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Manual: ABI 173A MicroBlotter

1. io 140D 50 um ID 24 in Mixing L Static Port2 Port3 0323 0003 Coma E Mixer Teflon sleeve Injector 50 ym ID 8 in 225060 5 pL Sample loop P N 201433 Coupling sleeve use a 3 4 in length of 0 012 x 0 062 tubing P N 225004 Teflon sleeve 225060 0323 0003 50 um ID 15 in CAUTION Do not position a connection directly over the solenoid If there is a leak it will drip into the solenoid This can short the solenoid and cause corrosion Teflon sleeve 225060 225118 30 um ID 15 in Detector Flowcell Use Peek sleeves over capillary Peek Sleeve Ferrule 225123 Capillary 225118 tubing 30 ym ID 15 in Microblotter Bushing solenoid GR1055 Teflon Foot use a 1 4 in length of 0 012 x 0 009 tubing P N 225119 C2 Plumbing Parts and Connections February 2001 Applied Biosystems Appendix D Accessory and Installation Kits February 2001 173A Voltage Accessory Kit P N 604030 Part Number Quantity Description 0117 0003 2 Fuse 1 A 220 240V 0117 0004 2 Fuse 2 A 250 V 3 AG Slo Blo 0117 0019 2 Fuse 1 25 A 250 V SB 1 1 4 x 1 1 4 0117 0020 4 Fuse 0 6 A SB 5 x 20 mm 0117 0024 8 Fuse 0 5 A SB 5 x 20 mm 0117 0025 4 Fuse 1 A SB 5 x 20 mm 0192 0005 1 Cord Power 15 A IEC320 Japan 0192 0006 1 Cord Power 10 A 240 IEC320 Austr 0192 0007 4 Power cord
2. Blockage between column inlet and the MicroBlotter 5 Reconnect the tubing to the column inlet 3 2 1 4 5 140D Column A oo le MicroBlotter 112A Figure 4 3 Blockage tracking guide 6 Disconnect the tubing from the column outlet If the pressure drops continue to step 7 If the pressure remains high the column is blocked Flush the column per the procedure listed on page 4 5 Then reinstall the column If the pressure remains high the column is still blocked Replace the column The system pressure should return to the normal range 7 Reconnect the tubing to the column outlet Then disconnect the tubing at the coupling sleeve between the column and the 785A 4 If the pressure drops reconnect the tubing and continue to step 8 If the pressure remains high replace tubing A and reinstall the coupling sleeve The blockage should no longer be present 8 Disconnect the tubing from the flowcell outlet 5 If the pressure drops continue to step 9 If the pressure remains high the flowcell is blocked Reverse the flow direction to dislodge the blockage If the pressure remains high replace the flowcell The blockage should no longer be present 9 Replace tubing B The blockage should no longer be present April 2002 4 Maintenance and Troubleshooting 4 19 Applied Biosystems Disconnect fitting from column inlet Still blocked
3. 1 4 1 Introduction April 2002 Applied Biosystems April 2002 A user s manual for each instrument and installation accessory kits are also included with this system The kits include e Consumables including solvents MicroBlotter trays a set of PVDF membranes and filters the cLC Dye Mark cLC Staining Dye and a cLC Protein Standard e A0 5 mmx 15 cm capillary peptide column e A capillary U shaped flowcell Appendix D contains a complete listing of the contents of each installation and accessory kit shipped with the instruments in this system Optional Equipment e The Kipp amp Zonen Dual Pen Strip Chart Recorder P N 400268 The 173A system was optimized using a chart recorder of this type therefore we recommend the use of the Kipp amp Zonen Dual Pen Strip Chart Recorder P N 400268 Throughout this user s manual generalized chart recorder instructions are listed along with specific instructions for the Kipp amp Zonen chart recorder 1 Introduction 1 5 Applied Biosystems 1 6 User Attention Words Four user attention words appear throughout this manual Each word implies a particular level of observation or action Note This word is used to call attention to information IMPORTANT _ This information is given because it is necessary for correct operation of the instrument Caution This word informs the user that damage to the instrument could occur if the user does not comply with this
4. Incubate the solution for 2 h at room temperature in the dark Add 2 uL of 4 vinylpyridine DU UGE sis WOO O Incubate the solution for an additional 2 h at room temperature in the dark February 2001 Sample Preparation Guidelines B 1 Applied Biosystems 10 11 Dialyze against DI water using a microdialysis technique 2 for 4 h to remove excess reagents and salts Add 1 M NH4HCOs to bring the final concentration to 200 mM NH 4HCOsz pH 8 0 Add trypsin or Lys C in an enzyme substrate ratio of 1 30 w w Incubate the solution at 37 C for 20 h in the dark Centrifuge the sample for at least 5 min immediately prior to injection onto the 173A system For Small Proteins with Few or no Disulfide Bridges Example Apomyoglobin In an Eppendorf tube l Prepare a 10 ug solution of protein in approximately 50 uL of a 200 mM solution of NH HCOsz containing 10 acetonitrile and 1 hydrogenated Triton X 100 pH 8 0 Add trypsin or Lys C in a 1 20 w w ratio of enzyme substrate Incubate the solution at 37 C for 20 h in the dark Centrifuge the sample for at least 5 min immediately prior to injection onto the 173A system 1 Ausebel F Brent R Kingston R E Moore D D Smith J A Seidman J G and Struhl K Eds 1994 Current Protocols in Molecular Biology John Wiley and Sons New York 2 Lau K L and Fujitaki J M 1981 An improved method of microdialysis Anal Biochem 110 144 145 3
5. Maintaining the Column on page 4 5 The following is a brief summary of what you will see during the different phases of this run In addition to the standard status information various status messages such as PRESSURIZE FLOWING RUNNING and EQUILIBRATE appear along the bottom of the liquid crystal display LCD on the 140D throughout the run The system prepressurizes to the target pressure As this occurs the PRESSURIZE status message is displayed When the target pressure is reached the pump ramps to the target composition during which time the TIME TARGET status message is displayed Note Even if the target pressure is not reached within 2 min the program still begins at time zero conditions Composition changes are displayed until the specified time zero value is reached At the end of the target time the status message changes to EQUILIBRATE The cLC Dye Mark should be injected into the 112A during the equilibration phase When the equilibration phase completes the following should occur e The 112A will move to the inject position e The 785A will autozero e The chart recorder will start to feed paper e The MicroBlotter solenoid will move to the start position over the membrane and begin the blotting action 2 Unpacking and Installation April 2002 Applied Biosystems At the end of the run the display on the 140D automatically returns to the Ready Screen The MicroBlotter has been designed to return t
6. References to the various parts of Figure 4 3 are given throughout this procedure References are indicated by a number placed in parenthesis such as 1 or a letter Note When replacing fittings always cut one half to one inch off the capillary tubing after feeding it through the PEEK sleeve Particles from the PEEK sleeve can block the column at the frit Once the column is blocked it must be replaced 1 Disconnect the fitting from the column inlet 1 If the pressure drops the blockage is somewhere between the column and the MicroBlotter Proceed to step 5 If the pressure remains high the blockage is between the 140D and the column inlet Proceed to step 2 Blockage between the 140D and column inlet 2 Disconnect the tubing from port 3 of the injector 2 If the pressure remains high continue to step 3 If the pressure drops replace the tubing from the injector to the column The blockage should no longer be present 3 Switch the 112A from the inject to the load position If the pressure remains high switch the 112A back to the inject position and continue to step 4 If the pressure drops the injector loop is blocked Replace the injector loop 4 Replace the tubing from 140D static mixer to the 112A 3 If the pressure remains high check for a blockage inside the 140D from the syringes to the static mixer or in the static mixer itself 4 Maintenance and Troubleshooting April 2002 Applied Biosystems
7. Label the capillary closest to the front of the detector as the outlet 6 Close the front panel of the detector Exit capillary tubing Handle flowcell on Avoid pressing or touching the outer edge only front of the flowcell housing and exit capillary tubing Figure 2 7 Capillary U shaped flowcell 2 14 2 Unpacking and Installation April 2002 Applied Biosystems Installing the Electrical Connections The electrical connections cables are in the 173A Voltage Accessory Kit unless otherwise noted Refer to Figure 2 8 while installing these cables 112A LT E 140D Terminal block Event 1 Event 2 Event 3 Event 4 Pressure Output Inject 12 Vac Chart Recorder 6000 0112 Event 1 112 Event 2 785 Event 3 Blotter Event 4 Recorder Inject Jumper D Figure 2 8 173A electrical connections To install the electrical connections l Remove the terminal block P N 0301 0006 from the 140D Accessory Kit Using a small screwdriver attach the 112A control cable 9 pin D connector P N 6000 0590 to terminals 1 and 2 Event 1 of the terminal block Attach the D connector to the 112A remote connection Attach the 785A control cable 25 pin D connector P N 0502 0077 to terminals 3 and 4 Event 2 by connecting both the white and red
8. Reconnect column inlet fitting and disconnect column outlet fitting Disconnect fitting from port 3 of injector Still blocked Switch injector to load position Replace tubing between injector and column inlet Still blocked Replace the injection loop Y Disconnect fitting from inlet of injector port 2 Still blocked Flush or replace injector Disconnect fitting from 140D mixing tee outlet Replace tubing between 140D and injector Locate blockage in 140D Still blocked Y Reconnect column outlet fitting and disconnect tubing from flowcell inlet coupler 4 Still blocked Reconnect coupler to flowcell and disconnect flowcell outlet coupler 5 Replace tubing A between column and flowcell inlet Wash column Still blocked y Replace the column Replace tubing B Still blocked Y Back flush or replace flowcell Blockage repaired Figure 4 4 Flow chart for locating a blockage References to Figure 4 3 are indicated by a letter or a number placed in parenthesis such as 1 4 20 4 Maintenance and Troubleshooting April 2002 Applied Biosystems April 2002 Using the
9. and a troubleshooting guide Appendixes Includes our warranty statement sample preparation techniques including protocols for protein digests recommendations for sample treatment prior to sequencing and plumbing diagrams Index A comprehensive index designed to help you locate the information you need quickly and easily In addition the ABI 173A MicroBlotter Capillary HPLC System Safety Summary P N 903918 is included in the front pocket of this binder It contains important information on the safe installation operation and maintenance of the instruments in this system The Safety Summary also contains Material Safety Data Sheets for the chemistry provided with this system 1 Introduction 1 3 Applied Biosystems System Description The ABI 173A MicroBlotter Capillary HPLC System 173A system is a capillary liquid chromatography sample preparation system It provides users with an integrated protein peptide mapping system for research requiring high sensitivity and high recoveries especially for protein sequence analysis The 173A system separates and prepares very small amounts of sample in one step with no significant sample loss The eluted protein peptide fractions at picomole or subpicomole levels are collected onto a PVDF membrane The surface area onto which the fractions are collected is very small 2 to 3 mm o d spots This eliminates the need for further concentration or transfer steps thus minimizing poss
10. 100 0 PURGE NO Figure 2 17 Purge Screen 4 Press the BEGIN gt soft key There will be a slight delay before the pump responds and begins the purge procedure Once the purge is complete the Ready screen is displayed To remove air from the injector 1 Disconnect the tubing from the column inlet and place the tubing in a small waste container such as a beaker 2 Press FILL gt then BEGIN gt Wait for the syringes to refill 3 Press MANUAL to place the pump in manual mode and display the Manual Status screen Figure 2 18 April 2002 2 Unpacking and Installation 2 29 Applied Biosystems TIME FLOW B FLOW gt PRESS EVENTS B gt CAP A CAP B PRESS gt REFILL gt Figure 2 18 Manual Status Screen Note Allow a few seconds for the screen to respond to commands when in manual mode 4 Press the PRESS gt soft key type in 1500 for NEW MAX PRESSURE then press ENTER to set the maximum pressure to 1500 psi Caution Pressures greater than 3500 psi may cause fittings to pop apart and fail 5 Press the B gt soft key type in 50 for NEW B then press Enter 6 Press the FLOW gt soft key type in 100 for NEW FLOW RATE then press Enter to set the flow rate to 100 yL min 7 Switch the injector to the Inject position 8 When air bubbles no longer elute and solvent flows continuously from the tubing press STOP to halt the flow and return to the Ready screen Note Ifthe system pressu
11. 14 conditions for proteins 3 14 equilibration 3 17 execution of 3 23 to 3 25 gradient step 1 3 18 gradient step 2 3 19 gradient step 3 3 20 gradient step 4 3 21 prepressurization 3 15 summary of gradient steps 3 14 target time 3 16 standard protein peptide gradient program 3 13 to 3 21 see standard gradient program start position MicroBlotter 3 8 STEP NO parameter 3 24 storage conditions for excised peaks of interest 3 28 for stained membranes 3 6 for the 173A system 4 4 4 23 syringe capacities displayed on 140D 3 24 system description 1 4 T Teflon couplers 4 8 Teflon foot see also foot and MicroBlotter temperature see 112A testing the system 4 4 TIME parameter 3 24 February 2001 Applied Biosystems time parameter wavelength explanation of 3 19 settings for proteins and timed steps programming 2 38 peptides 3 7 tray settings for standard gradient MicroBlotter 3 9 program 3 14 positioning notch 3 10 tray cover 3 10 tray covers how to clean and inspect 3 28 how to install 3 8 trifluoroacetic acid 3 4 see safety troubleshooting chromatographic problems 4 12 irreproducible chromatograms 4 13 see also blockages see also help troubleshooting guide 4 12 to 4 17 tubing MicroBlotter solenoid replacement of 4 6 MicroBlotter tube adjustment 4 4 Teflon couplers 4 8 tubing kit D 2 U user attention words caution 1 6 description of 1 6 important 1 6 note 1 6 warning 1 6 W warning descri
12. 17 to assemble and connect this fitting 2 Unpacking and Installation April 2002 Applied Biosystems Injector Valve rear view To Column _ From Pump Injection Loop In Figure 2 11 112A injector valve To install the capillary column and connect the 112A to the 785A l Cut a 15 in length of 50 um capillary tubing P N 0325 0003 Connect the tubing to the capillary column P N 5100 0005 outlet using a bushing P N 200249 ferrule P N 200248 and PEEK sleeve P N 225123 The outlet is the end furthest from label Follow the procedure listed under Assembling the Fittings on page 2 17 to assemble and connect this fitting Cover all but the last 3 inches of the tubing with a red Teflon sleeve P N 225060 Referring to Figure 2 15 on page 2 26 place the column inside the 112A and connect the column inlet to the 8 in capillary in the oven coming from port 3 on the injector Orient the column so the label faces the back of the instrument Route the tubing from the column outlet port to the 785A and couple it to the inlet tube of the U cell by following the procedure Connecting Tubing with Teflon Couplers on page 2 24 By convention the inlet tubing enters the rear of the cell April 2002 2 Unpacking and Installation 2 21 Applied Biosystems SS Soa To assemble the solenoid 1 Cuta 15 in piece of 30 uM i d capillary tubing P N 225118 in the 173A Tubing Kit 2 Unplug the solenoid electrical co
13. Biosystems To Reach Technical Support Through the Internet We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products You can also order technical 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 Documents on Demand Free 24 hour access to Applied Biosystems technical documents including MSDSs is available by fax or e mail or by download from our Web site To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Click the Index link for the d
14. Mixer 201553 1 Loop 504L Rheodyne 8125 225004 1 ft Tube TFE 012 x 062 225060 10 ft Tubing red Teflon 020 x 062 225118 2m Tubing 30 ym x 375 m o d Silica 225119 2 in Tube TFE 012 x 009 Wall Nat 225123 10 Sleeve 016 0 d PEEK tubing 604268 1 Asy Static Mixer 34L D 2 Accessory and Installation Kits February 2001 Applied Biosystems 173A Chemical Installation and Accessory Kit P N 402066 Part Number Quantity Description 400313 1 Acetonitrile Solvent B 120 1 L 400445 1 Trifluoroacetic Acid 10 mL 400905 1 Low pH Perf Eval Std 402009 2 Blotter Tray 402010 7 sets Blotter Paper Membrane Sets 402011 1 Tweezers 402057 1 cLC Blotter Protein Standard 402063 1 cLC Staining Dye 40 mL 402140 10 Blotter Tray Covers 402155 1 Blotter Staining Tray 402256 1 Dye Mark II 630118 2 Solvent bottles 200 mL with 28 400 caps 785A Accessory Kit P N 604091 Part Number Quantity Description 0162 0050 1 Conn D25 Male Solder Cup 1000 0547 1 Manual Operating 785A 1000 0555 1 Procedure Bumpon installation 2500 0968 4 Bumpon 81 x 52 2800 0997 1 Tool Wrench hex 3 32 L type 3700 0072 1 Windows extra set 6000 0186 2 Cable Asy Analog output 6 ft Pigt February 2001 Accessory and Installation Kits D 3 Applied Biosystems 140D Accessory Kit P N 603973 Part Number Quantity Descr
15. Orr A Ivanova S and Bonner W M 1995 Waterbug Dialysis BioTechniques 19 204 206 Sample Preparation Guidelines February 2001 Applied Biosystems February 2001 Sample Preparation Recommendations Prior to Sequencing Recommendations for Sequencing Peptides Once your peptide has been separated and blotted by the 173A system we recommend adding Biobrene Plus to the excised peaks of interest before sequencing Note Biobrene Plus is available from Applied Biosystems P N 400385 To prepare peptides for sequencing 1 Mix 1 vol Biobrene Plus stock solution with 1 vol 0 1 TFA and 2 vol MeOH The final concentration is 25 ug of Biobrene Plus uL of 50 MeOH 2 Apply 2 uL of this solution to each piece of PVDF membrane 3 Allow the pieces of membrane to dry completely Once completely dry your samples are ready for sequencing Sample Preparation Guidelines B 3 Applied Biosystems Appendix C Plumbing Parts and Connections 140D Plumbing Diagram B SYRINGE A SYRINGE PRESSURE TRANSDUCER B VALVE A VALVE MANIFOLD PURGE MIXING TEE FLOW MIXING TEE February 2001 Plumbing Parts and Connections Cl Applied Biosystems ABI 173A MicroBlotter Capillary HPLC System Plumbing Diagram Sleeve 225123 Bushing 200249 Sleeve 225123 Sleeve 225123 Ferrule 200248 Bushing 200234 Bushing 200249 Ferrule 200247 Ferrule 200248 0323 0003
16. or 2 mm min by pressing the toggle switch marked SPEED The corresponding speed indicator LED will light Solenoid The mechanism by which the sample is transferred from the flowcell to the PVDF membrane e Blotter Tray Holder Holds the blotter tray during a run The holder and the blotter tray are keyed so the tray fits into the housing one way only Reset Button Resets the solenoid to the home position by inserting the end of a paper clip into the hole marked reset Home position Reset button Speed Solenoid in the start position End position indicators Speed toggle switch ABI 173 MicroBlotter Blotter tray housing Figure 2 2 Front panel of MicroBlotter April 2002 2 Unpacking and Installation 2 7 Applied Biosystems Rear Panel CAUTION Hazardous Chemicals Read Material Safety Data Sheets before handling CAUTION Hazardous Waste Read Waste Profile before handling or disposal WARNING Risk of electric shock Disconnect power cord from ro LINE supply before removing power supply module from instrument 100 240 VAC 50 60 Hz USE ONLY WITH 250V FUSES 0 1 mA SINK CURRENT WARNING For continued protection 40W max DMA PUT against risk of fire replace with only Fuse Type ple li of the 250V 0 5 Amps SB T BEFORE REPLACING FUSES DISCONNECT POWER Start signal Power Powercord Fuse holder connection switch connector Figure 2 3 Rear panel of the Mic
17. page 3 27 position the membrane along the bottom of the chromatogram Use the pencil mark made on the membrane at the beginning of the run and the 3 dye marks if the cLC Dye Mark was used or staining dye marks as guides 8 Tape the membrane to the chromatogram Apply the tape carefully to the outer edges of the membrane where no sample is present 9 Using a razor blade excise each peak of interest Excise the stained area if the cLC Staining Dye was used or a piece of membrane the width of the peak 10 Place each piece of membrane in an Eppendorf tube one piece per tube Cap and label each tube 3 Operation April 2002 Applied Biosystems Dye Mark peaks IN II Chromatogram HN I l start point NN UNS IK LTE EW AA NIA N Vo af O PVDF __ membrane T l Pencil mark made Pieces of PVDF excised Dye Marks on at start of run for sequencing the PVDF Figure 3 16 Positioning the PVDF membrane with the chromatogram of a peptide map Numbered peaks are the peaks of interest excised from the PVDF membrane for sequencing Staining Blotted Proteins with the cLC Staining Dye To stain the PVDF membrane 1 Wet the membrane with 100 methanol for 3 to 5 sec 2 R
18. power off and on or allow the solenoid to finish traveling across the membrane and return to the home position on its own 3 Operation 3 25 Applied Biosystems 3 26 Processing Your Sample After a Run Once your sample has been separated and blotted onto the PVDF membrane the membrane is lined up with the chromatogram and the peaks of interest are excised for further analysis Follow the procedure listed below for removing the PVDF membrane from the MicroBlotter tray and excising the peaks of interest Before sequencing peptides we recommend the addition of Biobrene Plus to your sample Instructions are provided under Preparing Peptides for Sequencing on page 3 28 Excising Peaks of Interest From the PVDF Membrane Note To prevent contamination always wear gloves and use tweezers when handling filters and PVDF membranes To excise peaks of interest from the PVDF membrane 1 Remove the tray from the MicroBlotter 2 Using tweezers gently remove the blotter tray cover Set it aside for cleaning and inspection procedure on page 3 28 3 Again using tweezers gently remove the PVDF membrane and place it on absorbent tissue 4 Discard the bottom filter 5 Allow the membrane to dry completely 6 If desired follow the instructions on page 3 27 to stain the PVDF membrane with the cLC Staining Dye Remember the cLC Staining Dye is recommended for use with proteins only 7 Referring to Figure 3 16 on
19. protocols are provided in Appendix B IMPORTANT Never inject a sample containing visible particulates Particulates can block the column beyond repair Flushing the Column To flush the column 1 Disconnect the tubing from the column inlet and outlet ports 2 Remove the column flip it end to end and place it back in the 112A 3 Reconnect the tubing from port 3 of the injector to the column outlet port the port furthest from the label on the column 4 Place the column inlet port on absorbent tissue 5 Run 80 solvent B at 5 wL min for h through the column to flush out particles accumulating on the frit 6 Remove the column from the 112A flip it end to end and reinstall it in the proper position 7 Reconnect the tubing to the column inlet and outlet ports If the column was flushed to remove a blockage run the cLC Protein Standard using the sample gradient program to test the effectiveness of this procedure Instructions for using the protein standard are listed on page 4 21 The sample protein peptide gradient program is listed in Section 3 Operation If performance remains poor replace the column 4 Maintenance and Troubleshooting 4 5 Applied Biosystems 4 6 Maintaining the MicroBlotter The Teflon foot and coupler Figure 4 1 on page 4 7 should be replaced every 3 months The tubing that runs through the solenoid may require periodic adjustment or replacement Adjusting the Tubing Runnin
20. result in a full scale deflection Set the paper speed to the same speed as the MicroBlotter either 1 mm min or 2 mm min 3 12 3 Operation April 2002 Applied Biosystems D v H y cal var v 9 penl I MV up down range Int ext button zeo Range selector Cal var button m Paper speed controls Zero button Record button Zero adjust potentiometer Figure 3 6 Optional Kipp amp Zonen chart recorder control panel Programming the 140D This section describes how to program the 140D using the standard protein peptide gradient program The conditions listed for peptides are adequate for most enzymatic digests and peptide mixtures The conditions listed for proteins can be used for most protein mixtures Optimization of this program may be required however for your particular application or sample Note The standard protein peptide gradient program may require optimization for your particular application or sample April 2002 3 Operation 3 13 Applied Biosystems 3 14 The cLC conditions for the standard protein peptide gradient program are as follows e Solvent A composition is 0 1 TFA Hy0 e Solvent B composition is 0 085 TFA Acetonitrile e Flow rate is 5 wL min e Detection wavelength for proteins is 210 mm 1 5 AUFS with a 1 sec rise time for peptides use 210 mm 0 1 AUFS with a 1 sec rise time e B gradient consists of 4 steps B Gradient for Protein
21. sample gradient program to test any adjustments Instructions for using the protein standard are listed on page 4 21 the sample protein peptide gradient program is listed in section 3 on page 3 14 e Keep the system dust free e Ifthe system will be idle for one or more weeks store the components according to the procedure listed under Storing the 173A System on page 4 23 This will ensure continued optimal system performance 4 4 4 Maintenance and Troubleshooting April 2002 Applied Biosystems April 2002 Maintaining the Column Each time a new column is installed we recommend your record the column starting pressure once it is completely hydrated This can take 24 to 36 h of constant flow at 5 4L min Until then the pressure will be lower than normal Check the pressure weekly A gradual increase in pressure indicates the start of a blockage If this occurs flush the column per the instructions listed below Regular flushing can help extend the life of the column by removing particulates which may be accumulating at the frit As mentioned throughout this manual sample purity is critical when working with the capillary column and tubing used in this system Samples must be as particulate free as possible before injection Therefore we recommend you use only the highest quality reagents and solvents Also centrifuge your samples for at least 5 min before injection Sample preparation guidelines including protein digestion
22. the equilibration phase When the equilibration phase completes the following should occur e The 112A will move to the inject position e The 785A will autozero e The chart recorder will start to feed paper e The MicroBlotter solenoid will move to the start position over the membrane and begin the blotting action At the end of the run the display on the 140D will automatically return to the Ready Screen The MicroBlotter has been designed to return to the home position after 200 min has elapsed If you wish to stop the MicroBlotter when the run is finished either press the Reset button or cycle the power off and on April 2002 2 Unpacking and Installation 2 43 Applied Biosystems 2 44 Verifying the System Test 1 Remove the blotter tray 2 Gently remove the PVDF membrane with tweezers and place it on an absorbent tissue Discard the bottom filter 3 Stain the PVDF membrane by following the instructions listed in section 3 Staining Blotted Proteins with the cLC Staining Dye on page 3 27 The cLC Protein Standard consists of 3 proteins The chromatogram Figure 2 30 on page 2 45 should show three distinct peaks all baseline resolved In addition the marks from the staining dye should be well defined and should not exceed the width of the corresponding peak If the chromatogram is satisfactory your 173A system is performing correctly If the chromatogram is not satisfactory check the pump for a deli
23. they can be used as the basis for entering most gradient programs In addition the standard gradient program can be used as a template for creating your own programs 3 Operation April 2002 Applied Biosystems To enter the standard protein peptide gradient program From the Ready Screen Figure 3 7 press Edit to display the Edit Select Screen Figure 3 8 140D x xx CLC FILL gt PRESS EVENTS PURGE gt CAPA CAP B VALVE gt UTILITY gt Figure 3 7 Ready Screen 2 Entera 1 in the program number field PROG NO Leave the step number field STEP NO blank PROG NO 1 EDIT gt STEP NO ERASE gt Figure 3 8 Edit Select Screen 3 Press the EDIT gt soft key to display Edit Screen 1 Figure 3 9 The parameters for the prepressurization phase of the program are set using this screen Edit Screen 1 is identified by the word PRESSURIZE on the top line of the screen The star symbol x in a figure such as Figure 3 9 indicates the Note parameter can have more than one value for this particular program Refer to the corresponding table for guidelines on appropriate values PROG 1 PRESSURIZE NEXT STEP gt B 50 FLOW 100 MAX PRESS x TARGET PRESS 800 EXIT gt Figure 3 9 Edit Screen 1 Prepressurization Phase April 2002 3 Operation 3 15 Applied Biosystems 4 Modify the values of each parameter to those shown in Table 3 1 Table 3 1 Edit Screen 1 P
24. value that must be entered for the TIME in Step 3 is 85 min for proteins 75 min 10 min or 180 min for peptides 140 min 40 min 12 Press the NEXT STEP gt soft key to proceed to Step 3 Enter the values shown in Table 3 6 on page 3 20 April 2002 3 Operation 3 19 Applied Biosystems 3 20 Table 3 6 Edit Screen 4 Gradient Step 3 Parameter Choices Setting Step 1 150 Time 0 546 85 min for proteins 180 min for peptides B 1 99 65 for proteins 45 for peptides Flow 0 500 5 L min Max Press 0 3 500 psi 2 000 psi Min Press 0 3 500 psi 0 psi Only Y N N Events O for Open 1 C 2 0 3 0 4 C 1 2 3 4 C for Closed Comments 10 min isocratic hold 40 min isocratic hold The values for Time are cumulative Composition during the entire step Same as step 1 Same as step 1 Same as step 1 Same as step 1 Same as Step 1 Events defined in Table 3 3 on page 3 17 Events set in each step of the gradient occur at the beginning of the step This is converse to the equilibration phase 13 Press the NEXT STEP gt soft key to proceed to Step 4 Enter the values shown in Table 3 7 on page 3 21 3 Operation April 2002 Applied Biosystems April 2002 Table 3 7 Edit Screen 4 Gradient Step 4 Parameter Choices Step 1 150 Time 0 546 B 1 99 Flow 0 500 Max Press 0 3 500 psi Min Press 0 3 500 psi Only Y N Events O for Open 1 2 3 4 C for Closed Setting
25. 0 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 Warszawa 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 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 1 Introduction Applied Biosystems Telephone Fax Region Dial Dial 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 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 46 0 41 0 41 790 0676 31 0 180 331409 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 All other countries not listed Warrington UK 44 0 1925 282481 44 0 1925 282509 Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Lati in America Del A Obregon Mexico 305 670 4350 305 670 4349 1 Introduction April 2002 Applied
26. 00 psi Min Press 0 3 500 psi O psi Only Y N N Events O for Open 1 C 2 0 3 0 4 C Events defined in Table 3 3 1 2 3 4 C for Closed on page 3 17 Events set in each step of the gradient occur at the beginning of the step This is converse to the equilibration phase 11 Press the NEXT STEP gt soft key to proceed to Step 2 Enter the values shown in Table 3 5 on page 3 19 3 18 3 Operation April 2002 Applied Biosystems Table 3 5 Edit Screen 4 Gradient Step 2 Parameter Choices Setting Comments Step 1 150 Time 0 546 75 min for proteins The values for Time are 140 min for peptides cumulative B 1 99 65 for proteins Composition at the end of the 45 for peptides step Flow 0 500 5 L min Same as step 1 Max Press 0 3 500 psi 2 000 psi Same as step 1 Min Press 0 3 500 psi 0 psi Same as step 1 Only Y N N Same as step 1 Events O for Open 1 C 2 0 3 0 4 C Same as Step 1 Events 1 2 3 4 C for Closed defined in Table 3 3 on page 3 17 Events set in each step of the gradient occur at the beginning of the step This is converse to the equilibration phase Note The values entered in the TIME parameter for the gradient phase are cumulative The values entered in the TIME parameter for the gradient phase are cumulative For example the duration of Step 2 above is 75 min for proteins 140 min for peptides The duration of Step 3 is 10 min for proteins and 40 min for peptides However the
27. 006 140D 0478 0004 785A and 0478 0009 112A Fuses are also included in this kit Instructions for the MicroBlotter and chart recorder and are provided below MicroBlotter Fuse Installation The MicroBlotter contains a universal power supply and requires one 0 5 A fuse to operate Make sure the fuse is installed by following the instructions listed below WARNING ELECTRICAL FIRE HAZARD Improper fuses or power input can damage the wiring system and cause a fire Before turning on the MicroBlotter verify that the proper fuse is installed and that the voltage setting on the instrument matches the voltage in your laboratory WARNING ELECTRICAL SHOCK HAZARD Disconnect the power cord before resetting the line voltage or changing the fuse To install the fuse 1 Insert the blade of a small screwdriver into the slot on the left side of the fuse holder cover Figure 2 4 on page 2 10 on the rear panel of the MicroBlotter 2 Pry the cover forward until it snaps out April 2002 2 Unpacking and Installation 2 9 Applied Biosystems USE ONLY WITH 250V FUSES DISCONNECT POWER BEFORE REPLACING FUSES Slide screwdriver blade under cover here and lift up to remove fuse holder cover Figure 2 4 MicroBlotter fuse installation 3 Ifnot already installed insert a 0 5 A fuse P N 0117 0024 in the 173A Voltage Accessories Kit into the holder located on the cover 4 Snap the fuse holder cover back in place Chart Recor
28. 112A after first making sure the 112A is set to the load position At the end of equilibration the 785A is autozeroed the MicroBlotter and chart recorder are activated and the gradient phase begins 4 Check the blotting action of the MicroBlotter The tubing should lightly touch the membrane at the end of each downward stroke leaving a slight impression in the membrane If the membrane is being damaged adjust the position of the tubing by gently pulling it up 3 24 3 Operation April 2002 Applied Biosystems April 2002 IMPORTANT Record the starting pressure of each new column once it is completely hydrated This can take 24 to 36 h of constant flow at 5 uL min Until then the pressure will be lower than normal Then check the pressure weekly A gradual increase in pressure indicates the start of a blockage If this occurs flush the column per the instructions listed in section 4 See Maintaining the Column on page 4 5 At the end of the run the screen on the 140D automatically returns to the Ready Screen After 200 min the MicroBlotter automatically resets the solenoid to the home position Note The solenoid on the MicroBlotter will continue traveling across the membrane to the end position before returning automatically to the home position Travel time is 200 min If the length of your run is less than 200 min you can either press the reset button on the MicroBlotter control panel turn the MicroBlotter
29. 90 min for proteins 185 min for peptides 15 for proteins 5 for peptides 5 L min 2 000 psi 0 psi 1 C 2 0 3 0 4 C Comments 5 min gradient to return to the B composition specified in step 1 The values for Time are cumulative Composition at the end of the step Same as step 1 Same as step 1 Same as step 1 Same as step 1 Same as Step 1 Events defined in Table 3 3 on page 3 17 Events set in each step of the gradient occur at the beginning of the step This is converse to the equilibration phase 14 Press the EXIT gt soft key to display the Save Screen Figure 3 13 The program can be assigned a different program number at this time or abandoned without saving it by pressing the Stop key For this program make sure that 1 is displayed in the SAVE AS PROG field 15 Press the PERM gt soft key to save the program The volume of solvents A and B required will be displayed with the status message SAVING SAVE AS PROG 1 A VOLUME B VOLUME PERM gt TEMP gt Figure 3 13 Save Screen Once the program has been saved the display will automatically return to the Ready Screen 3 Operation 3 21 Applied Biosystems Preparing and Injecting Your Sample Because the i d of the capillary tubing is so small samples must be as free of particulates as possible before injecting them onto the column In addition to removing particulates we recommend you avoi
30. A position Event 2 UV VIS Detector 785A auto zero Event 3 MicroBlotter feed Event 4 Chart recorder paper feed Events set in Edit Screen 3 occur at the end of equilibration immediately before Step 1 of the gradient phase 9 Press the NEXT STEP gt soft key to display Edit Screen 4 Figure 3 12 and begin entry of the gradient phase of the program The gradient phase consists of a variable number of steps all of which use the same screen Edit Screen 4 Typically a different value for B and TIME is entered in each step For the standard protein peptide gradient program the gradient phase consists of four steps April 2002 3 Operation 3 17 Applied Biosystems Note When a step is created the default values displayed are those from the previous step PROG 1 STEP 1 TIME 0 1 NEXT STEP gt B x FLOW 5 PREV STEP gt MAX PRESS 2000 MIN PRESS 0 DELETE gt EVENTS 1 C 2 0 3 0 4 C ONLY N EXIT gt Figure 3 12 Edit Screen 4 Gradient Step 1 10 Enter the values for each parameter as shown in Table 3 4 Edit Screen 4 Gradient Step 1 Table 3 4 Edit Screen 4 Gradient Step 1 Parameter Choices Setting Comments Step 1 150 The purpose of this step is to reset events 2 and 3to open Time 0 546 min 0 1 min Isocratic hold B 1 99 15 for proteins Composition at the end of 5 for peptides the step Flow 0 500 yL min 5 4L min Flow rate at the end of the step Max Press 0 3 500 psi 2 0
31. ABI 173A MicroBlotter User s Manual Applied KS Biosystems Copyright 2001 Applied Biosystems ABI PRISM and the ABI PRISM design Applied Biosystems Aquapore Brownlee GeneScan INHERIT Masterpiece MicroCoat MPLC NEWGUARD OPC POLYPORE Precipitette ProBlott PROCISE ProSort ProSpin SeqEd SPHERI10 SPHERI5 SynthAssist and VeloSep are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries ABI Amplicover Anitron AutoAssembler BaseSprinter Biobytes CATALYST FastPhoramidite GeneAssist Genotyper HLP Hot Start MicroBlotter ONESTEP PCR MATE PDQ Phosphalink ProFocus ProSorb Sequence Navigator StockMarks Stretch and Synergy are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries All other trademarks are the sole property of their respective owners Applied Biosystems February 2001 Contents Introduction About This Manual System Description Optional Equipment User Attention Words Safety Information To Get Started Quickly Technical Support Contacting Technical Support To Contact Technical Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax To Reach Technical Support Through the Internet To Obtain Documents on Demand Unpacking and Installation Introduction Preparing the Site Unpacking the System Components Description of the MicroBlotter Fr
32. Applied Biosystems 1 9 home position automatic reset 3 25 see MicroBlotter I important description of 1 6 injection of samples 3 22 when to inject samples 3 24 injector blockages 4 12 membranes see PVDF membrane membranes and filters 3 9 Index noise note O adjusting the tubing 4 6 automatic reset to home position 3 25 cleaning blotter tray covers 3 28 control panel description 3 11 description of 1 4 end position 3 8 handling samples after separation 3 26 home position 3 8 improper tracking speed 4 16 maintenance of 4 6 preparation for a run 3 8 proper blotting action 3 24 replacing the tubing 4 6 speed settings 3 10 start position 3 8 Teflon foot adjustment 4 6 Teflon foot replacement 4 6 increased baseline noise 4 14 loss of signal to noise 4 14 description of 1 6 operation P peaks materials required 3 4 overview 3 3 see instrument setup for a run see materials required too small 4 12 4 14 plumbing 140D C 1 173A system C 2 February 2001 Applied Biosystems preparation of samples 3 22 of samples for sequencing 3 28 PRESS parameter 3 24 pressure drop 4 14 gradual increase of 4 10 monitoring system pressure 4 4 monitoring the column pressure 4 4 sudden increase of 4 12 pressure increases see blockages PROG NO parameter 3 24 programming the 140D 3 13 to 3 21 entering a gradient program 3 14 programming timed steps 2 38 protein standa
33. Connecting Tubing with Teflon Couplers To make coupling connections using Teflon couplers 1 Remove the 0 012 X 0 062 tubing P N 225004 from the tubing kit 2 Using a razor blade cut a 3 4in length 3 Using a pointed object such as a thumb tack slightly enlarge each end of the tube 4 Push the capillary tubing all the way through the coupler until approximately 1 2 protrudes Trim 1 2 1 of the tubing from the protruding end and then pull back into the coupler until the end is halfway through it Then push the other piece of capillary to be joined into the coupler Try to make the two capillaries touch as shown in figure 4 2 Note When making connections with the flowcell tubing start with the line being joined to that of the flowcell so that the length of the flowcell tubing is not decreased Figure 4 2 Capillary tubing inside a Teflon coupler 48 4 Maintenance and Troubleshooting April 2002 Applied Biosystems Maintaining the 140D The 140D requires the following routine maintenance e Replace the piston and head seals every 6 months e Replace the rheodyne valve rotor seals after every 6 to 24 months of continuous use Refer to the maintenance section of the 140D user s manual for instructions on replacing these seals April 2002 4 Maintenance and Troubleshooting 4 9 Applied Biosystems Maintaining the 112A e The capillary column can be flushed regularly to help preven
34. Dial 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 only or 1 781 271 0045 1 800 542 2369 U S 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 Eastern As ia 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 22358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 750 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 0
35. G ULTRAVIOLET LIGHT HAZARD Exposure to ultraviolet radiation can cause blindness or permanent eye damage To prevent eye injury adjust the detector sensitivity from the ultraviolet to the visible range 500 nm before beginning any detector maintenance procedures Always wear protective UV absorbing glasses when looking into the detector Set the 785A wavelength by pressing WAVE gt then 210 then Enter Set the 785A range by pressing RANGE gt then 0 2 then Enter Set the 785A rise time by pressing RTIME gt then 1 0 then Enter O ee 2 08 Prepare the MicroBlotter by following the instructions listed in section 3 Preparing the MicroBlotter for a Run on page 3 8 7 Prepare the chart recorder by following the instructions listed in section 3 Preparing the Chart Recorder for a Run on page 3 12 2 Unpacking and Installation April 2002 Applied Biosystems 8 Program the 140D per the instructions listed under System Installation Test Program on page 2 36 Note This must be done prior to step 12 below 9 Prepare a solution of Dye Mark II by adding 38 uL of 0 1 TFA in DI water to a clean Eppendorf tube Then add 1 uL of each Dye Mark and vortex the solution for 30 sec 10 Load the Dye Mark solution into the 25 uL syringe provided with this system Insert the needle of the syringe into the 112A 11 Ensure the 112A is in load position 12 Begin execution of the test program by pressing Run on the 140D The Run S
36. GHT HAZARD Exposure to ultraviolet radiation can cause blindness or permanent eye damage To prevent eye injury adjust the detector sensitivity from the ultraviolet to the visible range 500 nm before beginning any detector maintenance procedures Always wear protective UV absorbing glasses when looking into the detector Turn off the power to the lamp and allow it to cool before removing it from its fixture Check the flowcell for air bubbles dirt or leaks Clean the flowcell with 100 methanol Column is deteriorating This can be accompanied by an increase or decrease in system pressure Remove the column and flush it per the procedure listed on page 4 5 Reinstall the column If the pressure is normal run the cLC Protein Standard per the instructions listed on page 4 21 If the chromatogram is not correct the column is bad Replace the column Remember that new columns can take 24 to 36 h of constant flow at 5 L min to completely hydrate Until then the system pressure will be lower than normal If the operating pressure remains low check the entire system for leaks and repair them accordingly If the operating pressure remains high a blockage may be present Follow the procedure listed on page 4 18 to locate and remove the blockage 4 Maintenance and Troubleshooting 4 15 Applied Biosystems Problem Possible Causes Recommended Actions Injector is partially blocked Bypass the injector If the pressure returns
37. LIED BIOSYSTEMS Limited System Warranty February 2001 Applied Biosystems Appendix B Sample Preparation Guidelines Sample Preparation Guidelines Prior to Injection onto the 173A System To prevent blockages samples must be as particulate free as possible before injecting them onto the capillary column in the 173A system When preparing your samples we strongly recommend you e Use only the highest quality reagents for digestions etc e Minimize the content of reagents in your samples which may interact with solvents or react to temperature changes and precipitate inside the system Precipitation occurring after sample injection can block the capillary tubing or column e Never inject samples which contain visible particulates e Centrifuge every sample for at least 5 min immediately prior to injection Protein Digestion Protocols To minimize the occurrence of blockages we recommend you use one of the following protein digestion protocols to prepare your samples The use of volatile digestion buffers minimizes the possible occurrence of blockage due to salt elimination For Large Proteins with Multiple Disulfide Linkages Example Bovine Serum Albumin To prepare a 500 ng to 100 microgram solution 1 In an Eppendorf tube dissolve the protein to be analyzed in a 100 to 200 uL solution of 250 mM Tris HCl pH 8 0 containing 2 M Guanidine HCl Add 10 pL of 10 b mercaptoethanol or DTT Flush the tube with argon for 1 min
38. Period All repairs and replacements under this Warranty shall be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems 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 the printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems shall not be binding on Applied Biosystems Limited System Warranty A 1 Applied Biosystems A 2 Applied Biosystems shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties such products or parts will be subject to the warranties if any of their respective manufacturers 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 KNOWN TO THE SELLER AND OF ANY OTHER OBLIGATION ON THE PART OF APP
39. aks of Interest Cleaning Blotter Tray Covers 3 Operation 3 3 3 4 3 4 3 1 Applied Biosystems 3 2 3 Operation April 2002 Applied Biosystems April 2002 Introduction This section describes how to use the ABI 173A MicroBlotter Capillary HPLC System 173A system to separate and prepare protein and peptide samples for analysis Because cLC separations often require the use of solvent gradient programming for sample elution a standard protein peptide gradient program has been included for your use The gradient conditions listed for peptides are adequate for most enzymatic digests and peptide mixtures The conditions listed for proteins can be used for most protein mixtures Optimization of this program may be required however for your particular application or sample Note The standard protein peptide gradient program provided in this section may require optimization for your particular application or sample Step by step instructions are provided for setting up the instruments programming the 140D and preparing your sample for injection onto the column Because the i d of the tubing in this system is so small particulates in your sample or precipitation occurring inside the system can block the tubing or column IMPORTANT To avoid blocking the column and tubing samples must be as free of particulates as possible prior to injection Refer to Appendix B Sample Preparation Guidelines Recommen
40. ar protective UV absorbing glasses when looking into the detector WARNING PHYSICAL INJURY HAZARD The lamp can become very hot while in use Turn off the power to the lamp and allow it to cool before removing it from the fixture Always wear protective gloves when handling the lamp 4 Maintenance and Troubleshooting 4 11 Applied Biosystems Troubleshooting the 173A System The following troubleshooting guide provides detailed information on most of the problems you may encounter while using the 173A system Additional troubleshooting information can also be found in the user s manual for each instrument If you require further assistance contact the Technical Assistance Center at Applied Biosystems The telephone number for the center is listed under Technical Support in section lof this user s manual 173A System Troubleshooting Guide Problem Possible Causes Recommended Actions No chromatogram Improper injection late chromatogram Improper injection occurs when the 112A is set to the inject rather than the load position when injecting the sample If the 112A is set to inject the sample will be lost Be sure the 112A is set to the load position before injecting your sample Injector completely or partially blocked This is usually accompanied by a sudden increase in operating pressure and smaller peaks Bypass the injector If the pressure drops back to the normal the injector is blocked Remove the bloc
41. ark component may coelute with a peak of interest 2 Centrifuge the sample for at least 5 min in a bench top microfuge 3 Remove your sample from the tube using the 25 uL syringe provided with the system Once you have loaded your sample into the syringe continue to Executing a Programmed Gradient Run on page 3 23 3 Operation April 2002 Applied Biosystems Executing a Programmed Gradient Run The following instructions are based on the execution of the standard protein peptide gradient program As a reminder runs can be stopped one of two ways 1 Press Manual to enter manual mode operation Set the flow rate to zero by pressing the FLOWS soft key entering the value zero and pressing the Enter key Or 2 Press Stop This will terminate the programmed or manual run and return you to the Ready screen Note Whenever a programmed run is stopped the MicroBlotter must be manually reset to the home position Refer to page 3 25 for instructions on resetting the MicroBlotter To run the standard protein peptide gradient program 1 Press Run to display the Run Select Screen Figure 3 14 Position the cursor at 1 under PROG Enter the program number and 1 for RUNS number of runs PROG RUNS PROG RUNS BEGIN gt Us 1 1 4 DELETE gt 5 6 MANUAL N Figure 3 14 Run Select Screen 2 Press the BEGIN gt soft key to start the program The syringes fill automatically and the Run Status Sc
42. arks are blotted onto the membrane These marks coincide with two major peaks in the chromatogram When using the standard protein peptide gradient program provided in this section the dye marks are typically visible towards the end of the run Figure 3 16 on page 3 27 Note The cLC Dye Mark is recommended for use with peptide samples only since most peptides elute earlier than the dye mark Because the dye mark may co elute with larger proteinaceous material the cLC Staining Dye is recommended for use with protein samples Once your run is finished the dye marks are used in conjunction with the pencil mark made on the PVDF membrane before the start of the run Figure 3 3 and 15 on page 3 10 to align the PVDF membrane with the chromatogram The stock solution of cLC Dye Mark is 1 mg in 10 mL of 0 1 TFA WARNING CHEMICAL HAZARD The cLC Dye Mark solution contains hazardous chemicals and therefore requires special handling Do not store handle or work with this solution unless you have received appropriate safety training and have read and understood all related Material Safety Data Sheets MSDSs Comply with all federal state and local laws related to chemical storage handling and disposal The MSDS for this solution is located in the Safety Summary P N 903918 for this manual cLC Staining Dye The cLC Staining Dye is recommended for use with protein samples only since most peptides are not stainable Use the staining dy
43. burns upon contact Secondary containment of solvents and waste bottles should be provided at all times e Solvent A 0 1 TFA Hy0O Solvent B 0 085 TFA acetonitrile Before adding TFA degas solvents by helium sparge or sonication The relative concentrations of TFA should produce a flat baseline Purging the System For more information on the 140D control panel and the purge procedure refer to section 2 of the 140D user s manual Note Commands executed by pressing soft keys F1 F2 F3 and F4 are shown in boldface type and are followed by a gt PURGE gt for example Press the Stop key at any time to halt this procedure and return to the Ready screen 2 Unpacking and Installation April 2002 Applied Biosystems To remove air from the 140D syringes 1 Place the inlet and waste lines on the solvent manifold of the 140D into the appropriate reservoirs 2 From the Ready Screen which is shown in Figure 2 16 press the PURGE gt soft key to display the purge screen Figure 2 17 140D x xx CLC FILL gt PRESS EVENTS PURGE gt CAPA CAP B VALVE gt UTILITY gt Figure 2 16 Ready Screen This is also referred to as the main menu Note The values you change or enter are shown in this manual in bold face in the following screens 3 Enter 15 for OF PURGES BOTH for SYRINGE and 100 for OF SYRINGE PURGE RATE 2 500 BEGIN gt SYRINGE BOTH OF PURGES 15 OF SYRINGE
44. cLC Protein Standard The cLC Protein Standard consists of 25 ug each Ribonuclease A Lysozyme and Apomyoglobin Reconstitute the protein standard before use as follows Reconstituting the cLC Protein Standard To reconstitute the cLC Protein Standard 1 Uncap the cLC Protein Standard vial Add 250 uL of 0 1 trifluoroacetic acid TFA solution to the vial WARNING CHEMICAL HAZARD Although dilute solutions are not hazardous undiluted TFA is an extremely dangerous and corrosive liquid Always wear gloves a lab coat and eye protection when handling TFA 2 Blanket the vial with inert gas 3 Cap the vial and vortex thoroughly Allow 20 min for the contents to dissolve mixing several times during this period 4 Store at 15 to 25 C The yield is 0 1 ug of each protein 1 uL of stock solution To use the cLC Protein Standard 1 Perform a run using the standard protein peptide gradient program provided in Section 3 Operation 2 Inject 5 uL of the protein standard onto the column at the appropriate point in the protocol 3 Stain the PVDF membrane as described under Staining Blotted Proteins with the cLC Staining Dye on page 3 27 in section 3 Evaluating a cLC Protein Standard Run The cLC Protein Standard consists of 3 proteins Three distinct peaks all baseline resolved should appear in the chromatogram Figure 45 on page 4 22 In addition the marks from the staining dye should be well defined and shoul
45. can applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRism 877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI Prism9 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 Biolnformatics 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 acid 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 1 Introduction April 2002 Applied Biosystems April 2002 Product or Product Area Telephone Dial Fax
46. d not exceed the width of the corresponding peak 4 Maintenance and Troubleshooting 421 Applied Biosystems SF If the chromatogram is satisfactory the 173A system is performing correctly If the chromatogram is not satisfactory perform the following checks 1 Check the pump for a delivery problem Refer to the troubleshooting guide on page 4 12 2 Check the column for deterioration by replacing it with a new column and running the protein standard 1 Ribonuclease A 2 Lysozyme 3 Apomyoglobin Sequence of Ribonuclease A KETAAAKFGRQHMDSSTSAASSSNY Sequence of Lysozyme KVFGRCELAAAMKRHGLDNYRGYSL Sequence of Apomyoglobin GLSDGEWQQVLMVWGKVEADIAGHG Figure 4 5 cLC Protein Standard chromatogram and sequences 4 22 4 Maintenance and Troubleshooting April 2002 Applied Biosystems April 2002 Storing the 173A System If your system will be idle for one or more weeks it is important to store the instruments and column properly We recommend the column be stored separately with 90 B and the 140D be purged of solvents To store the 173A system 1 Run the 140D at 5 4L min for 30 min at 90 B 2 Stop the 140D 3 Remove the column from the 112A 4 Cap both ends of the column and place it in an appropriate storage area 5 Remove the 140D solvent inlet lines from the buffer reservoirs 6 Loosely cover the inlet filters to keep them free of contaminants 7 From the Ready Screen pre
47. d using reagents which may interact with solvents or react to temperature changes and cause precipitation Precipitation occurring after sample injection can also block the tubing or column IMPORTANT Never inject a sample which contains visible particulates This can block the column and damage it beyond repair Refer to Appendix B for sample preparation recommendations When preparing your samples we strongly recommend you e Use only the highest quality reagents for digestions etc e Minimize the content of reagents in your samples which could precipitate in the system e Centrifuge every sample for at least 5 min before injection Sample preparation recommendations including protein digest protocols are provided in Appendix B Sample Preparation Guidelines To prepare your sample 1 For peptide solutions only Add the Dye Mark II dyes A and B to the sample solution such that 125 200 nL of each are injected The amount to be added to your sample will depend upon the volume of the sample loop used with the injector For example with the 5 pL loop installed add 1 2 uL of each dye A B to the sample solution bringing the final volume to 40 uL and using 0 1 TFA in water for any additional volume necessary to reach 40 pL When injected this will load 125 250 nL of each dye Larger loops will require additional dilution of the dye in the solution to be injected Do not add the dye mark to protein solutions a dye m
48. dations for sample preparation prior to injection are detailed in Appendix B Sample Preparation Guidelines Protein digestion protocols are included 3 Operation 3 3 Applied Biosystems Materials Required The following materials are required for most runs on the 173A system Materials Supplied with the 173A System PVDF membranes and filters P N 402010 Blotter trays P N 402009 Blotter tray covers P N 402140 Dye Markll P N 402256 These supplies are included in the Blotter staining tray P N 402155 173A Chemical Installation and cLC Staining Dye P N 402063 a KIEF UN Ape0ee Tweezers P N 402011 Tweezers P N 100071 Acetonitrile P N 400313 Trifluoroacetic acid TFA P N 400445 Materials Required but not Supplied Disposable gloves Bench top microfuge Single edge razor blades g These supplies can be purchased Methanol from a major laboratory supplier Distilled deionized water DI water Absorbent tissue or paper Eppendorf tubes Biobrene Plus recommended for sample Available from Applied Biosystems preparation prior to sequencing P N 400385 3 4 3 Operation April 2002 Applied Biosystems April 2002 Dye Mark II The Dye Mark II is recommended for use with peptides only since most peptides elute earlier than the dye mark The dye mark is added to your sample prior to injection During the run two major dye m
49. der Voltage Configuration and Fuse Installation Configure the chart recorder for the local voltage and install the appropriate fuses Instructions for voltage configuration and fuse installation for the optional Kipp amp Zonen chart recorder are listed below To configure the voltage and install the fuses for the optional Kipp E Zonen Chart Recorder 1 Move the red power selection switch on the back of the instrument to the appropriate position 2 Remove the appropriate voltage kit from the 173A Voltage Accessory Kit and install the fuses into the holders on the left hand side of the instrument 250 mA for 115 V 125 mA for 230 V 2 10 2 Unpacking and Installation April 2002 Applied Biosystems Preparing the 140D Four solvent transfer lines must be installed on the 140D three on the solvent manifold and one on the solvent outlet port of the mixing tee The transfer line installed on the mixing tee with a static mixer in line will be used to connect the 140D to port 3 on the injector valve in the 112A and will be installed later in the system set up see page 2 19 All of the parts listed in the following instructions except the glass reservoirs and secondary reservoir container are in the 140D Accessories Kit Instructions for installing the solvent transfer lines are also listed in section 2 Unpacking and Installation of the 140D User s Manual To prepare the 140D 1 Place the two bottles of solvent sup
50. e separate corresponding dye marks on the PVDF membrane indicate a successful installation Figure 2 29 on page 2 41 illustrates the dye marks and chromatographic results of a successful run using the cLC Dye Mark Note We strongly recommend you perform at least one more run using a standard before you begin normal operation of the 173A system Guidelines for executing a run using the cLC Protein Standard supplied with this system are on page 2 42 If the chromatogram or the PVDF membrane is not satisfactory check the pump for a delivery problem Refer to the troubleshooting guide provided in section 4 Maintenance and Troubleshooting If you require assistance call Applied Biosystems Technical Support Information on Technical Support is provided in section 1 Technical Support on page 1 9 2 Unpacking and Installation April 2002 Applied Biosystems April 2002 Chromatogram with peaks corresponding to the cLC Dye Mark cLC Dye Marks on the GR1053 Figure 2 29 Results of a successful run using the Dye Mark not to scale 2 Unpacking and Installation Applied Biosystems Performing Additional System Tests Before normal operation we strongly recommend you conduct at least one additional test using a standard to e Reverify that your system is functioning properly e Gain experience working with the instruments setting up the MicroBlott
51. e when you wish to see the location of protein fractions on the PVDF membrane At least 200 ng of protein per spot must be present for the stain to appear and be of use Note The cLC Staining Dye is recommended for use with protein samples only since most peptides are not stainable The cLC Dye Mark is recommended for use with peptide samples 3 Operation 3 5 Applied Biosystems 3 6 The cLC Staining Dye stock solution is 0 05 dye in 0 1 TFA weight volume Stained membranes can be stored wet for up to 2 months at 4 C WARNING CHEMICAL HAZARD The cLC Staining Dye contains hazardous chemicals and therefore requires special handling Do not store handle or work with this solution unless you have received appropriate safety training and have read and understood all related Material Safety Data Sheets MSDSs Comply with all federal state and local laws related to chemical storage handling and disposal The MSDS for this dye is located in the Safety Summary P N 903918 for this manual 3 Operation April 2002 Applied Biosystems April 2002 Operating the 173A System The 173A system is easy to use The steps typically involved in a run are 1 Preparing the instruments for a run Programming the 140D Preparing and injecting your sample 2 3 4 Executing the run 5 Excising peaks of interest from the PVDF membrane 6 Preparing the excised peaks for further analysis or storage Preparing the Instrum
52. elect Screen Figure 2 19 will be displayed PROG RUNS PROG RUNS BEGIN gt ES 1 4 DELETE gt 5 6 MANUAL N Figure 2 19 RUN SELECT Screen 13 Position the cursor at 1 under PROG Enter 9 the program number and 1 for RUNS number of runs 14 Press the BEGIN gt soft key to start the program The syringes fill automatically and the RUN STATUS Screen Figure 2 20 is displayed TIME FLOW B RUN PRESS EVENTS PROG NO CAPA CAP B STEP NO Figure 2 20 RUN STATUS Screen 15 Watch the 140D during the prepressurization phase The pump should reach 800 psi in less than 1 min If it does not check the system for leaks or purge the 140D again as appropriate April 2002 2 Unpacking and Installation 2 33 Applied Biosystems 2 34 16 Inject the cLC Dye Mark solution during equilibration Make sure the 112A is in the load position before injecting the dye 17 Once the solenoid on the MicroBlotter begins the blotting action adjust the height of the Teflon foot by raising or lowering it through the solenoid assembly as appropriate When the solenoid is at its lowest point the foot should slightly touch the membrane and the effluent from the tubing will immediately be absorbed by the membrane 18 Record the starting pressure of the column and check it weekly A gradual increase in pressure indicates the start of a blockage If this occurs flush the column per the instructions listed in section 4
53. ents for a Run Preparing the 140D for a Run The 140D must be configured and primed with the appropriate solvents before a run If you are not familiar with these procedures refer to Section 2 Unpacking and Installation of the 140D user s manual P N 903586 A brief description of the 140D control panel how to move the cursor and how to move from one screen to another is also provided in this section To prepare the 140D for a run 1 Ensure adequate quantities of solvents A and B are present 2 Check the solvent lines for proper immersion in each solution 3 Ensure that all the air has been purged from the 140D by priming the system 15 times at 100 for both syringes Instructions for priming the 140D are provided in the 140D user s manual 4 Ifthe run is to be automated enter your program now Total run time should not exceed 200 min The standard protein peptide gradient program with entry instructions begins on page 3 15 Preparing the 785A for a Run Set the detection wavelength as desired For the standard protein peptide gradient program select one of the following settings For proteins 210 nm 1 5 Absorbance Units Full Scale AUFS with a 1 sec rise time For peptides 210 nm 0 1 Absorbence Units Full Scale AUFS with a 1 sec rise time 3 Operation 3 7 Applied Biosystems Control panel 3 8 LI mm min ABI 173 MicroBlotter Preparing the 112A for a Run To prepare the 112A for a run 1 S
54. er and handling samples after a run The cLC Protein Standard supplied with this system can be used to perform additional tests Instructions are provided below You will program the 140D using the standard protein peptide gradient program provided in section 3 Operation Again an explanation of the different phases of a programmed run prepressurization equilibration and gradient is provided in the Operation section of the 140D user s manual We recommend you thoroughly review these sections before proceeding with this test To test the 173A system using the cLC Protein Standard 1 Make sure you have all the materials required for a run These are listed in section 3 on page 3 4 2 Setup the instruments per the instructions listed under Preparing the Instruments for a Run section 3 page 3 7 through page 3 13 3 Program the 140D per the instructions listed under Programming the 140D section 3 page 3 13 through page 3 22 Be sure to use the values specified for proteins 4 Prepare the cLC Protein Standard per the instructions listed under Using the cLC Protein Standard in section 4 page 4 21 5 Load 5 uL of the cLC Protein Standard into the syringe provided with this system 6 Run the standard gradient program per the instructions listed under Executing a Programmed Gradient Run section 3 page 3 23 Be sure the 112A is set to the load position before injecting the standard 7 Watch the 140D duri
55. et the 112A to the load position This instrument resets to the load position automatically after every run 2 Set the temperature to 37 C IMPORTANT Ifa power failure occurs or if the power cord is disconnected the 112A will default to the inject position when power is returned If this occurs you must reset the instrument to the load position otherwise your sample will be lost Preparing the MicroBlotter for a Run A blotter tray cover is placed over the PVDF membrane to keep it from drying out during the run These covers can be reused until they crack break or become too contaminated for satisfactory sequencing results Cleaning instructions for the blotter tray covers are listed under Cleaning Blotter Tray Covers on page 3 28 To prepare the MicroBlotter for a run 1 Ensure the solenoid is in the home position as far left as possible in the recess behind the MicroBlotter control panel Figure 3 1 If it is not insert the end of a paper clip into the hole marked Reset and press the reset button 2 Make sure the end of the teflon foot and the end of the tubing Figure 3 1 are flush with each other This is critical for proper blotting action Home position End position Solenoid in the start position Teflon foot Blotter tray The end of the Teflon foot must be flush with the end of the tubing Figure 3 1 Front panel of MicroBlotter 3 Operation April 2002 Applied Biosystems 3 Wearing
56. fill rate To configure the 140D follow the configuration procedure listed in section 2 of the 140D User s Manual If you are not familiar with how to operate and program the 140D we strongly recommend you also review section 2 Unpacking and Installation and section 5 Software Reference of the 140D user s manual These sections contain detailed information on the front panel control keys and the functions of the various screens used to program and operate the 140D 2 Unpacking and Installation 2 27 Applied Biosystems 2 28 Priming the System Priming the system involves purging the solvent transfer lines of air and particulates from the 140D to the column in the 112A Trapped air can cause chromatographic anomalies particulates can block the tubing or column Once the system is operational purges are also performed to replace old solvent with new solvent The 140D must be primed e As part of the installation procedure e Whenever a solvent change is made e Each time a new run is started To purge the system first prepare solvents A and B if necessary then follow the procedures listed under Purging the System Preparing the Solvents Solvent purity is essential for the optimum operation of any LC system Impurities from dirty glassware can cause baseline anomalies Prepare solvents A and B in the following concentrations WARNING CHEMICAL HAZARD TFA is extremely dangerous and can cause severe acid
57. firmly at the same time This will force out air bubbles and excess fluid and will cause the bottom filter and membrane to adhere to each other 12 Gently lift off the top filter and discard 13 Place a clean blotter tray cover on top of the PVDF membrane as illustrated in Figure 3 4 Pressing down firmly run your gloved finger evenly across the length of the cover to create a seal Note If a blotter tray cover is not used the membrane could dry out before the end of the run Tray covers must be thoroughly cleaned before reuse See Cleaning Blotter Tray Covers on page 3 28 Blotter tray cover Figure 3 4 Blotter tray cover positioned on top of the PVDF membrane and filter 14 Place the blotter tray into the housing on the MicroBlotter The tray positioning notch Figure 3 3 ensures correct placement of the tray 15 Make a small mark on the PVDF membrane with a pencil directly below the chromatogram start position mark on the blotter tray Figure 3 3 16 Set the MicroBlotter speed to 1 or 2 mm min by pressing the toggle button on the control panel Figure 3 5 3 10 3 Operation April 2002 Applied Biosystems Reset button Speed indicator LEDs Toggle button for the speed setting Figure 3 5 MicroBlotter control panel April 2002 3 Operation Applied Biosystems Preparing the Chart Recorder for a Run Two sets of instructions are listed below One set is writte
58. g Through the Solenoid If adjustment is required gently pull up on or push down on the tubing Perform a run using the cLC Protein Standard and the sample protein peptide gradient program to test the adjustment Instructions for using the protein standard are listed on page 4 21 the sample protein peptide gradient program is listed in section 3 on page 3 14 Replacing the Tubing That Runs Through the Solenoid IMPORTANT Always turn the instrument power off before replacing the tubing To replace the tubing that runs through the solenoid 1 Disconnect the tubing from the 112A to the MicroBlotter by pulling it apart at the Teflon coupler between the two instruments 2 Unplug the solenoid electrical connector and unscrew the solenoid assembly from the drive arm 3 Unscrew the solenoid and remove the old tubing and Teflon foot 4 Cutanew 15 in piece of 30 uM i d capillary tubing P N 225118 in the 173A Tubing Kit 5 Thread the tubing through the sleeve inside the solenoid core until approximately 2 in of tubing protrudes from the blunt end of the core Figure 4 1 on page 4 7 6 Re assemble the solenoid 7 Cut anew 1 4 in length of 0 012 x 0 009 Teflon tube P N 225119 in the 173A Tubing Kit This will be used as the Teflon foot 8 Push the Teflon foot onto the end of the capillary protruding from the bottom of the solenoid assembly until it is flush with the end of the tubing IMPORTANT _ The Teflon foo
59. g and Removing a Blockage on page 4 18 Leakage in a system component or somewhere along the capillary tube route Visually check the entire system for leaks If leakage is spotted at a coupling sleeve push the tubing together if it has pulled apart If leakage continues replace the coupling sleeve If leakage is spotted inside an instrument at a seal or other connection replace the seal or PEEK sleeve and fitting Refer to the appropriate user s manual for replacement instructions New column not completely hydrated New columns can take 24 to 36 h of constant flow at 5 L min to completely hydrate Until then the system pressure will be lower than normal Loss of signal to noise resulting in smaller peaks or Increased baseline noise 4 14 Leakage in system or Faulty detector lamp or electronics First follow the instructions listed to the right to determine whether the problem is being caused by leakage or the detector To determine the cause of a noisy baseline set the flow rate to O and monitor the ABS trace on the chart recorder If the noise stops there is probably a leak in the system that is causing pressure problems If the problem remains the detector may not be functioning properly Each possible cause and the recommended action s are listed below individually Leaks 4 Maintenance and Troubleshooting Inspect all fittings for leaks If no leaks are apparent perform t
60. gloves remove one PVDF membrane with filters from the box Figure 3 2 The filters have a tendency to adhere to the membrane giving the appearance that one or both filters may be missing Note To prevent sample contamination always wear gloves and use tweezers when handling filters and PVDF membranes VA Filters PVDF membrane E Figure 3 2 PVDF membrane and filter sets Each membrane is sandwiched by a pair of filters Filters tend to adhere to the membrane 4 Using tweezers remove one of the filters and position it in the blotter tray as shown in Figure 3 3 5 Wet the filter thoroughly with DI water 6 Tilt the blotter tray and allow the excess water to drain onto an absorbent tissue 7 Separate the PVDF membrane from the second filter 8 Wet the membrane thoroughly with 100 methanol 9 Place the membrane directly on top of the filter in the blotter tray 10 Place the second filter directly on top of the membrane April 2002 3 Operation 3 9 Applied Biosystems Chromatogram start Tray positioning position mark notch Solenoid tracking references Pencil mark on PVDF membrane Figure 3 3 MicroBlotter tray top view The solenoid moves across the membrane between the solenoid tracking references depositing the sample and creating a slight impression in the membrane 11 Starting at one end run your gloved finger evenly along the top filter pressing down
61. h of 0 012 x 0 062 225060 tubing P N 225004 0323 0003 CAUTION Do not position a 50 um ID 15 in connection directly over the Detector solenoid If there is a leak it Flowcell will drip into the solenoid This can short the solenoid H and cause corrosion Teflon sleeve Use Peek sleeves over capillary 225060 I Peek Sleeve 225118 Ferrule 225123 30 um ID 15 in Capillary 225118 tubing 30 um ID 15 in E Bushing E Teflon Foot use a 1 4 in length of 0 012 x 0 009 tubing P N 225119 Microblotter El solenoid Co Figure 2 10 Plumbing diagram for the 173A system The tubing lengths suggested above are for the side by side configuration illustrated in Figure 2 1 on page 2 4 To connect the 140D to the 112A If not already installed replace the 20 uL sample loop in the 112A with the 5 pL loop P N 201433 in the 173A Voltage Accessory Kit Install the 5 uL loop between ports 1 and 4 of the injection valve 8 Twist together the pair of waste lines connected to ports 5 and 6 of the injector and route them via the 140D to the purge waste bottle 9 Cutan 8 in length of 50 ym capillary tubing P N 0323 0003 in the 173A Tubing Kit April 2002 2 Unpacking and Installation 2 19 Applied Biosystems 2 20 10 11 12 13 Connect the tubing to port 3 of the injector using a bushing P N 200249 PEEK sleeve P N 225123 and ferrule P N 200247 Follow the procedure listed under Assembli
62. he static pressure test provided in the 140D user s manual Tighten or replace fittings and seals as required Instructions for replacing seals in the 140D are listed in the 140D user s manual April 2002 Applied Biosystems Problem Possible Causes Recommended Actions April 2002 Detector lamp going bad Check the reference energy values for the detector If low 15 or higher replace the lamp Refer to the 785A user s manual for replacement instructions WARNING ULTRAVIOLET LIGHT HAZARD Exposure to ultraviolet radiation can cause blindness or permanent eye damage To prevent eye injury adjust the detector sensitivity from the ultraviolet to the visible range 500 nm before beginning any detector maintenance procedures Always wear protective UV absorbing glasses when looking into the detector Turn off the power to the lamp and allow it to cool before removing it from its fixture Detector lamp set at low power Detector not warmed up Detector lamp not seated properly Set lamp power to high Be sure the detector is warmed up before use Reseat the lamp Detector wavelength setting incorrect Air bubbles dirt or leaks in flowcell For most applications the wavelength should be set at 210 nM Wearing the proper protective glasses verify the detector is emitting the proper wavelengths green light at 555 nM fairly bright ruby red light at 656 657 nM WARNING ULTRAVIOLET LI
63. ible sample loss Once your sample has been separated and blotted onto a PVDF membrane you simply line up the membrane with the corresponding chromatogram and excise the fractions peaks you wish to analyze The excised membranes can then be directly loaded into an automated protein peptide sequencer for analysis 173A system components have been selected and designed to ensure maximum reproducibility sensitivity and sample recovery System components include e The ABI 140D Microgradient Delivery System P N 140D 01 The 140D accurately forms gradients at very low flow rates typically 3 to 10 wL min It eliminates the need for using a split technique which is required by other HPLC systems to achieve similar flow rates e The ABI 112A Oven Injector P N 0650 0016 The 112A is a column injection oven which loads samples onto a capillary peptide column for separation and analysis e The ABI 785A Programmable Absorbance Detector P N 604050 The 785A monitors the elution of the sample analytes proteins peptides as they pass through the capillary flowcell The ABI 173 MicroBlotter P N 173 0 The MicroBlotter is a membrane based sample collector which eliminates manual fraction collection Samples are collected directly onto a PVDF membrane after separation via a push pull solenoid Activated by the 140D the solenoid gently contacts the terminus of the capillary tubing to the membrane as samples are eluted from the column
64. in section 2 on page 2 42 The standard protein peptide gradient program provided in section 3 on page 3 14 can be used as the basis for your programmed gradient runs The conditions listed for peptides are adequate for most enzymatic digests and peptide mixtures The conditions listed for proteins can be used for most protein mixtures Optimization of this program will be required however for your particular applications and samples IMPORTANT To avoid blocking the column and system tubing samples must be as free of particulates as possible prior to injection Refer to Appendix B Sample Preparation Guidelines for more information on sample preparation and purification 1 Introduction April 2002 Applied Biosystems April 2002 Technical Support Contacting Technical Support You can contact Applied Biosystems for technical support by telephone or fax by 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 Support by E Mail Contact technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystem
65. information It also indicates a potentially hazardous situation which could result in minor or moderate injury to the user WARNING Serious physical injury to the user or other persons could occur if these required precautions are not taken 1 Introduction April 2002 Applied Biosystems Safety Information Safety warnings addressing chemical and high voltage issues appear throughout this user s manual and the ABI 173A MicroBlotter Capillary HPLC System Safety Summary P N 903918 The Safety Summary is a stand alone document located in the front pocket of this binder It contains detailed information provided to ensure the safe installation operation and maintenance of the 173A system The Safety Summary also contains Material Safety Data Sheets MSDSs for the chemicals that are shipped with this system The safety warnings listed throughout this manual appear in the following formats WARNING ELECTRICAL SHOCK HAZARD The ABI 173A MicroBlotter Capillary HPLC System is comprised of instruments containing high voltage power supplies Always disconnect the instrument s from their power source before changing a fuse adjusting the voltage selection or opening the instrument for maintenance or any other reason WARNING CHEMICAL HAZARD Some chemicals used with this instrument are considered hazardous Hazard warnings are prominently displayed on the bottle labels of all hazardous chemicals The Safety Summary for this manual contain
66. ing dye warning 3 6 trifluoroacetic acid 3 4 warnings see safety chromatograms baseline position 3 12 how to line up with membranes 3 26 problems with 4 12 start position 3 10 cLC dye mark see dye mark cLC Protein Standard see protein standard cLC staining dye see staining dye column 1 5 blockage indicator 3 25 blockages 4 5 flushing 4 4 how to flush 4 5 how to store 4 23 hydration of 3 25 45 maintenance of 4 5 monitoring column pressure 4 5 recording the starting pressure of new columns 3 25 4 4 replacement of 4 5 couplers teflon 48 Applied Biosystems D description of 173A system 1 4 dye mark description of 3 5 see safety stock solution 3 5 E electrical warnings 1 7 end position MicroBlotter 3 8 EVENTS parameter 3 24 F filters how to install in tray 3 9 filters and membranes 3 9 FLOW parameter 3 24 flow rate too low 4 12 flushing the column 44 45 foot instrument setup for a run 3 7 to 3 MicroBlotter 12 112A 38 140 3 7 785A 3 7 chart recorder 3 12 MicroBlotter 3 8 lamp 785A replacement of 411 785A testing of 411 M maintenance flushing the column 4 5 for the 173A system 4 4 of the 112A 410 of the 140D 49 of the 785A 4 11 of the column 4 5 of the MicroBlotter 46 adjusting the Teflon foot 4 4 material safety data sheets 1 6 see also MicroBlotter G gradient run see programming the 140D see runs see standard gradient program H help how to contact
67. ing the A blotter tray cover was either MicroBlotter for a Run on page 3 8 in Section 3 Operation not used or not properly If the membrane is still drying out check it halfway through an installed over the PVDF extended run Cautiously add small amounts of DI water dropwise membrane by pipette or squirt bottle to the right most corner of the membrane Membrane MicroBlotter tubing positioned Raise the tubing which runs through the MicroBlotter solenoid by damaged too low hand The Teflon foot should contact the PVDF membrane just enough to make a slight impression 112A injector valve Power resets of the 785A and Check for loose line connections on all devices activates out of 140D activate the injector Check the polarity of the control cables between the 140D and sequence or valve other devices without Make sure the injector is set to the load position before starting programmed each run direction Chart recorder or Speed on the chart recorder or Check the speed setting on the chart recorder and the MicroBlotter not MicroBlotter not set correctly MicroBlotter They should be identical tracking at the Check the voltage setting on the chart recorder proper speed 4 16 4 Maintenance and Troubleshooting April 2002 Applied Biosystems Problem Possible Causes Recommended Actions The speed of the MicroBlotter Check the calibration of the chart recorder and MicroBlotter by or chart recorder is off making a ma
68. inse with DI water for 1 min 3 Transfer the membrane to the Blotter staining tray and add enough cLC Staining Dye to cover the entire membrane Soak the membrane for 2 min 4 Wash the membrane 4 times with 50 mL of DI water each time 5 Discard the stain Fresh stain should be used for every membrane Stained membranes can be stored wet for up to 2 months at 4 C April 2002 3 Operation 3 27 Applied Biosystems 3 28 This stain will not produce additional junk peaks when the sample is sequenced However you may experience lower yields of histidine and arginine residues Preparing Peptides for Sequencing Before sequencing peptides we recommend you add Biobrene Plus to your sample Note Biobrene Plus is available from Applied Biosystems Ask for P N 400385 To prepare peptides for sequencing 1 Mix 1 vol Biobrene Plus stock solution with 1 vol 0 1 TFA and 2 vol MeOH The final concentration is 25 yg of Biobrene Plus 2 Apply 2 uL of this solution to each piece of excised PVDF membrane 3 Allow the pieces to dry completely Once dry your samples are ready for sequencing Storing Excised Peaks of Interest For samples that will not be subjected to further analysis the same day they are excised cap the tubes and store them at 4 C Cleaning Blotter Tray Covers The blotter tray covers supplied with this system can be reused until they crack break or become contaminated To clean and inspect bl
69. install the fittings Bad or improperly seated detector lamp If the lamp is still good reseat it if necessary Replace the lamp if it has expired Be sure the new lamp is properly seated April 2002 4 Maintenance and Troubleshooting 4 13 Applied Biosystems Problem Possible Causes Recommended Actions Drifting baseline either up or down during a run Solvents improperly balanced Replace solvents A and B with fresh solutions Make sure the proper amount of TFA has been added to both solvents Solvents aged or poorly prepared Replace solvents with fresh solutions and degas Residual air in solvents Degas solvents by helium sparge or sonication Significant drop in system pressure of 200 psi or more A fitting has popped apart Possible causes include blockage tubing accidentally pulled apart or too high a flow rate 1 Check the flow rate Lower the rate if it is too high Monitor the system pressure If the problem persists continue to step 2 2 Visually check all fittings and connection points along the tubing route If a fitting has popped apart replace the coupling sleeve or the PEEK sleeve and fitting as appropriate Reconnect the tubing Monitor the system pressure If the problem persists a blockage is most likely present at some point after the fitting that popped apart Track the blockage and replace the appropriate part per the instructions listed under Locatin
70. ipped in for signs of damage If any damage has occurred save all the packaging materials for inspection and contact the shipper 9 Verify that you have received the tubing and accessory kits listed below Check the contents of each kit against the corresponding packing list Packing lists are also included in Appendix D If any parts are missing contact your Applied Biosystems representative or Applied Biosystems technical support immediately Refer to section 1 Technical Support on page 1 9 for the technical support number Instrument Part Number 140D Accessory Kit P N 603973 785A Accessory Kit P N 604091 112A Accessory Kit P N 0602 0081 173A Voltage Accessory Kit P N 604030 173A System Tubing Kit P N 604066 inside kit 604030 2 Unpacking and Installation 2 5 Applied Biosystems 2 6 10 Place each instrument on the table or bench where the system will be used Figure 2 1 on page 2 4 illustrates the recommended configuration Allow 1 to 2 in between each instrument except those that are stacked and at least 5 in clearance behind the instruments to provide unobstructed air flow for ventilation 2 Unpacking and Installation April 2002 Applied Biosystems Description of the MicroBlotter Front Panel The front panel of the MicroBlotter is illustrated in Figure 2 2 e Speed Toggle Switch and Indicators The speed at which the solenoid moves across the PVDF membrane in the blotter tray The speed is set to 1
71. iption 0301 0006 1 Terminal Block 14 Position F 0381 0015 3 Ferrule 1 8 in SST 0392 0010 1 Tool Wrench 7 16 in Combo 0403 0063 2 Bushing 1 16 in SS 0403 0064 2 Ferrule 1 16 in SS 0403 0161 1 Plug 1 16 in Shut Off 200371 2 Bushing 1 8 in SS 3100 0167 120 in Tubing Teflon 1 16 x 1 8 5400 0010 2 Tool Wrench Open End 1 4 x 5 16 6000 0112 6 in Wire Bus 20 GA 604218 2 Assy Bottle Cap 903585 1 List w Instructions 140D 903586 1 Manual Users 140D T 6134 1 Tool Install Back up Ring 112A Accessory Kit P N 0602 0081 Part Number Quantity Description 200235 2 Bushing Ex long 200315 1 Syringe 100 yL 904069 1 Customer Letter CE Emission Immunity 0540 0009 1 Manual Users 112A Oven Inj 0954 0160 1 Inst Packaging 112A Accy Kit 0162 0049 1 Connector D9 Fem Solder Cup 0171 007 2 Ferrule 1 16 in Tube Rheodyne 2500 0985 2 pkg Fitting Nut Ferrule PEEK 5400 0010 1 Tool Wrench Open End 1 4 x 5 16 D 4 Accessory and Installation Kits February 2001 Applied Biosystems Index B parameter 3 24 Symbols 112A accessory kit D 4 activates on its own 4 16 activates out of sequence 4 16 checking the oven performance 410 description of 1 4 inject position default 3 8 maintenance of 4 10 preparation for a run 3 8 temperature setting 3 8 140D accessory kit D 4 description of 1 4 entering a gradient program 3 14 how to store 4 23 maintenance of 4 9 plu
72. it is flush with the front end of the PEEK sleeve Figure 2 9 Compression type fitting assembly using a PEEK sleeve 6 7 Cut 1 2 in off the end of the tubing to remove any particles from the bore A For the mixing T connection in the 140D it is critical that the tubing not bottom against the back of the mixing channel Blockages can result if this occurs Refer to step 13 on page 2 20 To properly position the tubing pull it back through the PEEK sleeve until it is flush with the end of the sleeve Re install the fitting into the mixing T outlet port This connection must be very tight To test the connection tug on the tubing If it slips out of the port repeat this procedure starting from step 4 B For all other connections insert the fitting back into the port and maneuver the capillary until it bottoms in the port Tighten the bushing To test the connection tug on the tubing If it slips out of the port repeat this procedure starting from step 4 2 Unpacking and Installation April 2002 Applied Biosystems Sleeve 225123 Bushing 200249 Sleeve 225123 Sleeve 225123 Ferrule 200248 Bushing 200234 Bushing 200249 Ferrule 200247 Ferrule 200248 0323 0003 _ 440D 50 um ID 24 in Mixing L Static Port2 Port3 0323 0003 Column E Mixer Teflon sleeve Injector 50 pm ID 8 in 225060 00000 5 pL Sample loop P N 201433 Coupling sleeve use a 3 4 in Teflon sleeve lengt
73. jumper 0478 0004 1 Instr Volt Selection 785 759 0478 0006 1 Instr Volt Selection 140B D 0478 0009 1 Instr Volt Selection 112A 0502 0077 1 Cable Asy Interconnect 2900 0078 2 Aperture Dry Cell 5100 0005 1 Fuse 1 A 250 V FB 1 1 4 x 1 4 6000 0074 1 Power Cord US Canada 120 VAC 10 A 6000 0250 1 Power Cord UK 240 VAC 10 A 6000 0251 1 Power Cord Ger France 220 V 10A 6000 0252 1 Power Cord Swiss 6000 0590 1 Control cable 172 251378 1 Connector Start Signal 401966 1 Capillary column 0 5 x 150 mm 603237 1 Socket Strip Asy Accessory and Installation Kits D 1 Applied Biosystems 604066 604103 Kit Tubing see ABI 173A Tubing Kit below Asy Cable Pump Rec twisted pair 903836 903886 Manual Users MicroBlotter Instr Pkg Chklst 173A Volt Kit 904069 Customer Letter CE Emission Immunity 173A Tubing Kit P N 604066 packed inside the 173A P N 604030 Part Number Quantity Description 5935 1 Capillary Flowcell 903887 1 Instr Pkg Chklst 173A Tube Kit 0323 0003 3m Tubing Silica 50 ym x 375 ym o d 0658 0001 1 pkg Pkg Capillary Cutters 200247 4 Ferrule 1 16 SS Tube Rheodyne 200248 6 Ferrule 1 16 in Tube SST 200249 6 Bushing 1 16 Tube Short SST 200234 4 Bushing 1 16 in SS long 201433 1 5 AL Sample Loop 201513 4 Elbow 1 16 in tubing 90 201514 3 Elbow 1 16 in tubing 180 201551 1 Frits Static
74. kage per the instructions provided in the 112A user s manual Use the cLC Protein Standard to retest the system after making the repair The normal initial operating pressure of every system will vary Your system s pressure should fall between 550 to 900 psi when using the standard protein peptide gradient program conditions listed in section 3 Operation The head or piston seals in the 140D are leaking resulting in lower flow rates than expected Leakage present between the column and detector Check the head and piston seal leak points Replace the seals if necessary Refer to the 140D user s manual for instructions on checking the leak points and replacing these seals Visually check for leaks between the column and detector If leakage is found repair as appropriate The detector is not on or the lamp has burned out First make sure the detector is turned on If so run the diagnostics listed in the 785A user s manual to test the lamp Replace the lamp if necessary WARNING ULTRAVIOLET LIGHT HAZARD Exposure to ultraviolet radiation can cause blindness or permanent eye damage To prevent eye injury adjust the detector sensitivity from the ultraviolet to the visible range 500 nm before beginning any detector maintenance procedures Always wear protective UV absorbing glasses when looking into the detector Turn off the power to the lamp and allow it to cool before removing it Bad electrical connec
75. leads to terminal 3 and the black lead to terminal 4 Attach the D connector to the 785A remote connection Attach the twisted pair cable P N 604103 to terminals 5 and 6 Event 3 Attach the connector on the other end of the cable to the start signal connection on the back of the MicroBlotter Figure 2 3 on page 2 8 If you are using the Kipp amp Zonen chart recorder P N 400268 attach the chart recorder cable 15 pin D connector P N 604151 to terminals 7 and 8 Event 4 Attach the D connector to the chart recorder If you are using a different chart recorder connect the appropriate cable to terminals 7 and 8 Event 4 April 2002 2 Unpacking and Installation 2 15 Applied Biosystems 6 Attach the 20 GA wire P N 6000 0112 in the 140D Accessory Kit to terminals 11 and 12 Inject of the terminal block 7 Plug the terminal block into the back of the 140D 8 If you are using the Kipp amp Zonen chart recorder remove one of the detector signal cables P N 6000 0186 from the 785A accessory kit Push the jackplug into the Record Out connection on the back of the 785A Attach the leads at the other end of the cable to the ve and ve inputs for the small pen on the back of the chart recorder Or use the cables supplied with the Kipp amp Zonen chart recorder If you are using a different chart recorder connect it appropriately to the Record Out connection on the back of the 785A 9 Ifadata system will be used to c
76. limit compatible with your column Min Press 0 3 500 psi Your choice Typically set to zero 7 Press the NEXT STEP gt soft key to advance to Edit Screen 3 Figure 3 11 3 Operation April 2002 Applied Biosystems PROG 1 NEXT STEP gt EQUILIBRATE TIME 30 PREV STEP gt EVENTS 1 C 2 C 3 C 4 C EXIT gt Figure 3 11 Edit Screen 3 8 Set the values in Edit Screen 3 to those shown in Table 3 3 The equilibration conditions defined in Edit Screen 3 are typically equivalent to the time zero composition and flow rate at which the gradient will begin During equilibration time zero conditions are held to achieve a steady state before beginning the gradient Equilibration times will differ based on the column sample type and solvents being used and must be adjusted to ensure the most reproducible retention times possible throughout a multigradient run Events are also set on this screen For the standard protein peptide gradient program all the events are set to Closed C This instructs the 140D to start the 112A 785A MicroBlotter and chart recorder Note Events in Edit Screen 3 occur at the end of equilibration Events designated in gradient steps occur at the beginning of the step Table 3 3 Edit Screen 3 Equilibration Parameter Choices Setting Equilibrate Time 0 546 min 30 min for both proteins and peptides Events 1 2 3 4 O for Open 1 C 2 C 3 C 4 C C for Closed Event 1 Injector 112
77. lter can be configured for helium sparging Simply pull the tubing down through the bottle cap until the length that will be inside the bottle matches that of the tubing with the filter Figure 2 6 Attach one of the solvent inlet filters from the 140D Accessory Kit P N 200270 to the end of the tubing Otherwise remove this line including the plastic fitting 8 Connect one bottle cap delivery line to port A on the 140D solvent manifold the other to port B 9 Screw the bottle cap assemblies onto the appropriate solvent bottles April 2002 2 Unpacking and Installation 2 13 Applied Biosystems Preparing the 785A A dry cell and capillary U shaped flowcell must be installed in the 785A To prepare the 785A 1 Open the front panel of the instrument 2 Ifnot already installed install one of the dry cells P N 0403 0174 from the 173A Voltage Accessory Kit on the left side of the detector monochromator Tape the spare dry cell to the inside of the front panel 3 Install the capillary U shaped flowcell P N 005935 in the 173A Accessory Kit in the right side of the detector monochromator IMPORTANT Handle the flowcell along the outer edge only Touching or placing pressure on the exit capillary tubing in the flowcell housing can cause it to break See Figure 2 7 below 4 Route the tubing through the slot in the detector head top plate Be careful not to break the capillary tubing when closing the compartment door 5
78. m Maintenance Maintaining the Column Flushing the Column Maintaining the MicroBlotter Adjusting the Tubing Running Through the Solenoid Replacing the Tubing That Runs Through the Solenoid Connecting Tubing with Teflon Couplers Maintaining the 140D Maintaining the 112A Maintaining the 785A Troubleshooting the 173A System 173A System Troubleshooting Guide Locating and Removing a Blockage Using the cLC Protein Standard Reconstituting the cLC Protein Standard Evaluating a cLC Protein Standard Run Storing the 173A System 2 44 3 7 3 13 3 22 3 23 3 26 3 26 3 27 3 28 3 28 3 28 4 3 4 3 4 3 4 3 4 4 4 5 4 5 4 6 4 6 4 6 4 8 4 9 4 10 4 11 4 12 4 12 4 18 4 21 4 21 4 21 4 23 February 2001 Applied Biosystems February 2001 A Limited System Warranty B Sample Preparation Guidelines Sample Preparation Guidelines Prior to Injection onto the 173A System Protein Digestion Protocols Sample Preparation Recommendations Prior to Sequencing Recommendations for Sequencing Peptides C Plumbing Parts and Connections 140D Plumbing Diagram ABI 173A MicroBlotter Capillary HPLC System Plumbing Diagram D Accessory and Installation Kits A l B 1 B 1 B 1 B 3 C2 D 1 Applied Biosystems April 2002 1 Introduction Contents About This Manual System Description Optional Equipment User Attention Words Safety Information To Get Started Quickly Technical Support Contacting Technical Support To Co
79. mbing diagram C 1 preparation for a run 3 7 programming 3 13 to 3 21 seal replacement 4 9 status messages displayed during a run 3 24 will not run 417 173 MicroBlotter see MicroBlotter 173A system chemical and accessory kit D 3 description of 1 4 locating and removing blockages 4 18 to 4 20 plumbing diagram C 2 storage conditions 4 23 storage guidelines 4 4 troubleshooting guide 4 12 tubing kit D 2 use after storage 4 23 voltage accessory kit D 1 warranty A 1 February 2001 785A accessory kit D 3 description of 1 4 lamp replacement 4 11 maintenance of 4 11 preparation for a run 3 7 wavelength settings 3 7 A accessory kits 112A D 4 140D D 4 173A chemical accessory D 3 173A tubing D 2 173A voltage accessory D 1 785A D 3 acetonitrile 3 4 see also safety B baseline abnormalities and shifts 4 13 drifting 4 14 increased noise 4 14 baseline chromatogram see chromatograms biobrene plus 3 4 3 28 B 3 blockages how to avoid 4 5 in the column 4 5 locating and removing 4 18 to 4 20 blotter tray covers see tray covers C CAP A parameter 3 24 CAP B parameter 3 24 caution description of 1 6 centrifugation recommendations 3 22 Index chart recorder control panel 3 13 improper tracking speed 4 16 pen positioning 3 12 preparation for a run 3 12 speed selection 3 12 chemical warnings 1 7 chemicals acetonitrile 3 4 biobrene plus 3 4 chemical installation kit D 3 dye mark warning 3 5 stain
80. n foot on the MicroBlotter Adjust the height of the Teflon foot by following the instructions not touching the PVDF listed on page 4 6 membrane sample diffused Ensure no stress develops on the tubing between the detector and the MicroBlotter as the solenoid travels across the membrane Low sample Teflon foot on MicroBlotter not Check the installation and positioning of the Teflon foot on the end recoveries installed and or positioned of the tubing that runs through the MicroBlotter solenoid The end correctly of the foot must be flush with the end of the tubing The Teflon foot should lightly touch the PVDF membrane each time it is lowered creating a slight impression in the membrane as it moves along its path Adjust the tubing Teflon foot by following the instructions on page 4 6 Baseline Problem with a fitting on the Turn the flow off If the problem continues proceed to the next abnormalities 140D mixing tee the column possible cause the detector lamp Otherwise visually check for and shifts injector valve the column inlet leakage at all the fittings If leakage is not apparent remove each port or the column outlet port fitting one at a time and inspect them On the mixing tee the end of the tubing must be flush with the end of the PEEK sleeve Adjust the position of the tubing and the sleeve as necessary Remember to trim the end of any tubing you push through PEEK sleeves to remove particulates which could block the system Re
81. n specifically for the Kipp amp Zonen Dual Pen Strip Chart Recorder P N 400268 The other set is generalized to accommodate most other chart recorders If you are using a chart recorder other than the Kipp amp Zonen you may need to refer to the user s manual supplied with your recorder for further instructions To prepare the Kipp amp Zonen chart recorder P N 400268 for a run 1 2 3 Load the chart recorder with paper Check and replace the pen if it has dried out Set the baseline to start on the first major grid by resetting the pen position Press the zero button turn the zero adjust potentiometer and press the zero button again Figure 3 6 on page 3 13 Set the range selector to 10 mV the record button to on the int ext button to the nt position and the cal var button to cal Set the paper speed to the same speed as the MicroBlotter by setting the mm s mm min button to mm min Then turn the paper speed control dial to 1 or 2 as appropriate To prepare other chart recorders for a run 1 2 3 Load the chart recorder with paper Check and replace the pen if it has dried out Set the baseline per the instructions listed in your chart recorder user s manual Set the range to 10 mV and turn the record control on Setyour chart recorder so the source of the pulses that moves the paper is internal If applicable set your chart recorder so that an input signal equal to the selected range will
82. n top of another instrument requires longer tubing This can adversely effect the precise timing required between sample placement onto the PVDF membrane in relation to the signals sent to the chart recorder to produce the corresponding chromatograph 112A 140D Solvent and 785A waste Strip Chart MicroBlotter reservoirs Recorder Figure 2 1 Recommended configuration for the 173A system 2 Unpacking and Installation April 2002 Applied Biosystems April 2002 Unpacking the System Components The hardware for the 173A system is shipped in six cartons The Chemical Installation Kit P N 402066 is shipped separately to arrive before the hardware To unpack the 173A system components and accessories 1 Check that the Chemical Installation Kit has arrived and that the contents were stored as directed WARNING BACK INJURY HAZARD Do not lift the 140D directly out of the carton Unpacking the 140D requires two people Use proper lifting procedures to avoid back injury 2 Set the carton with the 140D on the floor 99 Fold the flaps back tightly against the sides of the carton and carefully roll the box upside down Lift the carton off the instrument and packing materials Carefully roll the 140D right side up Remove the foam and packing materials Unpack the remaining instruments one at a time R ere Sh O S Carefully inspect the instruments and the boxes they were sh
83. ned to that of the flowcell so that the length of the flowcell tubing is not decreased Figure 2 13 Capillary tubing inside a Teflon coupler 2 24 2 Unpacking and Installation April 2002 Applied Biosystems ase d u Burqny ay pjoy 0 YY Buiqny WELZL y Ul VLGLOZ PUB ELGLOZ N d SMOq 3 sdt ay asn Burqny Aedes 10 weibeip Buynoy h z anba TEN VEL BY O a 2 25 2 Unpacking and Installation April 2002 Applied Biosystems CAUTION HOT Column inlet OS KSA UN A KS Ss Column outlet 2 Figure 2 15 Routing diagram for capillary tubing inside the 112A 2 26 2 Unpacking and Installation April 2002 Applied Biosystems April 2002 Configuring the 140D The 173A system is operated via the software located on the 140D During normal operation the Configure function is used to program the 140D for a variety of HPLC applications Prior to normal operation however the 140D must be properly configured and the system must be primed The configuration procedure consists of setting various parameters such as the pressure units and
84. ng prepressurization The pump should reach 800 psi in less than 1 min If it does not check the system for leaks or purge the 140D again as appropriate 8 Once the solenoid on the MicroBlotter begins the blotting action adjust the height of the Teflon foot by raising or lowering the tubing which runs through the solenoid as appropriate 2 42 2 Unpacking and Installation April 2002 Applied Biosystems When adjusted correctly the foot lightly touches the membrane at its lowest point and the effluent from the tubing is immediately absorbed by the membrane The following is a brief summary of what you will see during the different phases of this run In addition to the standard status information various status messages such as PRESSURIZE FLOWING RUNNING and EQUILIBRATE appear along the bottom of the liquid crystal display LCD on the 140D throughout the run The system prepressurizes to the target pressure As this occurs the PRESSURIZE status message is displayed When the target pressure is reached the pump ramps to the target composition and the TIME TARGET status message is displayed Note Even if the target pressure is not reached within 2 min the program still begins at time zero conditions Composition changes are displayed until the specified time zero value is reached At the end of the target time the status message changes to EQUILIBRATE The cLC Protein Standard should be injected into the 112A during
85. ng the Fittings on page 2 17 to assemble and connect this fitting Pay particular attention to step 7B on page 2 18 Connect the static mixer assembly P N 604268 to the 140D mixing tee using the PEEK tubing and fittings provided in the assembly The larger portion of the static mixer body is the inlet and an arrow indicating the flow direction is engraved in the body Cut a 24 in length of 50 uM i d capillary tubing P N 0323 0003 Connect this tubing to the static mixer outlet using a short bushing P N 200249 ferrule P N 200248 and PEEK sleeve P N 225123 Follow the procedure listed under Assembling the Fittings on page 2 17 to assemble and connect this fitting Pay particular attention to step 7A on page 2 18 IMPORTANT Follow the instructions listed on page 2 17 when assembling and connecting fittings which use PEEK sleeves Always trim 1 2 to 1 in off the tubing after sliding it through the sleeve This will remove particles which could permanently block the column 14 15 16 Cover all but the last 3 in of the tubing connected to the 140D mixing T with a red Teflon sleeve P N 225060 Route the tubing through the slotted trim of the 140D to the 112A Figure 2 14 on page 2 25 Connect the tubing to port 2 of the 112A injector Figure 2 11 usinga long bushing P N 200234 ferrule P N 200248 and PEEK sleeve P N 225123 Follow the procedure listed under Assembling the Fittings on page 2
86. nnector and unscrew the solenoid assembly from the drive arm 3 Unscrew the solenoid 4 Thread the tubing through the sleeve inside the solenoid core until approximately 2 in of tubing protrudes from the blunt end of the core Figure 2 12 on page 2 23 5 Re assemble the solenoid 6 Cuta 1 4 in length of 0 012 x 0 009 Teflon tube P N 225119 in the 173A Tubing Kit This will be used as the Teflon foot shown in Figure 2 12 7 Push the Teflon foot onto the end of the capillary protruding from the bottom of the solenoid assembly until it is flush with the end of the tubing IMPORTANT The Teflon foot must be flush with the end of the capillary to ensure proper blotting of samples onto the PVDF membrane 8 Adjust the tubing so the Teflon foot extends 2 mm from the bottom of the assembly 9 Screw the solenoid assembly back onto the drive arm and plug in the electrical connector 10 Cut an 8 in piece of red Teflon tubing P N 225060 in the 173A Tubing Kit and slide it onto the end of the tubing to be connected to the flowcell outlet 11 Route the tubing through the 112A so it protrudes vertically from the left hand trim strip Figure 2 14 on page 2 25 12 Following the instructions listed under Connecting Tubing with Teflon Couplers on page 2 24 couple the tubing to the output end of the flowcell 2 22 2 Unpacking and Installation April 2002 Applied Biosystems WARNING The tubing that connects the
87. ntact Technical Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax To Reach Technical Support Through the Internet To Obtain Documents on Demand 1 Introduction 1 3 1 5 1 6 1 7 1 8 1 9 1 9 1 9 1 9 1 10 1 13 1 13 Applied Biosystems 1 2 1 Introduction April 2002 Applied Biosystems April 2002 About This Manual This user s manual provides detailed information on the installation and use of the ABI 173A MicroBlotter Capillary HPLC System 173A system The manual is organized into the following sections 1 Introduction Includes a system description important safety information recommendations for getting started quickly and information on contactingApplied Biosystems for technical assistance with this product 2 Unpacking and Installation Describes how to unpack install configure program and test the instruments in the 173A system 3 Operation Provides detailed instructions on how to operate the 173A system Instrument setup prior to a run a standard protein peptide gradient program for use as a learning tool and as a template for designing your own programs and instructions on sample handling for subsequent analysis are included 4 Maintenance and Troubleshooting Provides general maintenance procedures and recommendations that will help you keep the 173A system performing optimally Also included are maintenance procedures for the column
88. o the home position after 200 min has elapsed If you wish to stop the MicroBlotter when the run is finished press the Reset button or cycle the MicroBlotter power off and on April 2002 2 Unpacking and Installation 2 35 Applied Biosystems 2 36 System Installation Test Program The following program is used with the cLC Dye Mark to test the installation of the 173A system To program the 140D l Press the Edit key on the 140D to display the Edit Select Screen Figure 2 21 2 Enter 9 for PROG NO then press the ERASE gt soft key PROG NO 9 EDIT gt STEP NO ERASE gt Figure 2 21 Edit Select Screen 3 Use the Prev Next keys to select Y yes then press Enter Edit Screen 1 Figure 2 22 is displayed The parameters for the prepressurization phase of the program are set using this screen Edit Screen 1 is identified by the word PRESSURIZE on the top line of the screen PROG 9 PRESSURIZE NEXT STEP gt B 50 FLOW 100 MAX PRESS 1500 TARGET PRESS 800 EXIT gt Figure 2 22 Edit Screen 1 IMPORTANT Do not change the default value of 50 B for this test 4 Type in 100 for FLOW 1500 for MAX PRESS the maximum pressure in psi and 800 for TARGET PRESS Caution Pressures greater than 3500 psi may cause the tubing to pop apart or fittings to fail 2 Unpacking and Installation April 2002 Applied Biosystems April 2002 5 Press the NEXT STEP gt soft key
89. ocument 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 April 2002 1 Introduction Applied Biosystems To order documents Then through the a Access the Applied Biosystems Technical Support Web site at Internet for fax or http www appliedbiosystems com techsupp e mail delivery 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 1 14 1 Introduction April 2002 Applied Biosystems 2 Unpacking and Installation Con
90. ollect data connect it to the Computer output connection on the back of the 785A detector 10 Place the four way power strip Socket Strip P N 603237 behind the 173A system 11 Using the jumper cords P N 0192 0007 connect the chart recorder 140D 785A and 112A to the power strip 12 Install the appropriate power cord for the MicroBlotter and plug the cord directly into a laboratory power source Caution If power is being supplied via an uninterruptable power supply UPS or a voltage stabilizer connect the power strip and all other system devices to this power source 13 Power up all the instruments and check for obvious malfunctions 14 Power down all the instruments before proceeding with the next section Installing the System Plumbing on page 2 17 2 16 2 Unpacking and Installation April 2002 Applied Biosystems Installing the System Plumbing The following plumbing procedure assumes that the system is configured using the side by side configuration illustrated in Figure 2 1 on page 2 4 To install the system plumbing you will need e The 173A Tubing Kit P N 604066 Two wrenches a 1 4 in wrench and a 7 16 in wrench both are in the 140D Accessory Kit e Capillary cutters P N 0658 0001 Extra Rheodyne fittings are in the 112A Accessory Kit P N 0602 0081 Refer to Figure 2 10 on page 2 19 during this procedure All capillary tubing lengths specified allow for trimming IMPORTANT Always u
91. olumn flush the column after every two weeks of continuous use Instructions for this procedure are listed under Maintaining the Column on page 4 5 e Prepare fresh solvents bi weekly to ensure consistent resolution retention times and baseline profiles with each run e Monitor system pressure routinely Investigate sudden drops or increases in pressure greater than 100 psi Refer to Troubleshooting the 173A System on page 4 12 for assistance e Record the starting pressure of each new column once it is completely hydrated This can take 24 to 36 h of constant flow at 5 wL min Until then the pressure will be lower than normal Check the pressure weekly A gradual increase in pressure indicates the start of a blockage If this occurs flush the column per the instructions listed on page 4 5 e Periodically check the degree of solenoid contact on the PVDF membrane The Teflon foot on the end of the tubing should lightly touch the membrane creating a slight impression in the membrane as it moves along its path Along with the cLC Staining Dye the cLC Dye Mark and the notches on the MicroBlotter tray this impression will help you locate the sample on the membrane after blotting is complete If adjustment is required gently pull up on or push down on the tubing Also it is critical the end of the capillary tubing be flush with the end of the Teflon foot that touches the membrane Perform arun using the cLC Protein Standard and the
92. ont Panel Rear Panel Operation Configuring the Voltage and Installing Fuses MicroBlotter Fuse Installation Chart Recorder Voltage Configuration and Fuse Installation Preparing the 140D Preparing the 785A Installing the Electrical Connections Installing the System Plumbing Assembling the Fittings Connecting Tubing with Teflon Couplers Configuring the 140D Priming the System Preparing the Solvents Purging the System Testing the System System Installation Test Program Verifying the System Test Performing Additional System Tests 1 1 1 3 1 4 1 5 1 6 1 7 1 8 1 9 1 9 1 9 1 9 1 10 1 13 1 13 2 3 24 2 5 2 7 2 7 2 8 2 8 2 9 2 9 2 10 2 11 2 14 2 15 2 17 2 17 2 24 2 27 2 28 2 28 2 28 2 32 2 36 2 40 2 42 iii Applied Biosystems Verifying the System Test 3 Operation Introduction Materials Required Materials Supplied with the 173A System Materials Required but not Supplied Dye Mark II cLC Staining Dye Operating the 173A System Preparing the Instruments for a Run Programming the 140D Preparing and Injecting Your Sample Executing a Programmed Gradient Run Processing Your Sample After a Run Excising Peaks of Interest From the PVDF Membrane Staining Blotted Proteins with the cLC Staining Dye Preparing Peptides for Sequencing Storing Excised Peaks of Interest Cleaning Blotter Tray Covers Maintenance and Troubleshooting Introduction Maintenance System Storage Troubleshooting 173A General Syste
93. ore the system during idle periods and how to troubleshoot most of the problems you may encounter while using the system Maintenance General system maintenance is described under 173A General System Maintenance on page 4 4 Included are recommendations for ensuring consistent results from run to run and tips for avoiding column and tubing blockages The required maintenance for each major system componentis listed under the component name for example Maintaining the 140D on page 4 9 System Storage If the system will be idle for one or more weeks the instruments and column should be properly stored Proper storage of system components will help ensure their continued optimal performance Refer to Storing the 173A System on page 4 23 Troubleshooting The troubleshooting portion of this section includes a troubleshooting guide instructions for locating and removing blockages and instructions for using the cLC Protein Standard 4 Maintenance and Troubleshooting 4 3 Applied Biosystems 173A General System Maintenance e All samples must be as particulate free as possible before loading them onto the system Therefore we recommend you use only the highest quality reagents and solvents when preparing your samples Sample preparation guidelines including protein digestion protocols are provided in Appendix B In addition centrifuge all samples for at least 5 min before injection e To help extend the life of the c
94. otter tray covers 1 Wash the cover with 100 methanol 2 Wipe the methanol and contaminants off the cover with an absorbent tissue 3 Inspect the cover for damage and contaminants If damaged or still contaminated discard the cover 4 If the cover is in good condition allow it to air dry before reuse 3 Operation April 2002 Applied Biosystems April 2002 4 Maintenance and Troubleshooting Contents Introduction Maintenance System Storage Troubleshooting 173A General System Maintenance Maintaining the Column Flushing the Column Maintaining the MicroBlotter Adjusting the Tubing Running Through the Solenoid Replacing the Tubing That Runs Through the Solenoid Connecting Tubing with Teflon Couplers Maintaining the 140D Maintaining the 112A Maintaining the 785A Troubleshooting the 173A System 173A System Troubleshooting Guide Locating and Removing a Blockage Using the cLC Protein Standard Reconstituting the cLC Protein Standard Evaluating a cLC Protein Standard Run Storing the 173A System 4 Maintenance and Troubleshooting 4 3 4 3 4 3 4 4 4 5 4 5 4 6 4 6 4 6 4 8 4 10 4 11 4 12 4 12 4 18 4 21 4 21 4 21 4 23 4 1 Applied Biosystems 4 2 4 Maintenance and Troubleshooting April 2002 Applied Biosystems April 2002 Introduction This section of the 173A user s manual describes how to maintain optimal performance of the ABI 173A MicroBlotter Capillary HPLC System how to properly st
95. plied 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 Warranty Period This Warranty does not extend to any Instrument on which any part 1 has been the subject of misuse neglect or accident 2 has been modified or repaired by any party other than Applied Biosystems or 3 has been 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 will be subject to separate warranties if any Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures by the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty
96. plied in the 173A Chemical Installation and Accessory Kit into a secondary container and set them to the right of the 140D Place an appropriate purge waste container in the secondary container as well Ferrule Tubing WASTE Bushing Figure 2 5 Compression fitting and solvent manifold on the 140D 2 Locate the 60 in length of 1 8 in o d Teflon tubing P N 3100 0167 in the 140D Accessory Kit This will be used for the purge waste line 3 Slip one compression nut P N 0403 0063 and one ferrule P N 0403 0064 onto the waste line and cut the tubing to the desired length 4 Connect the tubing to waste outlet port 5 Remove the two bottle cap assemblies from the 140D Accessory Kit P N 604218 6 Following the line from the filter cut the tubing 2 ft from the solvent cap Figure 2 6 on page 2 12 Keep the stainless steel bushing and ferrule on the line with the filter April 2002 2 Unpacking and Installation 2 11 Applied Biosystems i 4 Cut tubing here 3 Stainless steel bushing and ferrule Plastic bushing and ferrule 2 ft of tubing y Pull tubing through cap to this length and install a filter for helium sparge Filter Figure 2 6 Bottle cap assembly 2 12 2 Unpacking and Installation April 2002 Applied Biosystems 7 The remaining portion of tubing which has no fi
97. ption of 1 6 warnings chemical 1 7 electrical 1 7 see also safety warranty for the 173A system A 1 4 Index February 2001 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 D 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 02 2001 Part Number 903836D an Applera business
98. rd evaluating a protein standard run 4 21 how to reconstitute 4 21 how to use 4 21 protocols protein digestion B 1 purge 4 23 PVDF membrane 3 9 damaged 4 16 how to line up with chromatograms 3 26 how to stain 3 27 processing after a run 3 26 to 3 28 sample beading 4 16 storing excised peaks of interest 3 28 storing stained membranes 3 27 evaluating a protein standard run 4 21 runs execution of 3 23 to 3 25 status messages displayed during arun 3 24 safety dye mark warning 3 5 general safety information 1 7 material safety data sheets 1 7 safety summary 1 7 stalning dye warning 3 6 safety summary 1 7 sample beading on membrane 4 16 how to prepare 3 22 injection 3 22 low recoveries 4 13 preparation for sequencing B 3 preparation guidelines B 1 preparation of 4 4 prepration for sequencing 3 28 processing after a run 3 26 to 3 28 when to inject 3 24 sample preparation prior to injection 3 3 seal replacement 112A 4 10 140D 49 sequencing no results 4 13 preparing samples for 3 28 sample preparation guidelines B 3 service how to contact Applied Biosystems 1 9 solenoid adjust of 4 4 solvent A composition 3 14 solvent B composition 3 14 solvents preparation of 4 4 Index specifications 173A system 1 4 staining dye 3 26 description of 3 5 how to use 3 27 see safety stock solution 3 6 storage conditions 3 6 storing stained membranes 3 27 standard gradient program 1 8 conditions for peptides 3
99. rder We recommend the use of the Kipp amp Zonen Dual Pen Strip Chart Recorder P N 400268 with this system Each instrument has its own user s manual except for the MicroBlotter which is covered in this manual We recommend you review the installation and operation sections of the user s manual for each instrument before proceeding with these instructions At various points throughout this procedure you will be referred to other user s manuals for more detailed instructions The steps required to install the 173A system are e Preparing the site e Unpacking the instruments e Installing the electrical connections e Installing the plumbing capillary column and flowcell e Configuring the 140D e Priming the system e Configuring each instrument for operation e Testing the installation April 2002 2 Unpacking and Installation 2 3 Applied Biosystems 2 4 Preparing the Site Site preparation requirements are covered in detail in the ABI 173A MicroBlotter Capillary HPLC System Pre installation Manual P N 903829 Figure 2 1 illustrates the suggested instrument configuration for the 173A system This configuration requires a clear table or bench area at least 6 ft long and 2 5 ft wide In addition 2 power outlets one for the MicroBlotter and one for the power strip are required The MicroBlotter should always be placed on the bench or table surface never on top of another instrument Placing the MicroBlotter o
100. re exceeds 1500 psi during this procedure a blockage is present somewhere in the plumbing Locate and remove the blockage before proceeding For guidelines refer to Locating and Removing a Blockage in section 4 9 Reconnect the tubing to the column inlet To remove air from the column and injector loop 1 Disconnect the tubing from the column outlet and allow the effluent to run into a small waste container 2 Refill the syringes by pressing the FILL gt soft key then the BEGIN gt soft key 3 Press MANUAL to place the 140D in manual mode display the Manual Status screen and begin the purge 4 Observe the pressure increase When the pressure rises above 1000 psi press the FLOW gt soft key Type in 5 for NEW FLOW RATE then press ENTER to change the flow rate to 5 wL min 2 30 2 Unpacking and Installation April 2002 Applied Biosystems 5 After the pressure has stabilized continue to flush the column at 50 B for 20 min 6 Press the B gt soft key type in 95 for NEW B then press ENTER Flush the column and injection loop at 95 B for 20 min 7 Check all the fittings for leaks If you find a leak tighten or replace the fitting as appropriate 8 Reconnect the tubing to the column outlet and allow the flow to continue for 10 more min Again the pressure should not exceed 1500 psi Continue checking the entire system for leaks 9 Press Stop to halt the flow and return to the Ready screen You are no
101. reen Figure 3 15 is displayed TIME FLOW B RUN PRESS EVENTS 0000 PROG NO CAP A CAP B STEP NO Figure 3 15 Run Status Screen April 2002 3 Operation 3 23 Applied Biosystems The following status information is displayed during each run e TIME elapsed time e FLOW current flow rate e B composition of B solvent e PRESS current pressure e EVENTS current status of events e CAP A and CAP B remaining syringe capacities for solvents A and B e PROG NO number of the program being executed e STEP NO step number currently being executed Note Syringe capacities and the correct pressure are displayed after the syringes have been filled In addition various status messages such as PRESSURIZE FLOWING RUNNING and EQUILIBRATE appear along the bottom of the screen throughout the program The system will prepressurize to the target pressure As this occurs the PRESSURIZE status message is displayed Once the target pressure is reached the 140D ramps to the target composition The TIME TARGET status message is displayed as this occurs Note Ifthe target pressure is not reached within 2 min the program will still begin at time zero conditions Composition changes are displayed until the specified time zero value is reached At the end of the target time the status message changes to EQUILIBRATE and the equilibration phase begins 3 During equilibration inject your sample into the
102. repressurization Parameter Choices Setting B 1 99 50 Flow 0 1000 L min 100 L min Max Press 0 3 500 psi Your choice Target Press 0 3 500 psi Your choice Comments The starting point for prepressurization For most applications this value should be 50 The flow rate at which the pump prepressurizes In general 100 L min is adequate for most liquids Select an upper pressure limit compatible with your column For cLC operation a target pressure of 800 psi is recommended for both protein and peptide samples 5 Press the NEXT STEP gt soft key to display Edit Screen 2 Figure 3 10 This screen continues entry of the prepressurization parameters and establishes time zero target time conditions 6 Set the values in Edit Screen 2 to those shown in Table 3 2 PROG 1 TARGET TIME 1 NEXT STEP gt B FLOW 5 PREV STEP gt MAX PRESS x MIN PRESS x EXIT gt Figure 3 10 Edit Screen 2 Table 3 2 Edit Screen 2 Target Time Parameter Choices Setting Target Time 0 546 min 1 0 min B 1 99 15 for proteins Comments The time the 140D will take to ramp from the prepressurization B to the time zero B The time zero composition the 5 for peptides composition desired for the first step of the gradient Flow 0 500 yL min 5 yL min The time zero flow rate the flow desired for the first step of the gradient Max Press 0 3 500 psi Your choice Select an upper pressure
103. ril 2002 Applied Biosystems April 2002 PROG 9 STEP TIME 60 NEXT STEP gt B 55 FLOW 5 PREV STEP gt MAX PRESS 1500 MIN PRESS 0 DELETE gt EVENTS 1 C 2 0 3 04 C ONLY N EXIT gt Figure 2 27 Edit Screen 4 Gradient Step 3 17 Enter 60 for TIME and 55 for B 18 Press the EXIT gt soft key to display the SAVE Screen Figure 2 28 19 Press the PERM gt soft key to store the program as program number 9 The values displayed for the A and B VOLUME is the volume in microliters required for each syringe for one run of program 9 Note The SAVE Screen is displayed for a few seconds after which the Ready Screen is displayed automatically A VOLUME B VOLUME SAVE AS PROG 9 PERM gt TEMP gt Figure 2 28 Save Screen 2 Unpacking and Installation 2 39 Applied Biosystems 2 40 Verifying the System Test When the run is finished there should be three major peaks on the chromatogram and three corresponding dye marks on the membrane If necessary move the solenoid to the home position by pressing the Reset button or by turning the MicroBlotter power switch off and on To verify the system test 1 Remove the blotter tray 2 Gently remove the PVDF membrane with tweezers and place it on an absorbent tissue Discard the bottom filter 3 Line up the membrane along the bottom of the chromatogram and tape it in place Three separate peaks on the chromatogram and thre
104. rk on the PVDF membrane and chart recorder paper at 10 20 and 30 min Measure the distance between the marks to make sure both instruments are travelling at the designated speed Call Applied Biosystems if either instrument is not tracking properly 140D will not run No line power Make sure the 140D power switch is turned on Check that the 140D and other instruments in the system are properly plugged into the power mains Make sure the voltage of the power mains is correct Blown fuse Replace the fuse in the 140D with another of the proper rating Information on replacing the fuse is provided in the installation section of the 140D user s manual Pressure limit exceeded Check the minimum and maximum pressure settings on the 140D If necessary reconfigure these parameters for your particular application April 2002 4 Maintenance and Troubleshooting 4 17 Applied Biosystems 4 18 Locating and Removing a Blockage The following instructions will help you determine where a blockage is located and how to remove it The procedure begins by having you disconnect the tubing from the column inlet port If the pressure drops the blockage is somewhere from the column to the MicroBlotter If the pressure remains high the blockage is somewhere between the 140D and the column inlet port In addition to these instructions a flow chart illustrating this procedure is provided on page 4 20 To locate and remove a blockage
105. roBlotter Operation The MicroBlotter is activated by the 140D via a connection from the rear panel of the MicroBlotter to an event relay on the rear panel of the 140D During a programmed run the MicroBlotter and chart recorder are typically set to begin operation simultaneously at the end of equilibration The solenoid on the MicroBlotter is programmed to travel across the membrane to the end position Figure 2 2 on page 2 7 before returning automatically to the home position The travel time is 200 min If your run is less than 200 min you can either press the reset button on the MicroBlotter control panel turn the MicroBlotter power off and on or allow the solenoid to finish traveling across the membrane and return to the home position automatically 2 8 2 Unpacking and Installation April 2002 Applied Biosystems Configuring the Voltage and Installing Fuses The 140D 112A 785A and chart recorder are shipped with the voltage configured for 120 V Prepare each of these instruments for connection to a power source per the installation instructions provided in each instrument user s manual The steps required for each instrument include 1 Checking the voltage setting 2 Reconfiguring the voltage if necessary 3 Installing the appropriate fuse s Instructions for configuring the voltage on the 140D 112A and 785A are also provided in the 173A Voltage Accessory Kit The part numbers for the instruction sheets are 0478 0
106. s B Gradient for Peptides 1 Reset events 2 and 3 while 1 Reset events 2 and 3 while holding at 15 B for 0 1 min holding at 5 B for 0 1 min 2 Ramp to 65 B for 75 min 2 Ramp to 45 B for 140 min 3 Hold at 65 B for 10 min 3 Hold at 45 B for 40 min 4 Return to 15 B for 5 min 4 Return to 5 B for 5 min Total run time is 90 min Total run time is 185 min Protein separations can begin with a higher percentage of acetonitrile because they tend to be more hydrophobic than peptides The values for the parameters in this program are entered into a series of screens Unless specifically requested in the display the Enter key does not have to be pressed each time an entry is made If an improper entry is made the message INVALID will be displayed and the program will wait for you to enter another value If you enter a value incorrectly press the Clear key then enter a new value Use the right and left arrow keys to move the cursor from field to field on each screen The Prev and Next keys are used to display select or change the value of a parameter with a choice field To move from one screen to another press the NEXT STEP gt or PREV STEP gt soft key Note For detailed information on programming the 140D refer to sections 2 3 and 4 of the 140D user s manual P N 903586 Entering a Gradient Program Although the following instructions are based on entering the standard protein peptide gradient program described on page 3 13
107. s material safety data sheets MSDSs that provide information about the physical characteristics health hazards safety precautions first aid spill cleanup and proper disposal procedures Familiarize yourself with the information contained in the MSDSs before operating the instrument Additional copies of the MSDSs are available from Applied Biosystems at no charge April 2002 1 Introduction 1 7 Applied Biosystems 1 8 To Get Started Quickly Once your 173A system has been installed the 140D must be configured and primed and the entire system must be tested for proper performance These procedures are provided in section 2 Unpacking and Installation IMPORTANT Read the Safety Summary before installing operating or performing maintenance on any of the instruments in this system If you are not familiar with how to operate the 140D we recommend you read Sections 2 and 3 of the 140D user s manual to familiarize yourself with the instrument s control panel and operation Once your system has been configured primed and tested we recommend you perform at least one additional test run using a standard This will help you familiarize yourself with setting up the instruments and with handling the PVDF membrane before and after separation If desired the cLC Protein Standard supplied with this system can be used for the additional test Instructions for preparing and executing a run with this standard are provided
108. s com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing corelab appliedbiosystems com Peptide and DNA Synthesis Biochromatography PerSeptive DNA tsupport appliedbiosystems com PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers Applied Biosystems MDS Sciex api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com Hours for Telephone Technical Support 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 1 Introduction 1 9 Applied Biosystems 1 10 To Contact Technical Support by Telephone or Fax 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 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 GeneS
109. se one of the capillary cutters provided in the 173A Tubing Kit to cut the capillary tubing Caution Wear safety glasses when working with capillary tubing to avoid eye injury The compression type fittings used to connect the capillary tubing to the instruments must be carefully made to prevent blockages Instructions for the proper assembly and connection of these fittings is as follows Assembling the Fittings To assemble and connect fittings with PEEK sleeves 1 Slide the PEEK sleeve bushing and ferrule over the end of the tubing until the tubing protrudes slightly out the end of the sleeve Figure 2 9 on page 2 18 2 Holding both the larger end of the PEEK sleeve and the tubing so they do not move insert the fitting into the port as far as possible 3 Holding the sleeve and tubing in place use the 1 4 in wrench to slowly tighten the bushing until the ferrule starts to grip the PEEK sleeve At this point the sleeve will not pull out of the port but the tubing will still move within the sleeve 4 Loosen the bushing and remove the fitting from the port 5 Push the tubing through the sleeve April 2002 2 Unpacking and Installation 2 17 Applied Biosystems Ferrule Bushing P N 200248 P N 200249 i Cut tubing here PEEK sleeve Tubing P N 225123 After sliding the tubing through the PEEK sleeve cut 1 2 in off the tubing Then ease the tubing back into the fitting until
110. solenoid and flowcell can be cut and rejoined using a Teflon coupler This allows you to bypass the MicroBlotter and collect your sample in a container It is critical you do not cut and couple the tubing directly above the solenoid If the coupler leaks the liquid will drip into the solenoid This will damage and possibly short the solenoid Position the connection closer to the 785A Teflon coupler Capillary 30 ym ID Electrical m Solenoid connection Spring Washer C ring Core Capillary 30 pm ID Teflon foot Figure 2 12 Solenoid assembly diagram April 2002 2 Unpacking and Installation 2 23 Applied Biosystems Connecting Tubing with Teflon Couplers To make coupling connections using Teflon couplers l Remove the 0 012 X 0 062 Teflon tubing P N 225004 from the 173A Tubing Kit Using a razor blade cut a 3 4in length Use a pointed object such as a thumb tack to slightly enlarge both ends of the tube Push the capillary tubing all the way through the coupler until approximately 1 2 protrudes Trim 1 2 1 of the tubing from the protruding end and then pull back into the coupler until the end is halfway through it Then push the other piece of capillary to be joined into the coupler Try to make the two capillaries touch as shown in figure 2 13 Note When making connections with the flowcell tubing start with the line being joi
111. ss the PURGE gt soft key to display the Purge Screen Figure 4 6 PURGE RATE 2 500 BEGIN gt SYRINGE BOTH OF PURGES 7 OF SYRINGE 100 0 PURGE NO Figure 4 6 Purge Screen 8 10 11 Set the parameters as follows Purge Rate 2 500 Syringe Both of purges 7 of syringe 100 Press the BEGIN gt soft key When the purge is finished turn the power switches off on all the instruments Cover the instruments to protect them from dust IMPORTANT When you are ready to use the system again remember to reconfigure and prime the 140D and reinstall the column 4 Maintenance and Troubleshooting 4 23 Applied Biosystems Appendix A Limited System Warranty February 2001 Applied Biosystems warrants to the customer that for a period ending on the earlier of one year from completion of installation or fifteen 15 months from the date of shipment to the customer the Warranty Period the ABI 173A MicroBlotter Capillary HPLC System purchased by the customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the minimum performance specifications set forth 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 Ap
112. t blockages from occurring Instructions for flushing the column are provided under Maintaining the Column on page 4 5 e Each time a new column is installed record its starting pressure once the column is completely hydrated This can take 24 to 36 h of constant flow at 5 wL min Until then the pressure will be lower than normal A gradual increase in pressure indicates the start of a blockage If this occurs flush the column per the instructions listed on page 4 5 e Check the oven performance every 3 months Refer to the maintenance section of 112A user s manual for further instructions e Check for solvent leaks on a weekly basis Refer to the maintenance section of 112A user s manual for further instructions e Change the rotor seal when leakage is observed from the valve or when cross port contamination occurs Refer to the maintenance section of 112A user s manual for further instructions 4 10 4 Maintenance and Troubleshooting April 2002 Applied Biosystems April 2002 Maintaining the 785A Replace the lamp after every 1500 to 2000 h of normal use Refer to the 785A user s manual for instructions on how to test and replace the lamp WARNING ULTRAVIOLET LIGHT HAZARD Exposure to ultraviolet radiation can cause blindness or permanent eye damage To prevent eye injury adjust the detector sensitivity from the ultraviolet to the visible range 500 nm before beginning any detector maintenance procedures Always we
113. t Screen 3 occur at the end of equilibration Events designated in gradient steps occur at the beginning of the step Press the NEXT STEP gt soft key to display Edit Screen 4 Figure 2 25 and begin entry of the gradient phase of the program The gradient phase consists of a variable number of steps all of which use the same screen Edit Screen 4 Typically a different value for B and Time is entered in each step PROG 9 STEP TIME 0 1 NEXT STEP gt B FLOW PREV STEP gt MAX PRESS MIN PRESS DELETE gt EVENTS 1 C 2 0 3 0 4 C ONLY Y EXIT gt Figure 2 25 Edit Screen 4 Gradient Step 1 12 13 14 15 Step 1 in this program resets the detector autozero command Event 2 and resets the MicroBlotter start command Event 3 The ONLY command allows for activation of events without affecting the B or flow rate Enter 0 1 for TIME and Y for ONLY Move cursor to EVENTS Use the Prev Next keys to specify O open for Events 2 and 3 Press the NEXT STEP gt soft key to display another Edit Screen 4 and enter gradient step 2 Enter 30 for TIME and 55 for B PROG 9 STEP TIME 30 NEXT STEP gt B 55 FLOW 5 PREV STEP gt MAX PRESS MIN PRESS DELETE gt EVENTS 1 C 2 0 3 0 4 C ONLY Y EXIT gt Figure 2 26 Edit Screen 4 Gradient Step 2 16 Press the NEXT STEP gt soft key to display another Edit Screen 4 and enter gradient step 3 Figure 2 27 2 Unpacking and Installation Ap
114. t must be flush with the end of the capillary to ensure proper blotting of samples onto the PVDF membrane 4 Maintenance and Troubleshooting April 2002 Applied Biosystems Teflon coupler Capillary 30 pm ID Electrical connection Solenoid Spring Washer C ring Capillary 30 pm ID Teflon foot Figure 4 1 Solenoid assembly diagram 9 Adjust the tubing so the Teflon foot extends 2 mm from the bottom of the assembly 10 Screw the solenoid assembly back onto the drive arm and plug in the electrical connector 11 Cutan 8 in piece of red Teflon tubing P N 225060 in the 173A Tubing Kit and slide it onto the end of the tubing to be connected to the flowcell outlet 12 Insert the tubing from the flowcell outlet into the Teflon coupler following the instructions listed below under Connecting Tubing with Teflon Couplers on page 4 8 WARNING The tubing that connects the solenoid and flowcell can be cut and rejoined using a Teflon coupler This allows you to bypass the MicroBlotter and collect your sample in a container It is critical you do not cut and couple the tubing directly above the solenoid If the coupler leaks the liquid will drip into the solenoid This will damage and possibly short the solenoid Position the connection closer to the 785A April 2002 4 Maintenance and Troubleshooting 4 7 Applied Biosystems
115. tents Introduction 2 3 Preparing the Site 2 4 Unpacking the System Components 2 5 Description of the MicroBlotter 2 7 Front Panel 2 7 Rear Panel 2 8 Operation 2 8 Configuring the Voltage and Installing Fuses 2 9 MicroBlotter Fuse Installation 2 9 Chart Recorder Voltage Configuration and Fuse Installation 2 10 Preparing the 140D 2 11 Preparing the 785A 2 14 Installing the Electrical Connections 2 15 Installing the System Plumbing 2 17 Assembling the Fittings 2 17 Connecting Tubing with Teflon Couplers 2 24 Configuring the 140D 2 27 Priming the System 2 28 Preparing the Solvents 2 28 Purging the System 2 28 Testing the System 2 32 System Installation Test Program 2 35 Verifying the System Test 2 39 Performing Additional System Tests 2 42 Verifying the System Test 2 44 April 2002 2 Unpacking and Installation Applied Biosystems 2 2 2 Unpacking and Installation April 2002 Applied Biosystems Introduction This section describes how to install the ABI 173A MicroBlotter Capillary HPLC System 173A system and prepare it for operation The 173A system consists of five instruments e ABI 140D Microgradient Delivery System 140D e ABI 112A Oven Injector 112A e ABI 785A UV VIS Detector 785A e ABI 173 MicroBlotter MicroBlotter e Optional Kipp amp Zonen Strip Chart Recorder Chart Recorder IMPORTANT A chart recorder must be installed with the 173A system which was developed using a Kipp amp Zonen Strip Chart Reco
116. tion between the chart recorder and detector Check to see if the chart recorder responds to detector chart mark activation If not check the cable connection Adjust or replace if necessary 4 12 4 Maintenance and Troubleshooting April 2002 Applied Biosystems Problem Possible Causes Recommended Actions Irreproducible chromatograms Leaks Inspect all fittings for leakage If no leaks are apparent perform the static pressure test provided in the 140D user s manual Tighten or replace fittings and seals as required Instructions for replacing seals in the 140D are provided in the 140D user s manual Inadequate target time or equilibration time Increase the target and or equilibration time Inadequate target pressure Increase the target pressure No sequencing results good PVDF membrane not lined up correctly with chromatogram Check membrane alignment with the chromatogram Figure 3 16 in section 3 on page 3 27 A slight impression across the chromatogram or membrane indicates the path along which the sample has been a piece of PVDF membrane blotted If the sample isa protein stain the membrane with Ged with no sample was excised Staining Dye Refer to Processing Your Sample After a Run in section 3 on page 3 26 for guidelines Add the cLC Dye Mark to your peptide samples before injection Leak from detector to blotter Visually check for leaks and fix accordingly Teflo
117. to display Edit Screen 2 Figure 2 23 This screen continues entry of the prepressurization parameters and establishes time zero target time conditions 6 Enter 1 0 for TARGET TIME 25 for B and 5 for FLOW PROG 9 TARGET TIME 1 0 NEXT STEP gt B 25 FLOW 5 PREV STEP gt MAX PRESS MIN PRESS EXIT gt Figure 2 23 Edit Screen 2 Note Refer to the 140D user s manual for more information on prepressurization and target time 7 Press the NEXT STEP gt soft key to display Edit Screen 3 Figure 2 24 PROG 9 NEXT STEP gt EQUILIBRATE TIME 30 PREV STEP gt EVENTS 1 2 3 4 EXIT gt Figure 2 24 Edit Screen 3 8 10 Enter 30 for EQUILIBRATE TIME The equilibration conditions defined in Edit Screen 3 are typically equivalent to the time zero B composition and flow rate at which the separation will begin Events are also set on this screen Move the cursor to EVENTS Use the Prev Next keys to change Events 1 2 3 and 4 from O open to C closed Steps 11 and 12 designate an equilibration time of 30 min and set the event relays to be activated at the end of equilibration time zero The events activated at time zero are as follows Event 1 Switch sample valve to inject position Event 2 Autozero the detector Event 3 Activate the MicroBlotter Event 4 Start strip chart recorder feed 2 Unpacking and Installation 2 37 Applied Biosystems 2 38 Note 11 Events in Edi
118. to normal the injector This is typically accompanied is blocked Follow the instructions listed in the 112A user s by smaller peaks anda manual to remove the blockage sudden increase in operating pressure The 140D is not delivering Visually inspect the 140D for leaks The rotor and or piston seals solvent at the proper flow may have deteriorated and need to be replaced If a visual rates inspection reveals no leakage follow the leak test procedure in the troubleshooting section of the 140D user s manual to further check for leaks The maintenance section of the 140D user s manual includes instructions for replacing the rotor and piston seals Perform the flow calibration procedure listed in the maintenance section of the 140D user s manual to make sure the syringes are delivering flow at the proper rates Injector rotor seal is leaking Check the injector rotor seal for leakage replace if necessary per the instructions provided in the 112A user s manual Sample beads up The membrane is drying out Inspect the membrane A dried out membrane will change color on the PVDF before the run is finished This becoming more white than translucent First make sure blotter membranetowards can occur when runs are longer tray covers are being properly installed before starting each run the end of the run than 150 min The cover must be firmly pressed onto the PVDF membrane to create a seal Review the instructions listed under Prepar
119. very problem Refer to the troubleshooting guide provided in section 4 Maintenance and Troubleshooting If you require assistance call Applied Biosystems Technical Support Information on Technical Support is provided in section 1 Technical Support on page 1 9 Your 173A system is now ready for operation 2 Unpacking and Installation April 2002 Applied Biosystems 1 Ribonuclease A 2 Lysozyme 3 Apomyoglobin Sequence of Ribonuclease A KETAAAKFGRQHMDSSTSAASSSNY Sequence of Lysozyme KVFGRCELAAAMKRHGLDNYRGYSL Sequence of Apomyoglobin GLSDGEWQQVLMVWGKVEADIAGHG Figure 2 30 cLC Protein Standard chromatogram and sequences April 2002 2 Unpacking and Installation 2 45 Applied Biosystems April 2002 3 Operation Contents Introduction Materials Required Materials Supplied with the 173A System Materials Required but not Supplied Dye Mark II cLC Staining Dye Operating the 173A System Preparing the Instruments for a Run Preparing the 140D for a Run Preparing the 785A for a Run Preparing the 112A for a Run Preparing the MicroBlotter for a Run Preparing the Chart Recorder for a Run Programming the 140D Entering a Gradient Program Preparing and Injecting Your Sample Executing a Programmed Gradient Run Processing Your Sample After a Run Excising Peaks of Interest From the PVDF Membrane Staining Blotted Proteins with the cLC Staining Dye Preparing Peptides for Sequencing Storing Excised Pe
120. w ready to test the installation April 2002 2 Unpacking and Installation 2 31 Applied Biosystems 2 32 Testing the System Testing the system consists of executing a programmed gradient run using the Dye Mark II supplied in the 173A Chemical Installation and Accessory Kit The Dye Mark II is primarily used to help align the PVDF membrane with the chromatogram after a run For this use it is added to your sample prior to a run You can run the dye mark alone however to verify system operation Two dye marks will be visible on the membrane These dye marks must correspond to two major peaks in the chromatogram You will program the 140D with the System Installation Test Program on page 2 36 An explanation of the different phases of a programmed run prepressurization equilibration and gradient is provided in the Operation section of the 140D user s manual Thoroughly review this section before proceeding Note We strongly recommend you perform at least one more test using a standard before loading samples for further analysis on this system Refer to page 2 42 for additional information To test the 173A system installation 1 Make sure you have all the materials required for the run These are listed in section 3 on page 3 4 2 Set the 112A oven temperature to 37 C by pressing the Increment button on the inner panel until the correct temperature is displayed Then set the 112A to the load position WARNIN

Manual: ABI 173A MicroBlotter

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