one of our technical experts within 24 to 48 hours To Obtain Free 24 hour access to Applied Biosystems technical documents including MSDSs Documents on is available by fax or e mail or by download from our Web site Demand To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http
13. Each Safety label consists of a Labels 4 Signal Word panel which implies a particular level of observation or action e g CAUTION or WARNING If a safety label encompasses multiple hazards the Signal Word corresponding to the greatest hazard is used Message panel which explains the hazard and any user action required Safety Alert symbol which indicates a potential personal safety hazard See the Procise Procise cLC Protein Sequencing Systems Site Preparation and Safety 1 8 Introduction About Waste Profiles About Waste Disposal Before Operating the Instrument Safe and Efficient Computer Use Guide P N 4314377 for an explanation of all Safety Alert symbols provided in multiple languages A waste profile was provided with this instrument and is contained in the Procise and Procise cLC Site Preparation and Safety Guide Waste profiles list the percentage compositions of the reagents within the waste stream at installation and the waste stream during a typical user application These profiles assist users in planning for instrument waste handling and disposal which must be in accordance with local state provincial or national regulations Read the waste profiles and all applicable MSDSs before handling or disposing of waste IMPORTANT Waste profiles are not a substitute for MSDS information As the generator of potentially hazardous waste it is your responsibility to perform the actions listed below Characte
14. composed of four integrated modules the Procise Protein Sequencer the Model 140C Microgradient Delivery System the variable wavelength UV detector and a Microsoft Windows NT based computer equipped with Procise control software and SequencePro software The Procise system sequentially cleaves N terminal amino acids from protein peptide chains and analyzes the resulting phenylthiohydantoin PTH amino acid residues The chemical process used in the Procise system is derived from the technique developed by Pehr Edman in the 1950s for the sequential degradation of proteins and peptides In Edman degradation a protein s amino terminal amino acid is specifically reacted with phenylisothiocyanate PITC This derivatized amino acid is then selectively removed leaving the rest of the peptide chain intact Each cycle of the degradation removes the new amino terminal amino acid from the peptide chain The resulting PTH amino acids are analyzed sequentially to determine the amino acid sequence of the protein or peptide The Procise system completely automates Edman degradation and PTH amino acid analysis In all the system performs fully automated chemistry HPLC separations data quantitation and protein sequencing reporting The Model 140C Microgradient Delivery System the variable wavelength UV detector and SequencePro software are described in detail in separate manuals The Procise Protein Sequencing System Figure 1 1 controls precise deliv
15. minutes for the contents to dissolve mixing several times during this period 5 Store the stock solution vials at 20 C Preparing Fresh To prepare fresh working solution 1 pmol of each of the PTH amino acids uL Working Solution Step Action 1 Transfer 100 uL of each stock solution to a clean dry 10 mL volumetric flask or graduated cylinder 2 Bring to a total volume of 10 mL with R5 reagent 3 Mix thoroughly Transfer the dilution to a clean dry sequencer reagent bottle 4 Store the working solution at 20 C Preparing Fresh To prepare fresh dilution for loading Dilution for Loading Step Action 1 Add the appropriate volume of the working solution to a clean dry volumetric flask or graduated cylinder For a 10 pmol standard add 2 5 mL For a 5 pmol standard add 1 25 mL For a 2 pmol standard add 0 5 mL 2 Bring to a total volume of 10 mL with R5 reagent 3 Mix thoroughly Transfer the dilution to a clean dry polyethylene standard bottle 4 Use the Bottle Change procedure to load the new standard Sequencer Setup 2 9 Storing Chemical To store chemical solutions Solutions Step Action 1 Store the stock solutions at 20 C for up to 6 months 2 Store the working solution at 20 C for up to 1 month 3 On the system the standard may be used for peak identification for up to 1 week Several of the PTH amino acids P
16. not scratch or dent the column ends while handling them during installation Damage to the column ends may cause leaks which can be corrected only by replacing the column Care must be taken when handling columns Mishandling such as dropping or bumping can irreversibly damage the consistency of the packed bed and therefore impair the separation efficiency If the chromatography shows consistently broad peaks tailing peaks or poor separation which cannot be improved by adjusting Solvent A composition or preparing new Solvents A and B change to a new column or to a known good column If the separation dramatically improves with the new column discard the old column It is no longer suitable for analysis To remove or replace a column Step Action 1 If the pump is running press Stop on the front panel of the Model 140C pump If the column is to be reused use the manual mode to flush the column with 9096 Solvent B for 5 minutes at 300 uL min before removing Lift and remove the upper heating block Unscrew and remove the knurled end seal assembly closest to the injector valve by rotating it counterclockwise 4 Pull the cartridge column from the open holder If the cartridge is to be used again seal the ends with paraffin film To remove or replace a column continuea Step Action 5 Insert a new cartridge firmly in the holder Position the label to show through the holder window and screw t
17. page 1 5 CHEMICAL HAZARD Consider each sequencer chemical potentially harmful Become completely familiar with the MSDSs provided for each hazardous chemical See About MSDSs on page 1 8 When using hazardous chemicals wear appropriate safety attire listed in the MSDSs Minimize inhalation of or skin contact with chemicals Do not leave chemicals uncapped Work under a well ventilated hood when disposing of waste chemicals Dispose of waste in accordance with all applicable local and national laws and regulations Table 2 1 Storage Conditions for Chemicals Bottle Part Storage Number Chemical Contents Number Conditions 1 R1 5 phenylisothiocyanate PITC in heptane 400208 20 Ca 2 R2B N methylpiperidine water methanol MeOH 401535 4 C 3 R3 Trifluoroacetic acid TFA neat 400003 RT 4 R4A 25 TFA in water with 0 01 dithiothreitol 400028 4 C DTT 5 R5 acetonitrile with 0 001 DTT 400315 RT 6 S1 n heptane 400079 RT 7 S2B ethyl acetate 400854 RT 8 S3 n butyl chloride 400008 RT 9 S4B 20 acetonitrile in water 400314 RT 20 Amino Acid PTH standard 400879 20 Ca BioBrene Plus Reagent 400385 4 C B lactoglobulin 400979 4 C Supporting Solvents and Chemicals Solvent A3 401464 RT 3 5 THF Tetrahydrofuran in water 1000 mL Solvent B2 401570 RT 12 Isopropanol and acetonitrile 1000 mL Solvent B 4003
18. performed to verify that the Reaction Cartridges cartridge assembly is leak tight The cartridge leak test pressurizes the cartridge assembly at 3 8 psi and monitors the pressure drop for 30 seconds The result of the test is reported in the Event Log file at the end of the test IMPORTANT User intervention commands such as Jump Pause etc should never be used during leak procedures Interrupting the procedure may invalidate the test results or prevent the pressure regulator from resetting to the correct pressure Note The leak tests can only be performed while the sequencer is idle Testing To test for cartridge leaks Step Action 1 Select the Test view from the View menu 2 Select the Leak option Select the cartridge leak test to be used Hold down the Ctrl key to select more than one test Click the Start Test button If a cartridge fails the leak test troubleshoot the cartridge Troubleshooting To troubleshoot the cartridge Step Action 1 Remove the cartridge assembly cap 2 Disassemble the cartridge block holder and check that the glass fiber filter is properly installed in the recess of the upper cartridge block Check the cartridge seal for tears or unevenness in the sealing impression Using a new seal reassemble the cartridge block holder Check the Kel F ferrules of the cartridge assembly cap for damage or foreign materials Sequenc
19. the microprocessor controls the hardware During sequencing the sample is retained on a solid support such as a glass fiber disk in a temperature controlled glass reaction cartridge At the end of each degradation cycle the terminal amino acid is removed as an anilinothiazolinone amino acid ATZ AA derivative The ATZ AA derivative is automatically transferred from the reaction cartridge to a separately heated conversion flask for further derivatization to the more stable phenylthiohydantoin amino acid PTH AA The PTH AAs are then transferred from the conversion flask to the injection valve for subsequent separation and quantitation The Model 140C Microgradient Delivery System is a dual syringe gradient programmable microbore HPLC system connected to a low noise high sensitivity variable wavelength UV VIS detector A reversed phase analytical column in a temperature controlled heating block is used to separate the PTH AAs Because the different PTH AAs have unique relative affinities for the column the PTH AAs exit the column at different times The Procise Advanced Operation Manual describes how to use the HPLC system adjust the PTH AA separation prepare and change the mobile phase and change a column The output from the HPLC detector is collected by the Procise control software A 24 bit A D converter onboard the sequencer converts the analog signal to a digital signal and transmits the digital signal to the SequencePro software
20. warranty B 1 waste bottle 2 12 chemical hazard 1 4 disposal 1 9 WWW address Applied Biosystems A 2 Documents on Demand A 5 Index 2 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com AS Applied NP Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 05 2002 Part Number 4314376B an Applera business
21. 13 RT 100 Acetonitrile Acetone RT MeOH Methanol 400470 RT Premix Buffer Concentrate 401446 4 C a Allow this item to reach room temperature before opening If this bottle is opened while still cold water can condense inside it Check the bottle cap for tightness after placing this bottle at either 4 C 2 8 C or 20 C 15 to 20 C b RT Room Temperature 15 20 C in a dark dry place c Not used in current chemistry cycles Bottle position is empty Bottle Change Bottle Change The Bottle Change window Figure 2 3 is used to load chemicals onto the system Window The operator selects the bottle to be changed inputs the new bottle s lot number selects a bottle change procedure and then follows the prompts to remove and replace bottles The Procise system automatically depressurizes and backflushes the bottle to ensure operator safety during the bottle change process A log is automatically maintained showing when the bottle was last changed and the lot number of the new bottle Note Once argon is supplied to the Procise system the electronic pressure system attempts to pressurize all bottles to the settings in the Pressures amp Temperatures window All bottle positions must have a bottle installed to prevent excessive argon consumption E PROCISE Procise 1 0 File Edit Sequencer Help Bottle Change v Bottle_ Chemical 1 123455785 zl Rz 123245
22. 48 Injector Column oven in plumbing plate This length of peek tubing should be 0 01 in ID natural color Figure 2 2 Plumbing Connections Bumpons 2500 0968 Newguard holder 0715 0001 Dynamic mixer Peek red 0324 0020 ESE Bushing short 200249 Flowcell 12 uL 8 mm 2900 0542 Cap assembly 603651 Tubing 0 005 in To waste bottle s E Back To solvent pressure purge on assembly rear of 490 Note Bottles for Solvents A and B and the two waste bottles must be placed inside secondary containments Secondary containments are trays which hold free standing bottles and contain any spills 2 4 Sequencer Setup Pressures and Temperatures Setting Pressures Pressures and temperatures for the Procise system are set and adjusted from the and Temperatures Pressures 8 Temperatures window To set pressures and temperatures Step Action 1 Access the Pressures amp Temperatures window 5 PROCISE Procise 1 0 lel Es File Edit Sequencer Help a Idle a Temperst Pause Wavy Hause Later E Set Actual Off Ae I 25 25 kp I 35 35 Cartridge A J 35 24 v e F os os abl n 25 2 canider ff 24 pu MR FT 15 amay 30 30 canider 25 NW NUR a 3 a7 Flask p 18 canider FTT 2 NW o p 35 ss vel river os 10 Flask Fa sr Main Tank 80 Column Js sr Default
23. 678 2 E 12245678 4 RA 12245578 5 RS 12245578 6 s1 12245578 7 12345573 8 52 12345678 9 34 12245678 10 XL 123245678 iato Z 12245678 12 X3 123245676 Bottle Change Procedure v Bottle Change for 52 Figure 2 3 Bottle Change Window Lot Number Idle Stop Fun Changed Visa 1 1 94 1 1 94 1 1 34 1 1 34 141 94 Misa Misa Misa 1 1 94 Biel Es Pause How Pause Eater m Procedure Remaining Step Function Time Remaining Bottle Pressure kd Sequencer Setup 2 7 Changing a Bottle To change a bottle 2 8 Sequencer Setup Step Action 1 IMPORTANT The Procise system must be idle or paused prior to changing a bottle Select the pause function at the top of the window Click Pause Now or Pause Later to pause the cycle Select the Bottle Change window Select the bottle to be changed by clicking in the appropriate line in the reagent and solvent list Enter the new lot number Choose the appropriate bottle change procedure in the Bottle Change Procedure window Click Change Bottle A message appears when the bottle can be safely removed When prompted remove the old bottle and bottle seal Put a new bottle seal on the new bottle s rim and screw the new bottle into the bottle cap assembly Tighten until the bottle seal contacts the top of the bottle cap assembly and then turn approximately 1 4 turn more IMPORTANT It is not necessary to tighten a bottle unt
24. CAL HAZARD S4B 20 acetonitrile in water is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Sodium dodecyl sulfate SDS CHEMICAL HAZARD Sodium dodecyl sulfate SDS may cause an allergic respiratory reaction It is harmful if inhaled swallowed or absorbed through the skin Exposure causes eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Introduction 1 7 About MSDSs Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport a
25. Cart Begin Flask Standard Fast Normal 1 Gas Phase Starting Temp C 45 64 55 Table 4 1 Standard Methods continued Method Name Run Order Cartridge Flask Gradient PL PVDF Default Cart PL PVDF Flask Normal Fast Normal 1 Protein Protein 1 None Flask Prep Prep Pump Pump None Flask Blank Fast Normal 1 Cart Begin Flask Standard Fast Normal 1 Starting Temp C 48 64 55 GP PVDF Default Cart GP PVDF Flask Normal Fast Normal 1 Protein Protein 1 None Flask Prep Prep Pump Pump 2 None Flask Blank Fast Normal 1 Cart Begin Flask Standard Fast Normal 1 Gas phase Starting Temp C 48 64 55 PL PVDF Default Cart PL PVDF Flask Normal Fast Normal 1 Peptide Peptide 1 None Flask Prep Prep Pump Pump 2 None Flask Blank Fast Normal 1 Cart Begin Flask Standard Fast Normal 1 Starting Temp C 48 64 55 GP PVDF Default Cart GP PVDF Flask Normal Fast Normal 1 Peptide Peptide 1 None Flask Prep Prep Pump Pump None Flask Blank Fast Normal 1 Cart Begin Flask Standard Fast Normal 1 Gas phase Starting Temp C 48 64 55 Run Gradient Default None Run Gradient Fast Normal 1 Starting Temp C 35 64 55 PTH Standards Default None Flask Standard Fast Normal 1 Starting Temp C 35 64 55 Sequencer Operation 4 3 Filter Precycle Sequencing Liquid Samples Sequencing Blotted Membrane Bound Sample
26. Contact technical support by e mail for help in the following product areas Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax A 2 Getting Help Product Area E mail address Genetic Analysis DNA Sequencing galab O appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Biochromatography PerSeptive DNA PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers tsupport amp appliedbiosystems com LC MS Applied Biosystems MDS Sciex apisupport sciex com or api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700 DNA Analyzer
27. Procise Protein Sequencing System Basic Operation Manual Applied KS Biosystems O Copyright 2000 2002 Applied Biosystems For Research Use Only Not for use in diagnostic procedures Applied Biosystems and Procise are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries BioBrene and SequencePro are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation in the United States and or other countries All other trademarks are the sole property of their respective owners Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Contents Introduction Overview rece CP IER SES ERN E s USE ORG E 1 1 Product OVCRVICW 2 raid A e OA Oe ERES II UE T eg 1 2 Safety Leica sine awa c idee a kt t o E EUR Rc ep DAL ale 1 4 Sequencer Setup OVERVIEW occ eget A ra 2 1 Basic Connections i e e e Reb Saeed RE RR RR E Baa RE Re Hg 2 2 Pressures and Temperatures 0 0 e 2 5 Sequencer Reagents and Solvents 0 20 00 0 ccc eee rann 2 6 Bottle Change 42 vw vont SEE halde and E EV eee ee IE 2 7 PTH Amino Acid Standard o o oooooooooor eee 2 9 BioBrene Plus Solution soe sacs a reed deer Aaa 2 10 B lactogl
28. Revert al The pressures shown are not the defaults 2 To change the pressure settings a Highlight the appropriate numerical entry field under Pressures b Type in the new value c Click Execute Appropriate entries for pressures are values between 0 and 5 psi selectable to 0 1 psi 3 To change whether a heater is on or off a Click the Off checkbox w off blank on b Click Execute 4 To change temperature settings a Highlight the appropriate numerical entry field under Heaters b Type in the new value c Click Execute d Optional Click Revert to change temperatures back to their original settings Appropriate entries for temperatures are integer values between 30 and 70 C for the cartridge and column heaters and up to 78 C for the flask heater Only one cartridge heater can be activated at one time 5 Optional To return the system to its default settings click Default Default settings should be selected if the Procise system loses pressure or if the pressures and temperatures have been modified by using functions such as automatic leak testing Sequencer Setup 2 5 Sequencer Reagents and Solvents Overview All reagents and solvents supplied by Applied Biosystems are highly purified and 2 6 Sequencer Setup sequencer tested to ensure optimal performance A list of reagents solvents and other chemicals used with the standard sequencer cycles is given in Table 2 1 See Chemical Warnings on
29. TH Ser PTH Thr PTH Arg and PTH PE Cys are less stable in solution than the other PTH AAs at room temperature Accordingly if accurate quantitation of these residues is desired the standard should be changed more frequently BioBrene Plus Solution Overview BioBrene Plus reagent is a cationic polymer used to immobilize protein or peptide 2 10 Sequencer Setup samples on the glass fiber filter used for sequencing BioBrene Plus reagent is a dehydrated compound that must be reconstituted using 750 uL of distilled water This gives a final concentration of 1 5 mg polybrene and 0 1 mg NaCl per 15 uL Use 15 pl of this stock solution on the filter disc for each application B lactoglobulin Solution Overview B lactoglobulin is used as a standard for evaluating the sequencing efficiency of the Procise system Solutions are prepared as described below See Chemical Warnings on page 1 5 Preparing Dilution To prepare dilution solvent Solvent Step Action 1 Aliquot 40 mL of S4B 20 acetonitrile water P N 400314 into a clean 2 oz bottle 2 Add 40 uL of R3 trifluoroacetic acid P N 400003 to the bottle and mix well Preparing Stock To prepare stock solution Solution Step Action 1 Add 500 uL of dilution solvent to the vial 2 Vortex and or sonicate vial to dissolve the protein This may require 20 minutes of intermittent mixing Preparing Dilutions To prepare dilu
30. ation 4 11 Virtual A D File 4 12 Sequencer Operation When you start a sequencing run for a selected cartridge using the Procise control software a virtual A D file is created in the Procise folder The initial version of the Virtual A D file contains complete information on the Sequencer name and model the sample name the run date and time the sample and standard amounts and any other relevant information As the control software collects the data from the internal A D converter the data is appended to any existing data in the Virtual A D file When the Procise sequencer notifies the control software that the run is complete the control software sets the run finished flag in the Virtual A D file to indicate that the file contains a complete set of sequencing data At the end of the sequencing run the Virtual A D file contains the completed header information about the contents of the file as well as the raw data for the cartridge and sample during each cycle in the sequencing run Sequencer Idle Time Overview Idle Less Than One Week Idle One To Two Weeks Idle 14 28 Days Extended Shutdown When the sequencer is not used oxygen diffuses slowly into the system causing solvents and reagents to decompose and form byproducts which interfere with sequencing efficiency To minimize sequencing problems due to chemical decomposition during an inactive period use the procedures recommended in the following pages No spe
31. be achieved or restored by Overview adjusting the molarity or pH of Solvent A the B or the column oven temperature Figure 3 2 and Figure 3 3 summarize adjustments that can be made to improve the relative positions of certain amino acids pH Renew Solvent A Final Oven Temp iti Molarity Molarity B o Ba Molarity DPU Increase DNSQTGEAHYRPMVCWFIKL Figure 3 2 Separation Guidelines for the Original Solvent System Initial B Prony Renew 2 Solvent A Initial Initial Initial Premix B B B Increase j a DNSQTGEHARYPMVCW Figure 3 3 Separation Guidelines for the Premix Solvent System 3 12 HPLC Setup Separation of Figure 3 4 is a typical separation of 10 pmol of PTH amino acid standards with Premix Standards solvent system and Solvent B2 on the Procise system 2 Standard 1 1 00 0 00 1 00 2 00 Figure 3 4 Typical Separation of Standards HPLC Setup 3 13 Sequencer Operation Overview About This Chapter This section describes the common procedures needed for day to day operation of the Procise Protein Sequencing System You are instructed on how to precycle a glass filter with BioBrene Plus Reagent load samples on cartridges set up the run using the Procise control software and monitor the run using SequencePro software In This Chapter This chapter contains the following topics Topic See Page Standard Sequencing Methods 4 2 Loading React
32. block a Center the filter in the cartridge recess b Press the filter gently in place with the tamper tool Note An off center filter may cause cartridge sealing problems Any rips or holes in the filter reduce sequencing efficiency After preparing the solution according to the amount desired for your particular sequencing sample load the BioBrene Plus solution onto the center of the filter The typical loading amount is 15 pL Note Be sure that the volume of BioBrene Plus solution applied to the filter is sufficient to wet the entire filter Maximum liquid capacity of a dry filter is approximately 15 uL Additional fluid can be loaded if the filter is allowed to dry between loadings Dry the filter a Place the upper cartridge block in the cartridge drying assembly with the filter facing upwards b Lower the sample drying arm c Dry the filter for 5 minutes After 5 minutes the gas delivery is turned off For additional drying raise and lower the drying arm again Sequencer Operation 4 5 To precycle the BioBrene Plus solution treated filter continuea Step Action 8 Reassemble the glass cartridge blocks a Insert the upper cartridge block in the holder so that the filter side faces down toward the lower block and Zitex seal b Tighten the top of the cartridge block holder cap on the holder body 9 Reassemble the cartridge assembly a Insert the completed glass cartridge block hol
33. cial considerations are necessary if the sequencer has been idle for less than 1 week Before resuming sequencing after a short idle period run the Startup procedure to replace all reagents in the lines with fresh reagents from the bottles Purge the HPLC pump If the sequencer is to remain idle for 1 to 2 weeks use the Idle procedure to flush all the reagent bottles intermittently To initiate idling Step Action 1 From the Sequencer menu select Preferences 2 Click on the Execute Idle procedure check box and type a value for how often the procedure should be run ranging from once every hour to once every 999 hours Run the Standard Idle procedure once every 8 hours Select the Idle procedure from the menu and click OK The argon supply must remain connected to the sequencer in order for the Idle procedure to run The Model 140C Microgradient Delivery System should be put in Manual mode 5 10 uL min 90 B for free run or cleaned out and shut down Refer to the Model 140C User s Manual for additional information The computer can be shut down after you execute the Idle procedure and after all the information is downloaded to the sequencer If the sequencer is to remain idle for 14 28 days and is to remain connected to argon and electrical supplies with the instrument power on use the following procedure To initiate idling Step Action 1 From the Test view in the Procise software select and run th
34. d on c After replacing the buffers always run several Flask Standard cycles to check that the PTH AAs separate efficiently and reproducibly before sequencing an unknown sample If the separation is essentially the same as with the old buffers begin sequencing If the separation changes significantly with the new buffers then evaluate the separation and optimize it using the guidelines in Optimizing the PTH Amino Acid Separation on page 3 12 Always run at least four Flask Standard cycles and compare the last two before deciding that the separation needs to be optimized HPLC Setup 3 5 Changing the Column Description When to Replace a Column Removing or Replacing a Column 3 6 HPLC Setup The analytical column is a 22 cm long x 2 1 mm i d cartridge style column packed with a reversed phase support 5 micron PTH C18 The cartridge can be easily replaced when it no longer provides an acceptable separation of PTH AAs The Procise system is equipped with a cartridge type column for easy replacement Columns are held in place by knurled end seal assemblies see Figure 3 1 Grasp this end Unscrew this end and hold steady Cartridge Cartridge Knurled End Holder Seal Figure 3 1 Column Holder Assembly y Ne eh The knurled end seal assemblies must be handtightened only If they are overtightened the seals will be damaged The holder should be centered between the two knurled end seal assemblies Do
35. d Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855 PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North America Region Telephone Dial Fax Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 Getting Help A 3 A 4 Getting Help Region Telephone Dial Fax Dial Eastern Asia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2
36. der cap side up in the cartridge assembly b Place the cap on top of the assembly align the guide pins and slots then press down and turn the cap until it stops against the body of the assembly IMPORTANT The seal between the cartridge blocks and the Kel F ferrules is made by spring force Overtightening the cartridge assembly cap does not increase the sealing force Loading Samples To load samples 4 6 Sequencer Operation Step Action 1 Remove the glass cartridge block holder s a Unscrew and remove the cartridge assembly cap b Lift the glass cartridge block holder s from the cartridge assembly s 2 Remove the glass cartridge blocks a Unscrew and remove the cartridge block holder cap b Invert the holder body slowly to allow the cartridge blocks to slide out c Place the upper cartridge block precycled filter side up on a clean dry surface d Discard the used cartridge seal If samples are Then Liquid Proceed to step 4 Blotted membrane bound Place membrane piece s in the reaction chamber in the upper cartridge block Then skip to step 6 4 For liquid samples load the sample solution onto the center of the filter so that it is distributed evenly across the filter Note Maximum liquid capacity of a dry filter is approximately 15 uL With very dilute samples it may be necessary to load more than 15 uL total volume Additional fluid can be loaded if the filte
37. e 0 1 minute cannot be used for gradient programming HPLC Setup 3 9 Pump Control Two pump control functions are used with cycles Functions B Last Step Continuing Solvent Flow Changing Flow Rate External Events 3 10 HPLC Setup Function 227 Prepare Pump stops the pump if it is currently running refills and pressurizes the syringes and then runs the pump at the time zero conditions defined in the gradient Function 232 Start Gradient can be used to start a gradient without an injection Prepare Pump downloads the gradient to the pump Any changes made to the gradient on the Windows NT based computer are not implemented until the next time Prepare Pump is selected When programming a cycle Function 227 Prepare Pump must occur at least 10 minutes before the sample is injected on the column These 10 minutes allow for equilibration of the column at the time zero conditions defined in the gradient Inadequate equilibration results in variability in retention times and resolution To maintain constant B during a program segment set the B to the same value for the two steps spanning that segment To generate a gradient e change B during a segment simply set different B values for the two steps Positive or negative changes in B are generated depending on the relative B values at the two steps The time of the last step entered in a program is the end of run time At the last step in the progra
38. e Short Term Shutdown procedure to wash and flush all the valve blocks and backflush the reagents and solvents into the bottles Empty rinse and replace the waste bottle See Waste Bottle on page 2 12 Put the Model 140C system into Manual mode and run at 5 10 uL min 90 B Sequencer Operation 4 13 Idle gt 28 Days If the sequencer is to remain idle for more than 28 days and is to be disconnected from Extended argon and electrical supplies use the following procedure Shutdown 1 initiate idling Step Action 1 Replace the Model 140C Solvent A with HPLC grade or deionized distilled water Purge both pumps three times at 100 Run in Manual mode at 325 uL min 90 B for at least 30 minutes Remove all solvent and reagent bottles from the sequencer Place a bottle of HPLC grade methanol in the X3 bottle position Place empty bottles on all other bottle positions 4 From the Test view in the Procise software select and run the Long Term Shutdown procedure The sequencer washes all the valve blocks and reagent and solvent delivery lines with methanol and flushes all lines and valve blocks with argon 5 Empty rinse and replace the waste bottle 6 Prepare the Model 140C system for shutdown a Place both Model 140C solvent lines into a bottle of HPLC grade or deionized distilled water Purge both pumps three times at 100 b Place both Model 140C solvent lines into a bottle o
39. e of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties This Warranty is limited to the original location of installation and electrical power connection and is not transferable THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS Index A A D data collection 4 11 argon supply connections 2 3 ATZ derivative 1 3 B baseline noise 3 2 BioBrene solution loading 4 5 preparing 2 10 bottle change 2 7 buffer 3 2 C cartridge block 4 5 cartridges leak testing 4 7 loading 4 5 changing columns 3 6 changing solvents 3 4 column 3 6 communication connections 2 2 computer safety tips 1 9 connections 2 2 to 2 4 customer support e mail address A 2 help A 2 to A 5 Internet address A 5 regional sales offices A 3 telephone fax U S A 2 D data collection 1 3 4 11 Documents on Demand A 5 E Edman degradation 1 2 e mail address for technical support A 2 G gradient programming 3 8 typical program 3 9 H help e mail address A 2 Internet address A 5 regional sales offices A 3 telephone hours A 2 telephone fax U S A 2 HPLC mobile phase preparation 3 3 HPLC setup 3 1 I Internet address Docum
40. e phase Achange of peak shape i e broadening or tailing An increase in baseline noise or an unusual baseline rise A decrease in peak resolution that cannot be corrected by minor mobile phase component adjustments Precipitate in the mobile phase Note Before starting to sequence a sample be sure there are sufficient quantities of Solvents A3 and B2 to complete the run Changing mobile phase during sequencing can cause retention times to shift and make peak identification difficult Preparing the HPLC To prepare the HPLC mobile phase Mobile Phase Step Action 1 IMPORTANT Always use clean glassware for preparing Solvents A and B to minimize contaminants in the system Note Ifthe PTH column is new review the optimization information listed on the column insert on the line labeled Memo If the current column was used at installation and the separation is optimal use the conditions listed in the installation report Prepare 1 liter of Solvent A Add 25 mL Premix Buffer Concentrate to the 1 L bottle of Solvent A3 Invert the bottle several times to mix the contents 2 Optional Add up to 1 mL of HPLC grade 1 acetone in H O to 1 L of Solvent A3 Note Adding acetone to Solvent A3 increases this solvent s UV absorbance thereby reducing the baseline rise observed with increasing concentrations of Solvent B2 during gradient elution 3 Add 100 uL of 1 M NaH PO or KH PO reagent grade to 1 L of Solvent A3
41. e to divert the flow of solvent to waste a Purge the pumps and lines to remove all the old solvent Press PURGE gt The screen changes to the Purge Pump screen b Complete the screen as follows Line Prompt Description Action 1 Purge Rate at which the cylinder Enter a value between rate empties Maximum value is 7000 and 10 000 pL min 10000 or 10 mL min The Use the arrow keys to smaller the value entered move the cursor through the longer the purge takes the selections 2 Syringe Syringe to purge A B or Select Both Both Numberof Number of times to purge Use the numeric keypad to purges the pumps enter 2 3 Percent of Percentage of the syringe Enter 100 with the numeric syringe to empty refill and empty keypad Then press again BEGIN 4 Pump Appears while pumps are If necessary to stop a status purging purge press Stop 4 Place the solvent inlet line into the new bottle attach the cap and place the bottle in the bottle holder Repeat for each new bottle 3 4 HPLC Setup To change solvents continued Step Action 5 Purge the pumps and lines to rinse them with the new buffers Purge the pumps twice at 100 to thoroughly rinse all the tubing and the pump heads with fresh Solvent A and B and remove any bubbles in the system a Press PURGE gt b Complete the screen as follows Line Prompt Descriptio
42. ear appropriate protective eyewear clothing and gloves R2B n methylpiperidine in methanol and water CHEMICAL HAZARD R2B n methylpiperidine in methanol and water is a flammable liquid and vapor Exposure may cause eye skin and respiratory tract irritation and central nervous system depression and blindness Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R3 trifluoroacetic acid CHEMICAL HAZARD R3 trifluoroacetic acid causes severe burns to the eyes skin and respiratory tract Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R4 25 trifluoroacetic acid in water CHEMICAL HAZARD R4 25 trifluoroacetic acid in water causes severe burns to the eyes skin and respiratory tract Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves R5 0 00196 DTT in acetonitrile CHEMICAL HAZARD R5 0 00196 DTT in acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Sodium phosphate CHEMICAL HAZARD Sodium phosphate may cause eye skin and upper respiratory tract i
43. ents on Demand A 5 L leak testing reaction cartridges 4 7 loading BioBrene solution 4 5 reaction cartridges 4 5 samples 4 6 M mobile phase composition 3 2 preparation 3 3 MSDSs 1 8 O operation sequencer 4 2 optimization PTH amino acid separation 3 12 P peaks 3 2 plumbing connections 2 4 precipitate 3 2 preparation PTH amino acid standard 2 9 solvent 3 2 pressure setting 2 5 programming gradients 3 8 PTH amino acids analysis 1 2 detection 1 3 separation optimization 3 12 standard preparation 2 9 pump purge 3 4 R reaction cartridges leak testing 4 7 loading 4 5 reagent storage 2 6 reagents 2 6 restarting sequencer after extended shutdown 4 14 run sequencing 1 3 start 4 9 Index 1 S safety 1 4to 1 10 samples loading 4 6 SequencePro software 1 3 sequencer idle time 4 13 restarting after extended shutdown 4 14 setup 2 1 shutdown 4 13 sequencing methods standard 4 2 sequencing run 1 3 settings pressure and temperature 2 5 setup HPLC 3 1 sequencer 2 1 shutdown sequencer 4 13 software 1 3 Solvent A 3 2 SolventB 3 2 solvents changing 3 4 listed 2 6 preparation 3 2 storage 2 6 B lactoglobulin preparing 2 11 startingarun 4 9 storage reagents and solvents 2 6 T technical support A 2 to A 5 e mail address A 2 Internet address A 5 regional sales offices A 3 telephone fax U S A 2 temperature setting 2 5 trap bottle 2 12 y virtual A D data collection 4 11 W warning chemical 1 5
44. equencePro software Computer i i Procise Procise Virtual A D File i Control SequencePro EIE VE a SequencePro Software A D State P Software Data File Header Information Cycle 1 Data Cycle 2 Data File I O Cycle X Data File I O gt A EEG Procise Control Software Appends SequencePro Software Reads New Data to Virtual A D File Data from Virtual A D File Figure 4 1 Virtual A D Data Collection A D Converter There is an analog to digital A D converter contained within the Procise protein sequencer This A D converter is similar to previous A D converters from Applied Biosystems and is capable of storing a maximum of 90 minutes of data in its data buffer Procise Control The Procise control software is responsible for controlling the Procise sequencer and Software collecting the data from the sequencer s internal A D converter Unlike previous sequencers there is a high level of communication between the protein sequencer and the control software If the Procise sequencer determines that the control software is not collecting the data from the internal A D converter because the Windows NT based computer has malfunctioned the sequencer is paused automatically at the end of the current cycle until the control software resumes data collection This provides a very robust mechanism for preventing data loss due to overwriting the A D converter s data buffer Sequencer Oper
45. er Operation 4 7 4 8 Sequencer Operation Heating and Testing If a cartridge still fails the leak test you can try turning on the cartridge heater Expansion due to heat sometimes makes the seal leak tight To heat the cartridge and test for leaks Step Action 1 Turn on the cartridge heater and allow it to reach the setpoint 2 Select the Test view from the View menu 3 Select the Leak option Select the cartridge leak test to be used Hold down the Ctrl key to select more than one test Click the Start Test button Starting a Run Procedure MYILNGNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with and inhalation of chemical waste Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or clothing Seal the waste container with the cap provided after disposing of the contents Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and applicable environmental and health regulations Seal the waste container with the cap provided after disposing of the contents
46. ery of up to 12 different solvents and reagents Solvents and reagents are transferred to and from the reaction cartridge the conversion flask and the HPLC sample loop by a microprocessor controlled electromechanical pressure driven chemical delivery system The Procise system components include reagent and solvent bottles delivery valves the reaction cartridge conversion flask a vented waste bottle and interconnecting delivery lines GR1755 el 1 2 Introduction sel Q EEE EN SE SSE Sy The Procise Protein Sequencing System Figure 1 1 Software The Sequencing Run PTH AA Detection Data Collection The Procise control software regulates and monitors the functions of the Procise system The Procise control software allows the user to create and select sequencer functions gradients cycles and methods and constantly monitor the chemistry cycles and overall operation of the instrument Standard automated sequencing cycles and methods are supplied as part of the software package A cycle is a programmed series of steps designed to deliver reagents and solvents to carry out the Edman degradation The user can modify these cycles to create individualized protocols The Procise Protein Sequencing System Advanced Operation Manual P N 4314375 lists the cycles and describes how
47. es such as nearsightedness farsightedness astigmatism and the effects of corrective lenses When considering the user s distance from the screen the following are useful guidelines The distance from the user s eyes to the viewing screen should be approximately the same as the distance from the user s eyes to the keyboard For most people the reading distance that is the most comfortable is approximately 20 inches The workstation surface should have a minimum depth of 36 inches to accommodate distance adjustment Adjust the screen angle to minimize reflection and glare and avoid highly reflective surfaces for the workstation Use a well designed copy holder adjustable horizontally and vertically that allows referenced hard copy material to be placed at the same viewing distance as the screen and keyboard Keep wires and cables out of the way of users and passers by Choose a workstation that has a surface large enough for other tasks and that provides sufficient leg room for adequate movement Sequencer Setup Overview About This Chapter This chapter describes how to prepare the Procise Protein Sequencing System for operation including the necessary physical connections and reagent solvent preparation Detailed explanations of the sequencing chemistry can be found in the Procise Protein Sequencing System Advanced Operation Manual In This Chapter This chapter contains the following topics
48. f Solvent B2 Purge both pumps three times at 100 c Place both Model 140C solvent lines into clean dry bottles Purge both pumps three times at 100 7 Turn the instrument power off Me Neale If the reagent and solvent bottles are not removed before an extended instrument shutdown damage to the valve blocks may result Restarting After To restart the sequencer after an extended shutdown Extended Shutdown 4 14 Sequencer Operation Step Action 1 Turn the sequencer power and the argon supply on 2 Load fresh reagents and solvents See Sequencer Reagents and Solvents on page 2 6 3 Run the Startup procedure See Starting a Run on page 4 9 Prepare new HPLC solvents and purge the pump 5 Run a blank gradient on the HPLC and check for leaks Getting Help Overview About This This appendix describes how to get technical help from Applied Biosystems Appendix Getting Help A 1 Technical Support Contacting You can contact Applied Biosystems for technical support by telephone or fax by Technical Support e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below To Contact Technical
49. he customer that for a period ending on the earlier of one year s from the completion of installation or 15 month s from the date of shipment to the customer the Warranty Period the Procise or Procise cLC or Procise C purchased by the customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the minimum specifications set forth in set out in contained in the Instrument User s Manual and or the Instrument s Product Specification Sheet the Specifications During the Warranty Period if the Instrument s hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the Specifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense However if the valves or reagent lines become damaged or contaminated or if the chemical performance of the Instrument otherwise deteriorates due to solvents and or reagents other than those supplied or expressly recommended by Applied Biosystems Applied Biosystems will return the Instrument to Specification at the customer s request and at the customer s expense After this service is performed coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period This Warranty does not extend to any Instrument or part which has been a the subject of an accident misuse or neglec
50. he knurled end seal assemblies to the holder hand tight Do not use tools to tighten the holder assembly 6 In Manual mode use the softkeys on the pump to enter the flow rate FLOW gt 325 uL min solvent composition B gt 90 and the maximum operating pressure PRESS gt 4000 psi Press Enter after each selection 7 After a few minutes check around the fittings for any obvious leaks If leaks are found carefully tighten the knurled end seals and cartridge holder by hand Do not use tools to tighten these components Persistent leakage from the cartridge holder indicates damage either to the column or the knurled end seal assemblies Stop a leak at a fitting or bushing by tightening the fitting 1 4 turn with a small wrench 1 4 or 5 16 inch according to the size of the fitting Persistent leaking from a fitting indicates that the fitting has been damaged by overtightening and needs to be replaced 8 Periodically check all column connections for leaks Heating the column and holder may cause the fittings to expand and leak Continue to run until a stable baseline is achieved 9 Replace the column heater cover Continue to run until a stable baseline is achieved 10 Change the solvent composition to 50 B and continue to flush the column for 15 30 minutes 11 Equilibrate to the initial B of your gradient When there are no leaks and when the temperature baseline and operating pressure have stabilized the instr
51. il a snapping sound is produced by the bottle cap assembly Ratcheting the bottle cap assembly causes premature wear and may crack the bottle receptacle Click Continue and the remaining steps in the bottle change procedure are executed Note You are prompted to select Save to save the chemical data you have changed or Revert to return to the previous entry If a run was paused click the Resume button at the top of the Bottle Change window to continue the run PTH Amino Acid Standard Overview kit containing the PTH derivative of the 20 commonly occurring amino acids is provided with the Procise sequencer The standard mixture is reconstituted to a user specified concentration and loaded on the sequencer in the R5 position The PTH amino acid standard is required for peak identification and calibration of sequencer performance About R5 Note Be sure to use R5 acetonitrile reagent for all standard dilutions This reagent contains a small amount of DTT 0 001 which increases the stability of the PTH amino acids See Chemical Warnings on page 1 5 Preparing Stock To prepare stock solutions 1 nmol of each component 10 uL Solutions Step Action 1 Uncap each of the three vials If PTH PE Cys is not desired it should be omitted from the standard Add 1 0 mL of R5 reagent to each of the PTH standards 3 Blanket the vials with inert gas Cap the vials and vortex thoroughly Allow 20
52. ion Cartridges 4 5 Starting a Run 4 9 Data Collection 4 11 Sequencer Idle Time 4 13 Sequencer Operation 4 1 Standard Sequencing Methods 4 2 Overview Ten standard sequencing methods are available in the Procise control software For Sequencer Operation liquid sample sequencing you must use the Filter Precycle method for the Biobrene Plus solution treated filter prior to sample loading followed by either Pulsed Liquid or Gas Phase method sequencing For blotted membrane bound samples choose either a pulsed liquid method PL PVDF or a gas phase method GP PVDF The standard methods are listed in Table 4 1 Table 4 1 Standard Methods Method Name Run Order Cartridge Flask Gradient Filter Precycle Default Cart Flask Normal Fast Normal 1 Pulsed liquid 1 None Flask Prep Prep Pump Cycle Cart Precycle Flask Blank Fast Normal 1 Cart Precycle Flask Standard Fast Normal 1 Starting Temp C 48 64 55 Fast Precycle Default Cart Precycle None None 1 Cart Precycle Flask Standard Fast Normal 1 Starting Temp C 48 64 55 Pulsed Liquid Default Cart Flask Normal Fast Normal 1 Pulsed liquid 1 None Flask Prep Fast Normal 1 Pump None Flask Blank Fast Normal 1 Cart Begin Flask Standard Fast Normal 1 Starting Temp C 45 64 55 Gas Phase Default Cart Flask Normal Fast Normal 1 Gas Phase 1 None Flask Prep Prep Pump Pump None Flask Blank Fast Normal 1
53. llowing potential risk factors which include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors Use a seating position that provides the optimum combination of comfort accessibility to the keyboard and freedom from fatigue causing stresses and pressures Three basic requirements are Introduction 1 9 1 10 Introduction The bulk of the person s weight should be supported by the buttocks not the thighs Feet should be flat on the floor and the weight of the legs should be supported by the floor not the thighs Lumbar support should be provided to maintain the proper concave curve of the spine Place the keyboard on a surface that provides The proper height to position the forearms horizontally and upper arms vertically Supportfor the forearms and hands to avoid muscle fatigue in the upper arms Position the viewing screen to the height that allows normal body and head posture This height depends upon the physical proportions of the user Adjust vision factors to optimize comfort and efficiency by Adjusting screen variables such as brightness contrast and color to suit personal preferences and ambient lighting Positioning the screen to minimize reflections from ambient light sources Positioning the screen at a distance that takes into account user variabl
54. m the pump stops flow to the column This does not happen if the field Manual is set to Y on the Programs screen When the pump receives the Prepare Pump signal from the Procise system the syringes refill for the next analysis pressurize to the target conditions and pump solvent at time zero conditions Note At the last step in the program the pump stops flow to the column In the standard gradient Fast Normal 1 the pump stops at 21 5 minutes after the injection unless a subsequent sequencer cycle sends a Prepare Pump message Once the sample is injected onto the analytical column always continue the solvent flow until all of the sample components elute from the column and pass through the detector Sample components remaining on the column may elute during a subsequent run and interfere with peak identification and quantitation Change the flow rate by setting different flow rate values for the two program steps spanning a program segment Enter a B value for every step If no new flow rate value is entered for a step the value for the previous step is held throughout the segment spanning two steps In addition to changing B or flow rate during a program the gradient program can control external events These events include integrator start autozero detector chart recorder start stop additional A D start stop or additional data collection start stop Data collection by SequencePro software is started automatically by sample injec
55. mping 6 B Combined flow from pumps A and B is 325 uL min Turn on the chart recorder by selecting event 1 Autozero by selecting event 2 0 3 325 Autozero release by deselecting event 2 0 4 16 325 A quick jump in B from 0 3 to 0 4 minutes allows a lower starting B without loss of resolution of peaks S through G From time 0 4 to time 18 0 the pumps linearly increase the B from 16 B to 44 B 18 0 44a 325 Gives a start time for the pumps when they change the composition B In the 0 5 minute between steps 3 and 4 the pumps linearly increase the B from 44 B to 90 B Combined flow from pumps A and B remains constant at 325 pL min 18 5 90 325 Flow and composition remain the same 90 B and 325 uL min for 1 5 minutes This segment of the program washes contaminants and byproducts of the Edman degradation from the column in preparation for the next sample 20 0 90 325 Turn off the chart recorder by selecting the event 0 21 5 90 325 The pumps stop at 21 5 minutes after the injection unless another sequencer cycle tells the pumps to Prepare Pump a These B values are suggested starting values and may need adjustment to resolve all amino acid peaks See Optimizing the PTH Amino Acid Separation on page 3 12 for examples showing how to adjust gradient and Solvent A composition to correct poor resolution of PTH AAs b Tim
56. n Action 1 Purge Rate at which the cylinder Enter a value between rate empties Maximum value is 7000 and 10 000 pL min 10000 or 10 mL min The that does not smaller the value entered over pressurize your the longer the purge takes system 2 Syringe Syringe to purge A B or Select Both Both Numberof Number of times to purge Use the numeric keypad to purges the pumps in this case to enter 2 thoroughly rinse the pump cylinders 3 Percent of Percentage of the syringe Enter 100 with the numeric syringe to empty refill and empty keypad Then press again BEGIN gt 4 Pump Appears while pumps are If necessary to stop a status purging purge press Stop Note Before starting a run always purge as described in step 5 to remove old solvent from the pump cylinders The purge also clears any air bubbles that may have formed in the solvent supply lines while the instrument was idle 6 Equilibrate the column with the new buffers a Press Manual The pumps fill with the new solvents and the screen changes to display the Pump Control menu b Pump new solvent at 50 B and 325 uL min for approximately 5 minutes to allow the column to equilibrate with the new mobile phase s Use the softkeys to enter the flow rate FLOW gt 325 uL min solvent composition B gt 50 and the maximum operating pressure PRESS gt 4000 psi Press Enter after each selection Use the arrow keys and gt to select Events O open off or C close
57. nd dispose of the chemicals safely We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical NONIN CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order MSDSs Then Over the Internet Go to our web site at www appliedbiosystems com techsupport a Click on MSDSs b Enter keywords or partial words or a part number or the MSDSs Documents on Demand index number c Click on Search Click on the Adobe Acrobat symbol to view print or download the document or check the box of the desired document and delivery method fax or e mail By automated telephone Use To Obtain Documents on Demand on page A 5 service from any country By telephone in the United Dial 1 800 327 3002 then press 1 States By telephone rom Canada 7o order in Then dial 1 800 668 6913 and English Press 1 then 2 then 1 again French Press 2 then 2 then 1 By telephone from any other See Technical Support on page A 2 country For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Instrument Safety Safety labels are located on the instrument
58. nd inhalation of chemical waste Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or clothing Seal the waste container with the cap provided after disposing of the contents Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and applicable environmental and health regulations To empty the waste bottle Step Action 1 Raise the black bar above the waste bottle and carefully pull the waste bottle out of the waste container box Immediately cover the bottle to contain the vapors Discard the waste Add about 1 inch of water to the waste bottle Raise the black bar install the waste bottle and release the bar c1 OIN Inspect the top of the bottle where it seals against the O ring on the waste manifold The entire seal should be inside the bottle The O ring should be flattened against the bottle surface Rotating the bottle helps in properly seating the O ring Besides collecting waste the waste bottle assists venting by acting as a low pressure area Chemical deliveries flow from high pressure reagent or solvent bottle to low pressure vent or waste Therefore for flow to occur the waste bottle and its associated delivery and exhaust lines must be open to the vent only If the waste bottle is not effectively vented gas and liquid deliveries will be impeded Emptying the Trap To emp
59. obulin Solution lsseeeeeeeeeee III 2 11 Waste Bottle ois ind kn Ee as Coen NER LTS 2 12 HPLC Setup OVERVIEW p 3 1 Preparing Solvents eco ded a e abate 3 2 Replacmg SolventsS Le pelos o ey red LEGE adage aan Re ERES date ee 3 4 Changing the Column 2 2 00 6 he o A be eee eos 3 6 Gradient Programming os 32s pov rei geek eee DA ca LA ED de ERI 3 8 Optimizing the PTH Amino Acid Separation 0 0 02 eee eee eee eee 3 12 Sequencer Operation OVERVIEW sec eta dae set A ETE NORREWRRGMETENIUOSEN REIS eh ced 4 1 Standard Sequencing Methods 0 0 cc eee eee ae 4 2 Loading Reaction Cartridges 1 0 2 vara ra ven v varar varar rn ra raner annen 4 5 Starting 4 RUD cose ia nx stue Sea a he ak Gee sk dad sansen 4 9 Data Collect vs ansees Terek sr sere Adele Gera Ue Fed 4 11 Sequencer Idle Times to IS AAA SA 4 13 Getting Help OVELVI We Bea eee e o a EEE A 1 Technical Support eec torri RR ee A E aR A 2 B Warranty Applied Biosystems Limited Warranty Statement Index Introduction Overview About This Chapter This chapter provides an overview of the four modules of the Procise Protein Sequencing System as well as important safety information In This Chapter This chapter contains the following topics Topic See Page Product Overview 1 2 Safety 1 4 Introduction 1 1 Product Overview Introduction Hardware Components The Procise Protein Sequencing System is
60. only prepurified argon of 99 998 purity or greater Connections At least one size 1A cylinder of prepurified argon is required to supply inert gas to the Procise system A regulator and a CGA 580 cylinder adapter are required for each argon gas cylinder in order to control the inert gas supply to the instrument The exit side of the regulator should have Swagelok type end fittings for connection to 1 4 inch 6 355 mm o d tubing The regulator should be set between 65 and 75 psi 448 and 517 kPa If the input pressure drops below 60 psi during sequencing the system pauses EXPLOSION HAZARD Ensure that the pressurized gas cylinder is safely attached to the wall or table bench by means of approved brackets or chains Failure to do so could cause the cylinder to fall over and explode Always turn off cap and secure any cylinder that is not in use Sequencer Setup 2 3 Plumbing The Procise system is properly plumbed and ready for operation after the installation Connections Purge knob Solvent purge co Vent trap bottle Brass nut Tube Brass 1 4 in OD 10 ft 1 4 in ferrule 4 4 in 225016 110097 110096 Procise x Sequencer Y Push all tubing through slotted trim strips at corner Vent Trap Assy 603306 is complete Figure 2 2 shows the plumbing connections for this system X ag Rheodyne ferrule 200247 Bushing long 200234 Y HIN Ferrule 2002
61. ples onto cartridge s You choose the proper loading methods depending on the sample type Liquid samples require a precycled BioBrene Plus solution treated glass fiber filter Samples applied to the PVDF membrane by electroblotting the ProSorb device or the Model 173 MicroBlotter are placed in the cartridge with only a Zitex seal Loading PAAGI HOT Some components on the Procise sequencer may be hot Use caution when working near hot components to avoid injury BioBrene Solution To precycle the BioBrene Plus solution treated filter Step Action 1 Remove the glass cartridge block holder s a Unscrew and remove the cartridge assembly cap b Lift the glass cartridge block holder s from the cartridge assembly s Remove the glass cartridge blocks a Unscrew and remove the cartridge block holder cap b Invert the holder body slowly to allow the cartridge blocks to slide out Clean the cartridge blocks a Discard the used Zitex cartridge seal and glass fiber filter b Rinse the inner surfaces of both glass cartridge blocks with methanol c Dry the cartridge blocks with a stream of argon in the cartridge drying assembly Note Always handle cartridge seals and glass fiber filters with forceps Replace the lower glass cartridge block a Put the lower glass cartridge block in the holder b Place a new cartridge seal on top of the lower glass cartridge block Fit a new filter into the upper cartridge
62. pressure gas Released argon gas reduces the oxygen available for breathing Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves B2 12 isopropanol and acetonitrile CHEMICAL HAZARD B2 12 isopropanol and acetonitrile is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin Exposure may cause central nervous system depression and heart liver and kidney damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Biobrene Plus Reagent CHEMICAL HAZARD Biobrene Plus may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Dithiothreitol DTT I M Ye CHEMICAL HAZARD Dithiothreitol DTT may cause eye skin and respiratory tract irritation central In R4A R5 nervous system depression and damage to the kidneys Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Isopropanol NONIN CHEMICAL HAZARD Isopropyl alcohol can be harmful if inhaled ingested or absorbed through the skin It can cause CNS depression and be irritating to the eyes skin and mucous membranes Introduction 1 5 1 6 Introduction
63. r is dried between loading 5 For liquid samples dry the filter a Place the upper cartridge block in the sample drying station with the filter facing upwards b Lower the sample drying arm c Dry the filter for 5 minutes After 5 minutes the gas delivery is turned off For additional drying raise and lower the drying arm again 6 Reassemble the lower glass cartridge block a Put the lower glass cartridge block in the holder b Place a new cartridge seal on top of the lower glass cartridge block To load samples continuea Step Action 7 Reassemble the upper glass cartridge block a Insert the upper cartridge block in the holder so that the filter side faces down toward the lower block cartridge Note The easiest way to do this is by inverting the holder lower block seal group over the upper block until full contact is made with the seal b Tighten the cartridge block holder cap on the holder body Reassemble the cartridge assembly a Insert the completed block holder in the cartridge assembly b Place the cap on top of the assembly align the guide pins and slots then press down and turn the cap until it stops against the body of the assembly Note The seal between the cartridge blocks and the Kel F ferrules is made by spring force Overtightening the cartridge assembly does not increase the sealing force Leak Testing the Prior to starting a run a cartridge leak test should be
64. rize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial or national regulations Radioactive or biohazardous materials may require special handling and disposal limitations may apply Ensure that everyone involved with the operation of the instrument has Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs I M Willi Avoid using this instrument in a manner not specified by Applied Biosystems Although the instrument has been designed to protect the user this protection can be impaired if the instrument is used improperly Operating the computer correctly prevents stress producing effects such as fatigue pain and strain In order to minimize these effects on your back legs eyes and upper extremities neck shoulder arms wrists hands and fingers design your workstation to promote neutral or relaxed working positions This includes working in an environment where heating air conditioning ventilation and lighting are set correctly See the guidelines below MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by the fo
65. rritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Site Preparation and Safety Guide Table 1 1 Warnings on Chemicals Used in the Procise System continued Chemical Chemical Hazard S1 n heptane CHEMICAL HAZARD S1 n heptane is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness headache etc and irregular heartbeats Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S2B ethyl acetate CHEMICAL HAZARD S2B ethyl acetate is a flammable liquid and vapor It may cause eye skin and respiratory tract irritation Prolonged or repeated contact may dry skin It may cause central nervous system effects such as drowsiness dizziness headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S3 n butyl chloride CHEMICAL HAZARD S3 n butyl chloride is a flammable liquid and vapor Exposure may cause central nervous system effects such as drowsiness dizziness headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves S4B 20 acetonitrile in water CHEMI
66. s Screen Information Description Gradient Fast Normal 1 The gradient Fast Normal 1 can be modified or a new gradient can be created and stored under a unique name Max pressure 4000 psi Pressure above this value stops the pumps Choices are 0 through 4000 The default value is 4000 Min pressure O psi Pressure below this value stops the pumps Typical values are 0 to 100 psi The default value is O Target pressure 1000 psi The pressure the HPLC system should reach within the target time Choices are O through 4000 but must be within the limits set by the maximum and minimum pressure above The target pressue varies according to the flow rate column length and column packing The default value is 1000 psi Target time 1 0 min The amount of time the pumps use to reach target pressure Choices are 0 0 to 99 9 Typical values are 0 1 to 1 0 minute The default value is 1 0 minute Data collection time 20 min The length of time for which data is collected by the Procise control software Injection of the sample initiates data collection The default value is 20 minutes 3 8 HPLC Setup Typical Gradient Table 3 3 lists the steps of a typical gradient in the first four columns and describes Program the flow rate and composition for each step in the last column Table 3 3 Typical Gradient Program Time B HL min Event Description 0 0 6a 325 12 The pumps start pu
67. s 4 4 Sequencer Operation When sequencing a liquid sample you must load the glass fiber filter with BioBrene Plus solution a cationic polymer used to immobilize the sample on the filter during Edman chemistry and run the Filter Precycle method before loading the sample The Filter Precycle is necessary because BioBrene Plus solution may contain small amounts of compounds that could interfere with sequencing Filter precycling washes and conditions the BioBrene Plus solution coated filter by running several short cycles of the Edman chemistry Two methods are available for filter precycling The Filter Precycle method includes two filter conditioning cycles and a sequencing cycle allowing evaluation of the chemical and amino acid background level prior to sample loading This method requires at least 2 5 hours for completion The Fast Precycle method includes two filter conditioning cycles but does not include a sequencing cycle This method requires approximately 1 hour for completion but does not allowing evaluation of the chemical and amino acid background level prior to sample loading Two methods are available for sequencing liquid samples The Pulsed Liquid method delivers a small aliquot of liquid TFA to the cartridge for cleavage after coupling This method has a cycle time of 34 minutes and offers slightly higher repetitive yields The Gas Phase method delivers TFA vapor for the cleavage and has a cycle time of 41 minu
68. t b modified or repaired by a party other than Applied Biosystems or c used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems sole expense No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contained in Applied Biosystems printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems will not be binding on Applied Biosystems Warranty B 1 B 2 Warranty Applied Biosystems shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or us
69. tes The Gas Phase method offers lower background and reduced washout of hydrophobic samples especially peptides Four methods are available for sequencing samples on PVDF membrane Two of the four methods are for sequencing protein samples and include a delivery of 50 methanol from the X1 bottle during each coupling cycle This improves the coupling efficiency for membrane bound protein samples These methods should not be used for peptide samples because the 50 methanol addition increases washout of the peptide For peptide samples a small amount 3 6 uL of diluted Biobrene Plus solution 1 part 0 1 TFA 2 parts methanol 1 part Biobrene Plus solution should be applied to the sample before sequencing The PL PVDF Protein method delivers a small aliquot of 50 methanol to the cartridge during coupling and a small aliquot of liquid TFA to the cartridge for cleavage This method has a cycle time of 36 minutes The GP PVDF Protein method delivers a small aliquot of 50 methanol to the cartridge during coupling and TFA vapor to the cartridge for cleavage This method has a cycle time of 43 minutes The PL PVDF Peptide method delivers a small aliquot of liquid TFA to the cartridge for cleavage This method has a cycle time of 34 minutes The GP PVDF Peptide method delivers TFA vapor to the cartridge for cleavage This method has a cycle time of 41 minutes Loading Reaction Cartridges Overview This section describes how to load sam
70. tion Control of external events is accomplished through relay closures events 1 through 4 on the terminal strip located on the Procise rear panel These relay closures are controlled by selecting 1 2 3 and or 4 in the Event Column The events remain in effect until canceled by removing the appropriate number s from the Event Column or by the end of run signal which sets all external events to Off Time Limit Because the pump syringes have a limited volume there is a limit to the time between the Prepare Pump and Load Injector steps This time limit depends upon the time zero flow rate and B conditions and the volume of solvents required during the pressurization and analysis cycles The pump begins beeping when 5 minutes of equilibration time is left before injection If the limit is exceeded the pump continues pumping until one of the syringes empties or until the start signal arrives If the start signal is received late because of a hold or pause in the sequencer cycle there may not be enough buffer in the syringes to complete the analysis When the syringes empty the analysis terminates and the syringes refill in preparation for the next run before elution is complete Sample components remaining on the column may elute during a subsequent cycle and interfere with peak identification and quantitation HPLC Setup 3 11 Optimizing the PTH Amino Acid Separation Adjustment For certain PTH amino acids optimal position can
71. tions Step Action 1 Rinse a clean Eppendorf tube thoroughly with distilled water and then dry it 2 For 100 picomoles B lactoglobulin 20 uL BO picomoles B lactoglobulin 10 pL add 20 uL of the stock solution and 180 uL of the dilution solvent to the clean tube 3 For 20 picomoles B lactoglobulin 20 pL 10 picomoles B lactoglobulin 10 pL add 20 uL of the stock solution and 980 uL of the dilution solvent to the clean tube 4 Mix thoroughly by gently vortexing the tube Storing To store chemicals B lactoglobulin Step Action 1 Store the dilution solvent and dilutions at 4 C or below 2 Store the stock solution at 20 C 3 Discard the stock solution after 6 months 4 Discard any dilutions of the stock solution after 1 month Sequencer Setup 2 11 Waste Bottle Overview The waste bottle must be emptied when the liquid level rises to within 2 inches of the top of the bottle Do not empty the waste bottle when the instrument is sequencing FUZGNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Emptying the Waste Bottle Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with a
72. ty data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Do not leave chemical containers open Use only with adequate ventilation Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal NONIN CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Readand understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with and inhalation of chemical waste Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local
73. ty the trap bottle Bottle 2 12 Sequencer Setup Step Action 1 There is a polypropylene trap bottle mounted on the rear of the sequencer This bottle traps any condensate from the waste bottle vapor When the sequencer is operated using N methypiperidine as the coupling base this condensate is acidic and collects very slowly The trap bottle can be left empty or if desired place approximately 0 5 inch of sodium or potassium hydroxide pellets in the bottom of the bottle to neutralize the waste Empty the trap bottle when it is 40 50 full This is rarely required HPLC Setup Overview About This Chapter This chapter describes the procedures for preparing solvents changing solvents and columns modifying the gradient and optimizing the PTH amino acid separation These procedures give the menu prompts and the operator responses to the menus For complete descriptions of the menus used to control the pumps refer to the Model 140C Microgradient Delivery System User s Manual P N 903078 A general description of gradients and how gradient programming works is included Instructions for optimizing the PTH AA Phenylthiohydantoin amino acid separation are on page 3 12 In This Chapter This chapter contains the following topics Topic See Page Preparing Solvents 3 2 Replacing Solvents 3 4 Changing the Column 3 6 Gradient Programming 3 8 Optimizing the PTH Amino Acid Separation 3 12
74. ument is ready for sequencing To check the stability of the operating pressure watch line 2 on the Manual Control menu The pressure should vary no more than 10 psi If the baseline drifts after 15 minutes of equilibration continue delivering solvent until the baseline is steady Optional Monitor the baseline with the chart recorder 12 Run a few Flask Standard cycles to check the stability of the separation on the new column before sequencing an unknown sample 13 Document the column change by entering the change in the Bottle Change Log window on the Procise system HPLC Setup 3 7 Gradient Programming Overview Gradient programs change pump flow rates and composition on a time programmed basis A gradient program gradient consists of a series of steps Each step has a time a flow rate and a flow composition the output of the pumps in B Each step may also turn events on or off The time listed for the step is the time after the gradient is started either by injection of a sample or by activation of Function 232 Start Gradient The composition B and flow rate change as a linear function of time between steps Typical HPLC Table 3 2 lists the HPLC settings found in the Gradient View in the Screen Information Settings column together with the typical values The Description column explains what the entries mean and lists the range of accepted values as well as the default values Table 3 2 Typical HPLC Setting
75. y of 50 or 60 Hz The Model 140C pump and the UV detector come with the correct voltage configuration kit for the particular country either 100 120 220 or 240 Vac Communication connections for the Procise system are shown in Figure 2 1 Make sure that the line connecting the Procise sequencer and the computer is connected to the serial port 1 of the computer If a chart recorder with an external paper feed control is being used connect the respective pins to the two Event 1 terminals on the rear connection strip of the Model 140C pump Set the chart recorder to auto paper feed with a chart speed of 5 mm min Comp Out To Optional Chart Recorder Signal I P Record u UV Detector 0502 0077 UD F EEN ma 1 Event 1 Black 140C Event 2 Event 3 Event 4 Remote form feed l P not to scale Procise Instrument Ato D PumpCom 603341 Connect red and white wires to same terminal Black Auto zero Event mark CRM gel Computer Detector Input 201001 Monitor 4 RS232 t Pressure Output Inject 12 Vac Serial Port Computer 100390 Figure 2 1 Communication Connections Argon Supply PNI Use

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Procise® Protein Sequencing System