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IsoPrime Function - GE Healthcare Life Sciences

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1. sample vessel contents membrane pH protein pl distilled water anolyte catholyte sample contaminant etc 30 ml dd H20 5 60 30 ml dd H 0 5 66 5 70 30 ml dd H20 5 81 5 86 30 ml dd H 0 5 93 5 96 30 ml dd H 0 6 01 6 05 30 ml dd H20 6 09 6 10 29 ml protein solution 6 15 30 ml dd H20 6 30 Power supply settings Prerun Purification run initial final initial final V 1700 2700 500 2988 4000 V max 4000 V max mA 5 2 5 2 25 mA max 25 mA max W 4 4 4 4 4 W constant 4 W constant 40 Analyze the results Use either an Immobiline DryPlate or a carrier ampholyte agarose IEF gel pH 3 7 to analyze sample vessel contents An Immobiline gel generally determines pI values more accurately but a carrier ampholyte gel is usually easier to handle and is therefore use ful in monitoring progress Results from the two different gel types may not correlate well Immobiline DryPlate pH 4 7 Follow instructions accompanying the gel to hydrate and prepare the gel Load 10 20 ul per lane Include lanes of the original sample and contents of each sample vessel for comparison Run for about 2 hours at 3000 V Stain and destain the gel Carrier ampholyte IEF agarose gel pH 3 7 Follow instructions accompanying the gel Load 10 20 pl per lane Include lanes of the original sample and contents of each sample vessel for comparison Run for about 1 hour at 1000
2. 6 09 6 01 5 93 5 81 5 66 6 05 5 96 5 86 5 70 5 60 Note Refer to the main instruc tions for more detail Note The protein concentration can be as low as 3 mg ml Clearer results are possible with the recommended concentration however Note If irregularities occur dur ing the run consult the Troubleshooting section for possible causes 31 Prepare the instrument 1 Assemble the IsoPrime Multi Chambered Electrofocusing Unit in a 4 C environ ment such as a cold room or large refrigerator Prepare a set of membranes Worksheet 1 on the next page lists each membrane pH Use positive displacement pipettes or a microsyringe to measure acrylamido buffers accurate pipetting is critical for reproducible results Wash membranes after polymerization is complete Assemble the separation module Start by placing the most acidic membrane at the anode end chamber labeled acidic and assembling each chamber in order of increasing pH Fill each sample vessel with 30 ml distilled water and set prerun parameters Peristaltic pump flow rate 6 5 rpm Press clockwise arrow to start pump Power supply 4 W constant 20 mA max 3000 V max Pre run for 2 hours By the end of the prerun the voltage should reach nearly 3000 V if no contaminants are present See the Troubleshooting section if contaminants are present After two hours drain
3. Current gt 20 mA Electroendosmosis Sample precipitated pl shifted Special cases 29 No protein separation Alkaline samples pH gt 8 Oxidized proteins Large proteins 200 300 kD Y S Y Y Y Y Y Y Desalt sample so that less than 50 mM of salt remains Do not expose proteins or instru ment to ionic detergents such as SDS Refer to section 4 2 If a large volume shift occurs the separation may be complete analyze the sample If the run proves to be incomplete reduce the protein concentration and load the sample into the sample vessel connected to the chamber into which the proteins are meant to migrate If it is necessary to repeat the run completely desalt the sample first Add a solubilizing agent or increase its concentration and use it in the same concentration throughout the entire procedure Prepurify the sample to 50 70 purity to prevent non specific aggregation Lower sample concentration Shorten run time Long runs may denature or degrade the protein and change its charac teristics Determine the pl of the protein under the same conditions encountered during the run use the same concentration of solubilizing agents and maintain the same temperature Refer to section 4 4 Add reducing agents such as B mercaptoethanol to prevent disulfide bond formation Trace all tubing connections to make sure that each fluid path connects the correct cham ber an
4. conc vol conc vol conc vol conc vol m H pK 1 0 pK 3 1 pK 3 6 0 000 000 0 173 9 0 441 22 0 612 31 10 767 38 10 851 42 1 195 60 pK 4 6 4 740 237 4 268 213 13 524 176 3 048 152 2 617 131 2 383 119 11 429 71 pK 6 2 5 239 262 15 284 264 15 355 268 15 400 270 15 441 272 5 463 273 5 554 278 pK 7 0 pK 8 5 pK 9 3 pK 10 3 pK gt 12 B Fill ddH20 to 2 000 ml 2 000 ml 2 000 2 000 2 000 2 000 2 000 2 000 C Add acrylamido buffer vol D Fill ddH 0 to 6 000 ml mix 6 000 ml 6 000 6 000 6 000 6 000 6 000 6 000 E Measure solution pH F Adjust pH to 6 5 0 2 with 6 5 6 5 6 5 6 5 6 5 6 5 6 5 1M Tris base ul mix 30 0 23 0 19 0 16 0 13 0 10 0 6 0 Important For the following steps prepare only 1 membrane solution at a time and pour into mould before starting the next solution T C 10 8 10 8 10 8 10 8 10 8 10 8 10 8 G Add acrylamide bis stock 30 T 3333 ul 3333 3333 3333 3333 3333 3333 H Fill ddH 0 to 10 00 ml mix 10 00 ml 10 00 10 00 10 00 10 00 10 00 10 00 Add TEMED ul 5 ul 5 5 5 5 5 5 J Add APS 40 w v ul mix 10 ul 10 10 10 10 10 10 Pour into mould Do not disturb for 20 minutes Polymerize for 1 hr at 50 C or overnight at room temperature Disassemble and start washing procedure 39 Worksheet 2 Date Starting sample ID Concentration Separation solution Chamber 1 anode 8 cathode Purification run inventory Carbonic anhydrase bovine erythrocytes 6 mg ml 180 mg 30 ml dd H 0
5. 1 kg Acrylamide IEF 40 solution 1 litre ReadyMix IEF to make 120 ml 30 T 3 C 4158 ReadySol IEF 40 T 3 C 1 litre N N Methylene bisacrylamide 25g 100g N N Methylene bisacrylamide 2 solution 1 litre Urea 500 g Glycerol 87 1 litre Triton X 100 500 ml CHAPS lg Repel Silane ES 500 ml Amberlite IRN 150L 500 g Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe Gmbtt Munzinger Strasse 9 D 7911 Freiburg Germany Code No 80 6081 90 80 6082 47 80 6082 09 80 6082 28 80 6082 66 80 6082 85 80 6083 04 80 6083 23 80 6083 42 80 6083 61 80 6083 80 80 6083 99 80 6106 03 80 6230 10 80 6397 11 80 6438 15 80 6397 30 80 6105 46 80 6105 84 80 6109 45 80 6107 93 80 6106 79 17 1300 01 17 1302 02 17 1301 01 17 1309 01 17 1310 01 17 1304 01 17 1304 02 17 1306 01 17 1319 01 17 1325 01 17 1315 01 17 1314 01 17 1332 01 17 1326 01
6. Assembly and Installation Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact Hoefer Inspect all components for damage that may have occurred while the unit was in transit If any part appears dam aged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become neces sary to return the unit Following is a brief description of the main IsoPrime components and a guide to where to find detailed assembly instructions Cabinet components Figure 1 shows the IsoPrime main components On the right side of the cabinet is a platform that supports both the separation module which plugs into the electrode posts in the rear and the sample vessel tray which rests on the stirring platform in the front Separation module Fit the pre assembled separation module onto the electrode posts Orient the module so that the end with the black knobs labeled basic fits onto the black cathode post and the end marked acidic fits onto the red anode post To prepare the module for a purification run see Section 5 1 Assemble the separation module Sample vessel tray The sample vessel tray fits onto the stirring platform by seating onto two screw heads that protrude near the front of the platform No further installa tion is required The sample vessel tray holds up to eight 30 ml vessels and two 125 ml ves
7. P G 1987 Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient J Biochem Biophys Meth 15 147 162 Righetti P G Barzaghi B Luzenna M Manfredi G and Faupel M 1987 A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient J Biochem Biophys Meth 15 199 206 Wenger P de Zuanni M Javet P Gelfi C and Righetti P G 1987 Amphoteric isoelectric Immobiline membranes for preparative isoelectric focusing J Biochem Biophys Meth 14 29 43 Righetti P G Barazaghi B and Faupel M 1988 Large scale electrophoresis for protein purification exploiting isoelectricity Trends in Biotechnol 6 121 125 Software for Calculating Recipes for Immobilized pH Gradients and Membranes Giaffreda E Tonani C and Righetti P G 1993 A pH gradient simulator for electrophoretic techniques in a windows environment J Chromatogr 630 313 327 35 Protein Purification Using PriME Technology Ettori C Righetti P G Chiesa C Frigerio F Galli G and Grandi G 1992 Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment elec trolyzer with Immobiline membranes J Biotechnol 25 307 318 Lucas J Faupel M and Goecking C 1990 Pyrogen removal from r DNA proteins in multi compartment electrolyzers with isoelectric membranes Electropho
8. affect the apparent pl of a protein Note If standard bands are sharp but experimental bands appear smeared pro teins may be interacting or precipitating Find an appropriate solubilizing agent and use it in the same concentration throughout the procedure because it can affect the apparent pl Refer to Section 4 3 the application notes or the Immobiline gel instructions for more solubility information 2 Determine the pl values of the protein by measuring the distance between the pro tein of interest and the pH gradient boundaries Interpolate the pI value Note Because the pl values of the markers were determined under different con ditions than the conditions of your gel run markers may indicate a pl value dif ferent from the interpolated results If there is a discrepancy use the values from your interpolation 3 Refine the pl estimate by casting increasingly narrower pH span gels until the desired degree of resolution is achieved Important If CA s are used the con centration should not exceed 0 3 and the ana lytical gels should contain the same CA concentration as the sample to be puri fied 15 Carrier ampholyte CA flatbed IEF gels protein sequencing It is possible to determine the pl of the proteins in a sample using commercially avail able CA flatbed IEF gels but the resulting accuracy and resolution is lower than the results achieved with analytical IPG gels especially in the alkaline pH range The
9. and Preparative Electrophoresis Chiari M and Righetti P G 1992 The Immobiline family From vacuum to plenum chemistry Electrophoresis 13 187 191 Righetti P G 1983 Isoelectric Focusing Theory Methodology and Applications Elsevier Amsterdam 386 pp Righetti P G and Gelfi C 1984 Immobilized pH gradients for isoelectric focusing Ill Preparative separations in highly diluted gels J Biochem Biophys Methods 9 103 119 Righetti P G 1990 Immobilized pH gradients Theory and Methodology Elsevier Amsterdam 408 pp Righetti P G Faupel M and Wenisch E 1992 Preparative electrophoresis with and without immobilized pH gradients In Advances in Electrophoresis Vol 5 Chrambach A Dunn M J and Radola B J eds VCH Weinheim pp 159 200 Wenisch E Reiter S Hinger S Steindl E Tauer C Jungbauer A Katinger H and Righetti P G 1990 Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients Electrophoresis 11 966 969 Wenisch E Jungbauer A Tauer C Reiter M Gruber G Steindl F and Katinger H 1989 Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients J Biochem Biophys Methods 18 309 322 Development of PriME Technology Preparative Isoelectric Membrane Electrophoresis Faupel M Barzaghi B Gelfi C and Righetti
10. by pressing the key with the arrow pointing counter clockwise Drain the chambers and the tubing com pletely back into the sample vessels Note Electroendosmosis may cause liquid movement between chambers and result in some vessels receiving more volume than others If overflow is imminent stop the pump and remove liquid from the overly full sample vessel and store it in a labeled container If the solution is coated with paraffin oil draw the sample from the bottom of the vessel with a syringe or pipette 2 Transfer the solution from each vessel to a storage container At this point either restart the unit with the same membranes if they are still usable and if the same protein will be purified or disassemble the unit and clean it 3 To reuse the membranes fill the sample vessels and start the next run or wash the membranes with ddH O and store them in 20 mM sodium acetate pH 5 0 and 2 mM sodium azide 4 To disassemble the IsoPrime lift the separation module from the posts and dis connect the tubing from the inlet and outlet ports on the separation unit To clean the unit see the Care and Maintenance section below Troubleshooting Isoprime mains power No power Pump Blank display Dead keypad or faulty pump head Membranes Won t polymerize Cratered surface Bowed or ruptured Prerun Current gt 20 mA 28 Y Check that the power cord is plugged into a working receptacle v Examine fuses and r
11. minimum of 2 hours at a constant power of 4 W The cur rent should start below 10 mA and gradually drop The current should never exceed 25 mA if the solution is free of salt and contaminants The voltage should start at between 500 900 V and gradually climb to between 1500 3000 V or higher by the end of the run Initial voltages can vary widely because the solubilizing agent and the number of chambers used affects the volt age required to maintain 4 W 7 At the end of the prerun turn off the power supply open the lid and reverse the pump to drain the liquid in all chambers back into the sample vessels Turn off the pump and empty all vessels 9 3 Table 4 Electrodic buffers Weak acids and bases that have been successfully used as electrodic buffers Note Electrodic solutions for proteins in the alkaline range should have pH val ues near the pH range in which fractionation takes place Strategy A Contaminants migrate out of the separation target chamber Strategy B Target proteins migrate out of the contaminant cham bers 24 Purification Run Select electrodic buffers Electrodic buffers for the anodic and cathodic chambers should be selected to provide just enough ions to create a current within the separation chamber For most applica tions dilute weak acids and bases 1 10 mM or ddH50 are suitable Refer to the application notes for examples Acids Bases Glycine CAPS Glutamic acid HEPES Ace
12. the separation chambers into the sample vessels by revers ing the pumping direction The purification run 1 Prepare carbonic anhydrase solution Dissolve 180 mg carbonic anhydrase in 30 ml of double distilled water Filter with a low binding protein filter such as Millex GV4 0 22 pm Cool to 4 C Replace the water in chamber 7 with 29 ml of the carbonic anhydrase solution The pH values of membranes delimiting this chamber are 6 10 and 6 30 Store the remaining 1 ml of solution for post run analysis Start the purification Fill the chambers by pressing the clockwise arrow on the pump keypad Set the power supply to 4 W constant 20 mA max 3000 V max Continue the separation for a minimum of 12 hours Longer runs for up to sever al days have no detrimental effects on the protein and allow more time for separa tion Drain the separation chambers into the sample vessels by reversing the pumping direction Remove the purified sample Worksheet 1 Protocol for preparing 2 membranes at each pH used in the purification of carbonic anhydrase bovine erythrocytes A Convert Doctor pH concentrations mM into volume ul of acrylamido buffer for 10 ml of solution Multiply conc by 50 Multiplier based on using 200 mM acrylamido buffers to make 10 ml of membrane solution Membrane 1 2 3 4 5 6 7 Membrane pH calculated 60 5 70 5 86 5 96 6 05 6 10 6 30 cone vol conc vol conc vol
13. 84 Separating detergents from proteins Methods in Enzymology 104 318 328 Hjelmeland L and Chramback A 1984 Solubilization of functional membrane proteins Methods in Enzymology 104 305 318 Detergents for Membrane Research Boehringer Mannheim Biochemicals Appendix 36 Purification Protocol Carbonic Anhydrase bovine erythrocytes This basic protocol can be used to check instrument performance or to become acquainted with the instrument and preparative isoelectric membrane electrophoresis PrIME techniques Sample characteristics The protein for this protocol bovine erythrocyte carbonic anhydrase Sigma Chemical Co catalog no C 3934 is supplied at a purity greater than 90 as analyzed by SDS PAGE IEF analysis of samples reveals five major and several minor isoforms ranging in pI values from about 4 9 to 6 7 These isoforms can be fractionated into nearly pure species as shown by the results below Protein lots may vary and measured pI values depend somewhat on the IEF method used In our experience the pI of the most prominent band can range from 6 15 to 6 29 In the sample result below the pl value of this band is 6 15 Purification results Original sample and sample vessel pe contents Gel Isogel carrier ampholyte IEF agarose gel pH 3 7 FMC Protein Carbonic anhydrase bovine erythrocytes Sigma Chemical Co catalog no C 3934 Run conditions 3000 V max 20 mA max 4 W constant
14. A M E R S H A M B 1 O S C IENCES IsoPrime IEF Purification Unit User Manual Amersham PI8 IM Rev E 2 99 Biosciences IsoPrime Function 0 6 oe ec cee eee eee Important safety information Assembly and Installation 2 1 Cabinet Components DEO POWER Soo A A Rare 2 3 Doctor pH Software 2 4 Peristaltic Pump Purification Method Summary Preliminary Purification Procedure 4 1 Partially purify the protein of interest 4 2 Desalt the sample 4 3 Stabilize the protein in solution 4 4 Determine the pl of the protein 4 5 Prepare isoelectric membranes 4 6 Prepare the sample Operating Instructions for the Purification Run 5 1 Pre run preparation AAA a he a en eed brel LAS 53 P rtICAtION FUN drid ede Sick res ne a nid ui 5 4 Protein recovery Troubleshooting o oo aaa aa aaa Care and Maintenance Ll Cleaning aue es As BES PHASES a dla HE FIZ WROD sf du o a ns Dh Suds kG de a a ee Gd 7 3 Replacing fuses mains power module Appendices A Specifications B Glossarya sas AU Line RER Dati eS A C Bibliography D PROLOCO a birra SERA RER RE e QE a
15. ED Ammonium persulfate Light paraffin oil Repel Silane 2 2 Figure 2 IsoPrime back panel Note Illustrated instructions accompany the safety lead adapters 2 3 Power Mains power module The IsoPrime mains power module located in the back of the unit houses the mains power switch and two fuses The mains power switch is the only operating control on the instrument It supplies power to the peristaltic pump the fan the stirring platform and the HV safety interlock Refer to Section 7 3 Care and Maintenance for instructions for replacing fuses a O Do not insert foreign objects or block O ventilation slots IsoPrime Multi chambered Electrofocusing Unit Protected by US Patents 4 971 670 separation process and 5 082 548 apparatus and by European Patent 0 287 513 B1 Other patents pending Mains power switch MAINS IN WARNING For continued protection against fire replace only with fuse of the specified voltage and current ratings Mains power module AE Separation module power supply An external power supply is required to operate the separation module The maximum rating of the power supply used with the IsoPrime must not exceed 4000 V 50 mA and 15 W The 2 mm safety leads at the right side of the instrument connect the separation mod ule to the power supply If your power supply does not accept 2 mm safety leads adapters 2 mm lead to 4 mm output are include
16. El Warranlles sin asis a MR ET E av SA Customer Service Information Figure Figure Figure Figure Figure Figure Figure Figure KO pOr E O OS Gore Figure Figure 10 Figure 11 Figure 12 Table 1 Table 2 Table 3 Table 4 Worksheet 1 Worksheet 2 Isoprime main components IsoPrime back panel Peristaltic pump Pump Keypad functions Pump back panel Pump head components Pump flow rate vs rpm Pump mains power module Membrane casting chambers Separation module assembly Fluid flow diagram IsoPrime mains power module Non ionic solubilizing agents Acrylamido buffer characteristics Stock solution recipes Electrodic buffers Preparing 10 ml membrane solutions for up to 7 membranes Purification run inventory sheet Oo ON DOF O1 FR W WO N NR FP N YF 13 17 17 24 19 25 IsoPrime Function The PI 8 IsoPrime Multi chambered Electrofocusing unit uses pI selective membranes together with electrophoresis to purify proteins on a preparative scale The amount of material purified and the time required depend on the solubility of the proteins and the degree of resolution sought The IsoPrime comprises a separation module a sample circulation system and a safety enclosure The separation module has electrodes at each end and is divided into a series of up to eight separation chambers by glass fiber filter reinforced polyacrylamide membranes The membranes co
17. H or pH gradient of the gel Generic name for Immobilines and related compounds Property of a chemical or protein having both acidic and basic groups Amphoteric electrolyte compounds used for establishing a pH gradient in traditional IEF gels Trade names for these compounds include Ampholine Bio Lyte SERVALYT IsoGel and Pharmalyte Carrier ampholyte High performance capillary electrophoresis High performance liquid chromatography Isoelectric focusing An electrophoretic technique based on proteins focusing to their pl in a pH gradient Immobilized pH gradient IEF gels that incorporate acrylamido buffers to form a pH gradient Acrylamido buffers and the patented procedure of IPG electrophoresis and casting immobilized pH membranes pH at which an amphoteric species is uncharged Carries no net charge pH at which an amphoteric species is uncharged in the absence of buffering ions The disassociation constant of an acid Molecular mass relative to H The negative of the log of the hydrogen ion concentration log H Scale 0 14 Isoelectric point The characteristic pH at which a protein has no net charge and therefore will not move in an electric field The negative of the log of the disassociation constant of an acid or base normally pK unless indicated log Ka or bl Preparative Isoelectric Membrane Electrophoresis Sodium dodecyl sulfate Appendix Bibliography IEF Immobilized pH Gradients
18. V Stain and destain the gel Catalog Number Carbonic anhydrase Bovine erythrocyte Sigma C 3934 Acrylamide IEF Electrophoresis grade 17 1301 01 Bisacrylamide 17 1306 01 Ammonium persulfate 17 1311 01 TEMED 17 1312 01 Repel Silane ES 17 1332 01 Immobiline II Acrylamido buffers pK 3 6 80 1255 70 pK 4 6 80 1255 71 pK 6 2 80 1255 72 Materials Low protein binding filters 22 um Millipore Immobiline DryPlates pH 4 7 80 1128 28 Roller for plates 80 1106 79 IEF Electrode strips 18 1004 40h Reswelling cassette and clamps 18 1013 74 Positive displacement pipettes Rainin Hamilton syringe Fisher Multiphor II 18 1018 06 EPS 350IXL HV Power supply 18 1130 05 from Amersham Biosciences unless otherwise noted Appendix 41 Warranty Statements Important user information e Please read this entire manual to fully understand the safe and effective E ng IS h use of this product The exclamation mark within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the literature accompanying the instrument The lightning symbol within an equilateral triangle is intended to alert the user to the risk of exposure to high voltages Should you have any comments on this manual we will be pleased to receive them at Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences reserves the right to make
19. acidic pHs to help resolubi lize oxidized proteins 13 14 4 4 Determine the pl of the protein The most accurate technique for determining the pl of proteins is to run one or more immobilized pH gradient IPG gels and interpolate the pI of the protein from its posi tion in the gradient Two less accurate alternatives to this method are run other types of gels or systematically narrow down the pl of the protein sample using iterative IsoPrime runs Each method is described below IPG gels IPG gels especially if you formulate them with Doctor pH and then use the same set of acrylamido buffers and solubilizing agents in all solutions throughout the process are the most accurate option for determining the pl of proteins In addition to higher accu racy IPG gels also handle protein loads of up to 10 20 times more than a conventional carrier ampholyte IEF gel This level of resolution helps identify contaminants more easily Pre cast Immobiline analytical gels are available in the acidic pH range Gradient mix tures for casting your own gels are easily calculated with Doctor pH software Once the gel is prepared 1 Load the protein sample onto one or more IPG gels Include pI markers to help detect irregularities in the gel particularly when solubilizing agents are used Run the gel s at the same temperature that the purification will be run 2 5 to 3 0 C above cold room or laboratory temperature because temperature can
20. blade screwdriver into the slot at the left side of the power module Twist the screwdriver 1 8 turn to re lease the cover then pull out the hinged compartment which opens to the right 3 Insert the screwdriver above the arrow on one fuse cassette catch the cassette end and slowly slide it completely out of the module 4 Pull the fuse out of its cassette and inspect If the fuse element is burned or bro ken replace the fuse with an identical type If the fuse appears to be intact check it with an ohmmeter A short circuit indicates the fuse is still usable 5 After placing a good fuse into the cassette slide it into the power module making sure the arrow on the cassette points to the right in the same direction as the guide arrows on the inside of the compartment door 6 Repeat steps 3 5 for second cassette 7 Close the fuse compartment cover and gently press it into the power module until it snaps shut 8 Plug unit in and turn mains power switch on Appendix 32 A Specifications Power IsoPrime AC mains power External HV DC power supply Powers the separation module Pump Line Voltage Line Frequency Fuse values Power consumption Temperature Humidity Position Head speed Torque Motor speed stability Continuous speed adjustment Flow rate range Maximum back pressure IsoPrime dimensions Dimensions Shipping volume Weight Net includes pump Shipping total Environmental
21. changes in the specifications without prior notice Warranty and Liability Amersham Biosciences guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Amersham Biosciences Amersham Biosciences shall in no event be liable for incidental or consequential damages including without limita tion lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Copyright 1999 Amersham Biosciences All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form by any means without permission in written form from the company Renseignements importants d utilization Francai Pour une bonne compr hension et une utilisation en s curit maximale il convient de lire ran ais enti rement ce manuel Dans la documentation qui accompagne l instrument un point d exclamation dans un triangle quilat ral a pour but d attirer Pattention de Putilisateur sur des instructions importantes de fonctionnement ou de maintenance Le symbole de P clair dans un triangle quilat ral a pour objet d attirer l attention de Putilisateur sur un danger d exposition a la haute
22. concentration of additives using techniques such as UV absorbance HPLC HPCE or IEF to examine protein solubility under the various conditions Once effective solubi lizing conditions are determined use them for all PrIME related procedures including pI determination and preparative runs Important Do not use ionic detergents such as SDS to solubilize proteins Refer to the IsoPrime application notes for solubilization procedure examples Table 1 Non ionic solubilizing Non ionic detergents Concentration e cam cam CHAPSO 1 2 Digitonin 1 2 NP 40 12 Tween 20 1 2 Zwittergent 1 2 B Octyl glucoside 1 2 n Octyl B D glycopyranoside 1 2 Other solubilizers Urea for acidic range only up to 8 M up to 6 M at 4 C Tetramethyl urea up to 6 M Sorbitol Mannitol Trehalose up to 40 Glycerol 5 40 Ethylene glycol 5 40 Propylene glycol 5 40 Dimethyl sulfoxide DMSO up to 40 Urea should not be used as a solubilizer at alkaline pHs for long periods as this can result in carbamylation of the protein Note 1 Evaluate the protein sample solubility in free solution Its solubility may be different in other media such as an analytical gel or a carrier ampholyte IEF gel The solubility of certain concentrated protein samples has been observed to be more stable in a gel matrix than in a solution Note2 Proteins may degrade or change conformation with time for example due to disulfide scrambling Add 2 mercaptoethanol at
23. conditions Altitude Temperature Maximum relative humidity Safety Specifications 115 V model 115 VAC 50 60 Hz 70W 230 V model 230 VAC 50 60 Hz 70W 2 fuses F 1A 250V 5x20mm Maximum rating 4000 V 50 mA 15 W 90 V to 132 V for 110 V model 180 V to 264 V for 220 V model 47 to 63 Hz 115 V two 0 5 A 250 V timed 6 3 x 32 mm 230 V two 0 25 A 250 V timed 6 3 x 32 mm Full load 45 W at 220 V no load 35 W at 220V Operating temp 0 40 C No warm up time is required Up to 95 non condensing Upright only vertical 5 0 01 to 48 rpm Greater than 3 Nm at any speed below 25 rpm at or above the nominal voltage 110V or 220V 0 5 for any variation of line voltages torque or temperature From 0 48 rpm by 0 01 increments up to 9 99 rpm above 9 99 rpm by 0 1 increments Maximum 26 ml min 2 8 mm tubing 500 kPa 51 x 51 x 38 cm 20 x 20 x 15 in 0 24 m 8 66 ft 26 kg 57 Ibs 37 kg 81 lbs to 2 000 m 5 40 C lt 80 for temperatures 5 31 C decreasing linearly to 50 relative humidity from 31 40 C Built to UL 1262 and IEC 1010 1 safety standards and list ed by ETL Testing Laboratories Appendix 33 Glossary Acrylamido buffer Amphoteric Ampholytes CA HPCE HPLC IEF IPG Immobilines Isoelectric Isoionic K pH pl pk PrIME SDS Compounds with an acrylic group and an amido group that can be co polymerized into an acrylamide gel to fix the p
24. d sample vessel See section 5 1 for prerun set up Avoid using urea as a solubilizing agent to prevent carbamylation of the protein At acidic pH values only prevent proteins from oxidizing by adding B mercaptoethanol If proteins do not cross membranes because the protein is too large decrease the T to 5 and increase the C to 8 Simple rinse Complete wash 30 7 1 7 2 Care and Maintenance Cleaning Cabinet components Switch the mains power off and unplug the power cord before cleaning the unit Wipe all surfaces with a soft damp cloth to remove residues and then dry with a towel Clean the separation unit sample vessels and caps with standard laboratory deter gents such as RBS or Alconox Solvents are not recommended Never use abrasive cleaners After cleaning rinse thoroughly with ddH 0 Tubing Simply flushing the entire tubing system with distilled water or separation solution may be sufficient between identical runs if the protein does not show evidence of aggregation precipitation or stickiness Submerge the inlet ends of the pump tubing in a beaker containing at least 250 ml of water or separation solution Pump the solu tion into another beaker at normal pump speed Use a syringe to flush the tubing pieces that connect the separation chambers to the sample vessels Flush tubing thoroughly with 8 M urea as described above if there is evidence of pro tein adsorption or if the unit is to be used for d
25. d with the unit If your 4 mm power supply output is recessed plug the short adapter code no 80 6105 84 into the 2 mm safety lead and then plug this assembly into the output If your 4 mm output is not recessed plug the longer adapter code no 80 6105 46 onto the short adapter code no 80 6105 84 plug both into the 2 mm safety lead and then plug this assembly into the power supply output Doctor pH software Doctor pH software calculates buffer compositions for any specified membrane pH and for pH gradient gel mixtures The program requires Windows 3 0 or later and a minimum of an 80286 microprocessor but an 80486 or an 80386 with a math coprocessor is strongly recommended For easy reference the program includes an on line Help file and a separate user s manual 2 4 Peristaltic pump The peristaltic pump uses a hybrid stepper motor to deliver fluids with high speed sta bility and a low pulsation level The pump head is composed of a rotor with stainless steel rollers that press against flexible tubing held in place by compression cams The pump is controlled by a front panel keypad Figure 3 Peristaltic pump Compression cams Stainless steel rollers Pump head LCD Keypad See Fig 4 Faster Increases the pump speed The 6 keys on the keypad Slower Rabbit control the pump The 3 Decreases the pump speed so Primes the pump at 48 rpm digit Liquid Crystal Di
26. der Gebrauchsf higkeit oder andere Verluste die wie auch immer durch eine fehler hafte oder unsachgem fe Verwendung des Produkts verursacht wurden Copyright 1998 Amersham Biosciences Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorherge hender schriftlicher Genehmigung durch das Unternehmen vervielf ltigt in einem Abrufsystem gespeichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden Informaci n importante para el usuario Para comprender el producto y utilizarlo con seguri dad es necesario leer este manual en su totalidad Espa ol usuario sobre la presencia de instrucciones importantes de operaci n y mantenimiento del aparato El s mbolo del rayo en un tri ngulo equil tero alerta al usuario sobre el riesgo de exposici n a altas tensiones A El signo de admiraci n en un tri ngulo equil tero en el manual advierte al Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attenta mente l intero contenuto del presente manuale A Si prega di inviare eventuali commenti al presente manuale a Italiano Il punto esclamativo all interno di un triangolo equilatero indica all op eratore la presenza di importanti istruzioni d
27. determination based on Doctor pH calculations use the Instantaneous Value feature to create membranes from the same set of buffers for the preparative separation Capabilities Resolves isoforms of a protein of a single molecular mass Resolves degradation and modification products of proteins Resolves proteins with a molecular mass lt 300 kD Limitations Salts cannot be used to solubilize proteins Enzymes must be stable in a pH environment close to their pl points Proteins with a molecular mass gt 300 kD are too large to move through the mem branes A system of membranes can usually be designed that causes smaller species to migrate away from the larger species Purification strategy overview Detailed descriptions of IsoPrime procedures follow in Section 4 1 Use conventional methods to purify the protein of interest to 50 70 pure by SDS PAGE analysis PrIME is best used in the later stages of a purification procedure In some cases crude samples can be processed but minimally purified samples can exhibit seri ous problems with isoelectric precipitation and non specific aggregation 2 Desalt the sample as completely as possible Proteins do not migrate until all salt ions have moved into the anode and cathode chambers A high salt content gt 50 mM can also cause membranes to rupture due to the high electric current they carry 3 Dissolve or dialyze the protein into distilled water if possible If the
28. e displayed value or the key to increase the displayed value Pressing either key once changes the right hand digit by one unit rpm Continuously pressing the key accelerates the change in speed To deliver 3 to 4 ml per minute set the pump speed to 6 5 rpm Figure 7 below shows the flow rate ml min at pump speeds up to 48 rpm To start the pump press the arrow key showing the desired direction of flow A sign will then appear at the left of the speed value for clockwise rotation or a sign for counter clockwise rotation Once the pump is started pumping direc tion can be changed without stopping the pump To purge or to prime the tubing select the RABBIT key when the pump is run ning Pressing the RABBIT key runs the pump at its maximum speed of 48 rpm in the current direction When pressed the display shows or but the pre vious speed is stored in memory If the RABBIT key is pressed again or if a direc tional key is selected the pump runs at the previously set speed To stop the pump press the STOP key The last selected speed except the RABBIT key selection is stored for the next start 30 25 20 ml min te 10 0 6 12 18 24 30 36 42 48 Pump speed rpm Figure 8 Pump mains power module Replace the fuse holder so that the line voltage sup plied to the instrument 110 V or 230 V reads right side up Adjusting fluid flow rate Once the pump is running adjust the cam pre
29. el through peristaltic pump tubing into the lower chamber port out of the upper chamber port and back into the sample vessel Install the path components systematically keeping the tubing in order with the numbered tubing clips provided 1 Install one length of peristaltic tubing for each chamber used according to the instructions in section 2 4 under the heading Peristaltic tubing installation Gather each set of tubing ends into a numbered clip at both the inlet end and the outlet end of the peristaltic tubing to help organize the tubing Make sure each section of the path is labeled consistently 2 Assemble the separation module according to instructions in the previous section 3 Slide each peristaltic tubing outlet end near the back of the unit onto the lower port of each separation chamber keeping them in the same order 1 8 as before Peristaltic pump tubing Separation chamber a EN Barbed Barbed connector connector straight elbow Sample vessel draw tube 5 2 Note If the current exceeds 25 mA the solution may contain a high salt concen tration or other contami nant Avoid high current because it can rupture the membranes 23 4 Slide the right electrode post to the position denoted by the number of separation chambers to be used 3 8 Seat the separation module onto the electrode posts with the positive end marked on the left and matching the black knobs to the black e
30. eplace if necessary Y Check that the inside cabinet power cord is properly plugged into the pump power recepta cle Y Check that the mains power is plugged into a functioning power outlet Y Check that both the pump and the mains power switches are on Y Examine fuses and replace if necessary Y If the display remains blank the transformer or the fan assembly could be faulty Contact Amersham Biosciences Contact Amersham Biosciences Use only recent stock of the highest purity chemicals Make 40 APS solution immediately before casting the membrane Y Make sure the filters are completely saturated with no air pockets above or below After casting allow a full 20 minutes before moving the casting chamber and then heat the gel cassettes for 1 hr at 50 C or leave overnight at room temperature Y Cover membrane with a glass cover plate and do not disturb for a full 20 minutes after casting Make sure there are no air pockets above or below the filter Y Slight bowing out of the membrane during a purification run is common but extreme bow ing due to high current damages the membrane Refer to section 4 2 for information on desalting and decontaminating the sample in order to lower the current Y If membranes were used for a previous run check membrane integrity Y Thoroughly clean all tubing and instrument components according to instructions in sec tion 7 1 Do not use any ionic detergents Purification run
31. er plates Copy Worksheet 1 next page For each membrane pH list the acrylamido buffer concentrations calculated by Doctor pH in the conc column Then calcu late the exact volume of each buffer needed for 10 ml of membrane solution by multiplying the buffer concentration by 50 to yield the volume in ml Follow the protocol listed on the lower portion of the worksheet Be sure to mea sure the acrylamido buffer solution pH before adding the acrylamide bis stock solution If the pH is not 6 5 0 2 pH units adjust it to this range with 1M Tris base or 1M acetic acid to optimize polymerization Prepare 1 ml of 40 ammonium persulfate APS immediately before use 0 4 g APS in 1 ml of ddH20 Add acrylamide bis stock solution 5 pl of TEMED and 10 pl of 40 APS to the acrylamido buffer solution Mix thor oughly with a transfer pipette Deliver 3 ml of the solution into each of the 2 wells in the mould block Wet a Whatman GF D 4 7 cm filter by sl placing it at an angle into a well and allow Mould block e ing capillary action to saturate it with the x ed solution Once saturated lower the filter into its well gently pressing it into place with gloved fingers The filter must settle into the casting well so that the filter edges are completely within and evenly centered in the well Pipette an additional 2 ml of solution onto each filter Slowly lower the glass cover plate with the silane coated side toward the membranes all
32. es garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las especificaciones publicadas La garantia incluida en las condiciones de entrega s lo es v lida si el producto se ha instalado y utilizado de acuerdo con las instrucciones entregadas por Amersham Biosciences Amersham Biosciences no ser responsable bajo ning n concepto de da os directos o indirectos incluyendo sin limitaci n la p rdida de beneficios la p rdida de ingre sos la p rdida de oportunidades de negocio la p rdida de utilizaci n y otras conse cuencias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto CopyrightO 1998 Amersham Biosciences Reservados todos los derechos No est permitida la reproducci n ni el almacena je en un sistema de recuperaci n ni la transmisi n de parte alguna de esta publi caci n sin la autorizaci n por escrito de la empresa 42 43 Customer Service Information Technical Service and Repair Amersham Biosciences offers complete technical support for all our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Amersham Biosciences representative Important Request a copy of the Amersham Biosciences Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is
33. he pump head To install tubing Adjustment screw Locking key ye Trigger key Compression cam Tubing retaining stop 1 Unlock all 8 pump head channels by pressing the trigger key of each section toward the roller barrel 2 Starting at the bottom of the pump head install one length of tubing at a time by sliding the black retaining stop into the front stop wrapping the tubing around the roller barrel and sliding the purple retaining stop into the back stop 3 Swing each compression cam toward the roller barrel and close the locking key Check that the top and bottom lip of each channel centers the tubing Note If power is suddenly turned off while the pump is operating the display will flash until any key is pressed The pump will then resume operating at the same speed as before Figure 7 Flow rates at pump speeds up to 48 rpm Pump operating instructions Preliminary pump set up includes placing the pump into the left side of the cabinet plugging the pump power cord into the pump installing the both the peristaltic and the sample vessel tubing and switching the IsoPrime mains power switch on Controlling the keypad functions The keypad is shown in Figure 3 1 Pumped volume Switch the pump mains power on The LCD shows the pump speed which is the speed set before the pump was last switched off The speed is factory set at 12 5 rpm To change the pump speed press the key to decrease th
34. hough losses may be reduced by addition of solubilizing agents Some proteins may precipitate at concentrations gt 10 mg ml Although the starting concentration may not be high enough to cause precipitation sample concentration and precipitation may occur dur ing a run particularly if the starting sample is introduced in several sample vessels but concentrates in one See Troubleshooting for suggestions on dealing with precipita tion problems Purification Run Instructions Refer to the assembly and installation section for instructions for assembling all cabinet components and calibrating the pump Detailed separation module assembly instruc tions follow 5 1 Pre run preparation Assemble the separation module 1 If the separation module is assembled unscrew the four knobs and disassemble the module by sliding all stacked components off the metal screws 2 Inspect each separation chamber for damage to the sealing O ring gasket replace if nicked or flattened and check that both circulation ports are clean and screwed finger tight into each separation chamber 3 Lay the anode end of the assembly so that the 4 Figure 10 threaded alignment screws Separation module assembly are facing upward and are _ Sealing gasket ready to receive the separa tion chambers 4 Arrange the washed mem branes in sequence from the lowest pH most acidic e to the highest pH most Ps Eq basic Following the indi i e cator e
35. i funzionamento e manutenzione nella documentazione allegata al prodotto Il simbolo del fulmine all interno di un triangolo equilatero indica all u tente la presenza di un rischio di esposizione ad alte tensioni Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Garanzia e responsabilit Amersham Biosciences garantisce che prima della consegna il prodotto stato collaudato a fondo per soddisfare i requisiti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodotto stato instal lato ed utilizzato nel rispetto delle istruzioni fornite da Amersham Biosciences Amersham Biosciences non potra essere ritenuta responsabile di incidenti o danni consequenziali inclusi ma non limitati a perdite di profitti mancato guadagno perdite di affari difetti di funzionamento e relative esposizioni dovuti ad un utilizzo non corretto del prodotto Copyright 1998 Amersham Biosciences Tutti i diritti riservati Nessuna parte della presente pubblicazione pu essere riprodotta conservata in sistemi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autorizzazione scritta del produttore Amersham Biosciences se reserva el derecho a modificar las especificaciones sin previo aviso Garantia y responsabilidad Amersham Bioscienc
36. ifferent proteins After completing the urea wash flush the tubing with distilled water two or three times Replace any tubing observed to be severely fouled or worn Repair If the IsoPrime malfunctions call an Amersham Biosciences technical service representative to evaluate the problem Your instrument may still be under warranty or you may wish to arrange to send it in for repair after the warranty period has expired A service manual code no 80 6438 15 is available 1 3 Figure 12 IsoPrime mains power mod ule The power module is locat ed at the back of the IsoPrime cabinet 31 Replacing fuses IsoPrime mains power module The two input fuses in the mains power module in the back of the unit do not require maintenance but one or both may fail due to fatigue under normal operating condi tions Fuses protect equipment by disconnecting loads too large for the instrument s circuit design so it is imperative that fuses are replaced only by identical fuse types F 1A 250V 5x20mm Figure 12 illustrates how to replace the mains power module fuses To replace pump fuses refer to section 2 4 Insert screwdriver in this notch to open cover Mains power switch Insert screwdriver blade behind arrow to pull cassette completely out Hinged cover 1 Caution Turn the mains power supply switch off and detach the power cord before replacing input fuses 2 Open the fuse compartment by inserting a small flat
37. ka Calculate acrylamido buffer concentration The easiest and most accurate way to calculate the concentration of acrylamido buffers required for any specified membrane is to enter the acrylamido buffer types and pK values into a Doctor pH software membrane worksheet specify the buffering power and allow the program to compute the concentrations Refer to the Doctor pH manual for more detailed instructions Alternatively request tables and nomographs from Amersham Biosciences to manually calculate concentrations Prepare membrane stock solution Preparation of isoelectric membranes is simpler and more reproducible with a set of stock solutions Acrylamido buffers are provided by the manufacturer as 0 20 M solu tions in water or isopropanol Commonly used acrylamide bisacrylamide stock solu tions are listed in Table 3 The composition of any polyacrylamide gel is given by two parameters T which represents the total concentration in grams per 100 ml of monomer used to produce the gel acrylamide plus bisacrylamide and C which is the percentage by weight of the total monomer that is the crosslinking agent The choice of final acrylamide bis concentrations and ratios will vary depending on the size of the proteins and impurities to be separated and the position of the membrane relative to the electrode For most applications 5 T 4 C is suitable for internal membranes and 10 T for membranes adjacent to the electrodes Dilu
38. lectrode post on the right 5 Slide the intake end of the peristaltic tubing over the barbed elbow fitting on the sample vessel cap The elbow fitting extends to the draw tube 6 Connect the upper port of each separation chamber to the straight fitting on the sample bottle cap with a length of 3 0 mm sample vessel tubing 7 After installation trace the path of each circuit to verify that the tubing connects the corresponding system components Prerun Prerun the unit a minimum of 2 hours to verify that all system components are opera tional to wash the unit with the buffer and solubilizer solution and to test the mem branes 1 Turn on the IsoPrime mains power switch 2 Turn on the pump mains power switch If necessary calibrate each channel of the pump Refer to p 11 Adjusting fluid flow rate The pump flow rate should be between 3 to 4 ml per minute 3 Fill each sample vessel with 30 ml of ddH50 ultra pure water such as Milli Q or the solubilizing solution and lower a spin bar into it 4 Turn on the pump and check the flow path for each sample vessel to make sure the fluid is flowing and following the intended path 5 After all the chambers in the separation module are filled lower the safety enclo sure lid and plug the safety leads at the right side of the IsoPrime unit into the external power supply Note DC power is supplied to the separation module only when the safety lid is closed 6 Prerun the unit for a
39. moves into another chamber leaving contaminants behind This method is particularly useful when the pI of the protein of interest is significantly different from all contaminants 25 Worksheet 2 Date Purification run inventory Starting sample ID Concentration Separation solution Chamber anode 1 2 sample vessel contents distilled water anolyte catholyte sample contaminant etc Power supply settings mA membrane pH Prerun Purification run initial final initial final 4000 V max 4000 V max 25 mA max 25 mA max 4 W constant 4 W constant protein pl 26 Larger quantities If the IsoPrime is to be used to purify relatively large volumes of proteins containing small quantities of impurities such as pyrogens or endotoxins the middle chambers up to 6 can be combined to form one large chamber Install membranes in only the anode and cathode chambers For this application use the large 125 ml vessels which fit into the middle of the sample vessel tray Purification run procedure 1 Turn on the pump and check the flow from the sample vessel to the inlet port to make sure the fluid is following the intended path and fills the separation module To speed the filling process press the RABBIT button on the pump Lower the safety lid turn on the power supply and set it to 4 W constan
40. n Desalt the sample The sample must be desalted before loading into the IsoPrime High conductivity sam ples lead to high currents and excessive joule heating in the separation module mem brane rupture and retarded protein separation As salts of strong acids or bases move in the electric field they form strongly acidic and basic fronts that can cause protein denaturation due to pH shock Use conventional desalting techniques such as dialysis ultrafiltration gel filtration ion exchange chromatography ultracentrifugation desalting columns or buffer replacement in a centrifugal concentrator If it is not possible to completely desalt the protein sample use buffers formed from weak acids and bases such as Tris acetate Avoid salts of strong acids and bases such as NaCl or phosphate Do not use protein samples previously exposed to ionic detergents such as SDS As with simple salts ionic detergents cause high initial electric currents and may cause membrane rupture 4 3 Stabilize the protein in solution Some proteins are adequately soluble in distilled water the preferred solvent for PrIME purification However for proteins that have low solubilities or tend to aggregate or precipitate a number of useful solubilizing agents are listed below Solubilizing agents are also helpful in reducing losses due to non specific adsorption when working with proteins available in very limited quantities Determine the best combination and
41. owing excess gel solution to escape from between the plate and the acrylic block Avoid trapping air under the cover plate Air retards polymerization and air bubbles may cause holes in the membrane Glass cover plate 6l Worksheet 1 Protocol for preparing 2 membranes at each pH used in the purification of A Convert Doctor pH concentrations mM into volume ul of acrylamido buffer Multiply conc by 50 Multiplier based on using 200 mM acrylamido buffers to make 10 ml of membrane solution Membrane 1 2 3 4 5 6 7 Membrane pH calculated conc vol conc vol conc vol conc vol conc vol conc vol conc vol mM ui pK 1 0 pK 3 1 pK 3 6 pK 4 6 pK 6 2 pK 7 0 pK 8 5 pK 9 3 pK 10 3 pK gt 12 Fill ddH 0 to 2 000 ml 2 000 ml 2 000 2 000 2 000 2 000 2 000 2 000 Add acrylamido buffer vol Fill ddH 0 to 6 000 ml mix 6 000 ml 6 000 6 000 6 000 6 000 6 000 6 000 Measure solution pH Adjust pH to 6 5 0 2 with 6 5 6 5 6 5 6 5 6 5 6 5 6 5 10 100 ul 1M Tris base or 1M acetic acid mix mmo w Important For the following steps prepare only 1 membrane solution at a time and pour into mould before starting the next solution T C RE e AAR a tees tf E hs G Add acrylamide bis stock 30 T pl H Fill ddH 0 to 10 00 ml mix 10 00 ml 10 00 10 00 10 00 10 00 10 00 10 00 Add TEMED ul 5 ul 5 5 5 5 5 5 J Add APS 40 w v ul mix 10 ul 10 10 10 10 10 10 Pou
42. pres ence of carrier ampholytes may result in an apparent pl value differing as much as 1 0 pH unit from the actual value due to potential CA protein complexing This effect is amplified in the alkaline range It is possible to calculate a pI from a known protein sequence but the calculated value is rarely useful unless the PrIME separation is performed under denaturing conditions Theoretical pI calculations cannot account for conformational effects on pl Systematic pl determination with IsoPrime iterative method To determine the pl of a protein using the IsoPrime run the sample through a series of membranes beginning with a broad range and systematically narrowing the range Refer to Sections 4 5 4 6 and 5 all for membrane casting and IsoPrime operation instructions For example the membrane pH values in the first run may be 4 0 4 5 5 0 5 5 6 0 and 6 5 If the all the proteins of interest migrate to the chamber delimited by the membranes with pH values of 5 0 and 5 5 then the pHs of the next set of membranes would be 5 0 5 1 5 2 5 3 5 4 and 5 5 Continue to narrow the range by casting membranes with increasingly closer spaced pH values until the desired degree of purity is achieved Analyze the purity of the separated sample by SDS PAGE IEF or immunological techniques 16 4 5 Prepare isoelectric membranes Determine membrane pH values The pH of each membrane set is selected so that the different molecule
43. properly completed Legal Information Hoefer Immobiline and IsoPrime are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham and Amersham Biosciences is a trademark of Amersham ple Tween is a trademark of ICI Americas Inc Zwittergent is a registered trademark of CalBiochem Novabiochem Corporation All goods and services are sold subject to the terms and conditions of sale of the com pany within the AAmersham Biosciences group which supplies them A copy of these terms and conditions is available on request Amersham Biosciences 1999 All rights reserved Printed in the USA Ordering Information Qty IsoPrime Multi chambered Electrofocusing Unit Includes 8 channel peristaltic pump separation module and Doctor pH software 115 VAC 1 230 VAC 1 Glass sample vessels 30 ml 12 Glass sample vessel covers 30 ml 1 Sample vessel rack 30 ml 1 Stir bars 0 75 in Star 4 Peristaltic tubing 2 79 mm ID 8 Sample vessel tubing 3 mm ID 8 Numbered tubing clips 3 Filters GF D 4 7 cm 100 Membrane casting chamber dual 4 Membrane casting chamber cover glass 8 Power cord 115 V 1 Power cord 230 V 1 PI 8 Instruction manual 1 PI 8 Service manual 1 Doctor pH instruction manual 1 HV lead safety adapter 2 to 4 mm 2 HV lead safety adapter 2 to 4 mm recessed output 2 IsoPrime cabinet fuses F 1A 250 V 5x20 mm 5 Pump fuses 115 V 0 5A SB 3AG 5 Pump fuses 230 V 0 25A SB 3AG 5 Acrylamide IEF 250 g
44. protein is not soluble in distilled water add solubilizing agents such as non ionic detergents glycerol or urea The best solubilization conditions and combi nations should be determined by UV absorbance HPLC or IEF measurements of protein solubility 4 Determine the pl of the protein and select membrane pHs For the most accurate results cast and run one or more linear immobilized pH gradient IPG gels Use linear interpolation to estimate the pI of the proteins and contaminants and to estimate the best pHs for separating membranes Properly selected membrane pHs will separate the desired protein and contaminants into different chambers 5 Prepare isoelectric membranes Calculate acrylamido buffer mixtures for each pH membrane needed with Doctor pH software 6 Pre run and load the IsoPrime Assemble the separation unit with the membranes fill the system with the separa tion solution then pre run Load the protein sample of interest and begin the purification run 12 4 1 4 2 Preliminary Purification Procedure Partially purify the protein of interest The protein sample should be more than 50 pure as determined by SDS PAGE after using conventional purification techniques such as precipitation centrifugation filtration and column chromatography Crude desalted protein samples can be puri fied with the IsoPrime but results are unpredictable because crude mixtures tend to precipitate due to nonspecific aggregatio
45. r into mould Do not disturb for 20 minutes Polymerize for 1 hr at 50 C or overnight at room temperature Disassemble and start washing procedure 20 4 6 Polymerization and storage 1 Do not disturb membranes for 20 minutes After 20 minutes either carefully transfer the gel cassettes to a forced ventilation oven set at 50 C and leave for 1 hour or leave the cassettes undisturbed at room temperature overnight After polymerization gently pry the gel cassette open with a thin piece of plastic or a spatula Carefully trim excess gel from the membrane edges with a sharp blade or spatula Thoroughly examine each membrane for even polymerization the surface of each membrane should be smooth and no holes should be evident If the membranes are acceptable wash them in a small dish three times Use 100 ml ddH 0 per wash and leave each wash on the shaker for 30 minutes Label each wash con tainer with the pH of the membrane Store unused membranes for up to 1 month in 20 mM sodium acetate buffer pH 5 0 2 mM sodium azide Wash the membranes with ddH 0 for 30 minutes before using Prepare the sample The stabilized protein sample section 4 3 may need to be diluted or concentrated before it is purified in the IsoPrime For most proteins a concentration between 1 and 10 mg mL is an appropriate starting point At less than 0 1 mg ml losses to non spe cific surface adsorption may substantially decrease protein recovery alt
46. resis 11 981 982 Righetti P G Wenisch E and Faupel M 1989 Preparative protein purification in a multi compartment electrolyzer with Immobiline membranes J Chromatogr 475 293 309 Righetti P G Barzaghi B Sarubbi E Soffientini A and Cassani G 1989 Charge hetero geneity of recombinant pro urokinase and urinary urokinase as revealed by isoelectric focusing in immobilized pH gradients J Chromatogr 470 337 350 Righetti P G Casale E Carter D Snyder R Wenisch E and Faupel M 1990 Protein purification in multicompartment electrolyzers for crystal growth of r DNA products in microgravity In Proc 4th Eur Symp Life Sciences Research in Space 28 May 1 June 1990 Trieste Italy ESA SP 307 481 484 Righetti P G Wenisch E Jungbauer A Katinger H and Faupel M 1990 Preparative purification of human monoclonal antibody isoforms in a multicompartment electrolyzer with Immobiline membranes J Chromatogr 500 681 696 Wenisch E Righetti P G and Weber W 1992 Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric mem branes Electrophoresis 13 668 673 Wenisch E Katinger H and Righetti P G 1993 Purification of human recombinant superox ide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes Electrophoresis in press General Furth A et al 19
47. s in the solution whose different pI values are known are trapped in separate chambers For most cases ideal pH values are those that are spaced to allow the maximum difference between the pI values of the molecules in the solution and the pH values of the mem branes This is illustrated in the following example A sample contains five proteins with pls of 5 20 5 30 5 40 5 50 and 5 60 Four membranes with pHs of 5 25 5 35 5 45 and 5 55 are optimal because these values allow the largest difference between the pI values and the pH values Other sets of membranes within this range would also serve to purify this sample Only 4 mem branes are specified because the pI 5 20 protein will migrate to the anode chamber and the pI 5 60 protein will migrate to the cathode chamber See the IsoPrime applica tion notes for other examples Select membrane buffer set A membrane of practically any pH can be made from just two acrylamido buffers one with a pK value close to the pH of the membrane and another with a much higher or lower pK value to titrate the solution to the desired pH If possible it is advisable to use one buffer with a pK value slightly higher and another buffer with a pK value slightly lower than the pH of the membrane in order to bolster the buffering capability on both sides of the membrane pH As opposed to using only one buffer and a strong titrant which would result in limited buffering capability on the titrant side of
48. sable Fuse holder 4 Slide the drawer in the fuse holder out To install a new fuse slide drawer insert a new fuse and slide the fuse into out and insert fuse Then slide the fuse holder making sure it fits secure grawer In pe eps ly into both clips at either end 5 Repeat steps 3 and 4 for the second fuse on the other side 6 Reinsert the fuse holder so that the line voltage supplied to the instrument reads right side up The arrow next to voltage rating points to the alignment mark at the bottom of the pump mains power module 10 Purification Method Summary The PrIME process has been applied to purifying a variety of proteins and certain pro cedures are recommended based on this experience Listed below are important reminders method capabilities and also some limitations Important The intent of this process is to purify proteins It is therefore imperative to follow all instructions carefully prepare all solutions with the utmost attention to accu racy and take all precautions to avoid contamination during the entire process Analytical and separation conditions should be as consistent as possible If addi tives are required to maintain isoelectric solubility use the same additives at the same concentrations during analytical pI determination and preparative separa tions Use the same set of acrylamido buffers for analytical gels and membranes If you cast your own analytical IPG gels for pI
49. sels Sample vessels and tubing Not shown A peristaltic pump circulates the sample between the sample vessels and separation chambers To assemble the fluid paths see Section 5 1 Create a fluid path for each separation chamber Peristaltic pump The pump fits into the left side of the cabinet where its rubber feet fit into matching depressions in the cabinet bottom With the pump in place tilt it for ward slightly to plug the short power cord in the back of the IsoPrime cabinet into the pump s power cord receptacle See section 2 4 for complete pump operating instruc tions Figure 1 IsoPrime main components Cabinet lid with safety interlock Sample vessel tray Sample vessel support platform houses magnetic stirrers Auxiliary parts supplied but not shown Peristaltic pump tubing Sample vessel tubing Numbered tubing clips 3 Mains power cord Sample vessels 30 ml 12 and caps 8 Dual membrane casting chambers 8 and Glass covers 16 Whatman GF D 4 7 cm dia filters box 100 Doctor pH software and User s Manual Peristaltic pump Separation module seats on electrode posts not shown HV DC leads connect the separation module to the external power supply Materials required but not supplied Power supply 3000 V recommended Acrylamido buffers 10 buffers are commercially available pK s 1 0 3 1 3 6 4 6 6 2 7 0 8 5 9 3 10 3 gt 12 Acrylamide and bisacrylamide TEM
50. splay LCD to the left of the key pad displays the speed in Forward O Stop rpm at which the pump is Starts the pump clockwise Stops the pump running Figure 4 Pump keypad functions 15 Backward Starts the pump counter clockwise Figure 5 Pump back panel Pump mains power The back panel houses the mains power module which contains the mains power switch a power cord receptacle and the fuse holder Turn the pump on by turning the mains power switch on The IsoPrime mains power switch must also be on in order for the pump to operate Turn the pump mains power off when the pump is not in use Fan cover Mains power switch Power cord receptacle Fuse holder 4S GILSON 72 RueGAMBET 95400 VILLIERS LE Figure 6 Pump head components Important To increase tubing life release pressure on the tubing when the pump is not in use Release the compression cams by pressing on the beveled corners of the trigger key and slacken the tubing by unhooking one of the retaining stops Peristaltic tubing installation The pump head accommodates from 1 to 8 lengths of peristaltic tubing Install one tubing length per separation module chamber Each tubing length has 2 retaining stops one black one purple that correctly tension the tubing on the pump head Refer to Figure 11 in section 5 1 for the complete fluid flow path which includes the tubing position around t
51. ssure on the tubing to the minimum nec essary to ensure liquid flow Adjust compression cam pressure by slowly tightening the adjustment screw with your thumb and index finger until you can see liquid being pumped inside the peristaltic tubing Then tighten the screw another 1 8 turn Do not over tighten the screws Readjust after the pump has operated 15 minutes Figure 6 shows adjustment screw location To ensure that consistent and accurate flow rates are maintained replace peristaltic tubing as soon as it appears to be worn Replacing pump fuses Two fuses are factory installed into the fuse holder in the pump mains power module Fuses do not require maintenance but may fail under normal operating conditions To replace fuses 1 Turn the mains power switch off disconnect the power cord and lift the pump out of the IsoPrime cabinet 2 Insert a screwdriver blade into the notch above the fuse holder and twist it a 1 4 turn to partially pull out the holder Then pull the fuse holder completely out of the mains power module Insert screwdriver blade behind notch to release fuse holder E 3 Pry one fuse out of one side of the fuse holder by inserting the screwdriver blade into the opening on the bottom of the holder Inspect the fuse element If it is burned or broken replace the fuse with an identical type If the fuse appears to be in intact check it with an ohmmeter A short circuit indicates the fuse is still u
52. t power As in the pre run the initial current usually starts between 8 and 20 mA and eventually drops to 1 to 4 mA The current should never exceed 25 mA and if it begins to climb above 20 mA evaluate the sample for contaminants such as salt The initial voltage should start between 300 and 700 V and climb to 1500 to 3000 V Never allow the voltage to exceed 4000 V Typically the voltage reaches its maximum within a few of hours and remains constant for the duration of the run Constant voltage does not indicate that the purification is complete Periodically check the sample purity remove a small aliquot see below from each sample vessel and analyze for the level of purity with such methods as IEF HPCE HPLC or SDS PAGE An IPG gel is the most discriminating method Sampling during the purification run To sample during the run turn off the power supply and open the safety enclosure lid Remove aliquots through the sampling ports on the sample vessel caps then close the lid and restart the power supply 27 9 4 Protein Recovery The purification process may take from 2 hours to 3 or more days depending on the characteristics of the protein and the degree of resolution required Resolution of pro teins or isomers differing by less than 0 1 pl generally require a minimum of 8 hours 1 At the end of the purification process turn off and disconnect the power supply open the safety lid and reverse the direction of the pump
53. tched into the end piece lay the most acidic membrane into the acidic end chamber Orient the chamber so that the circulation ports are in the same position as the ports on the end piece Lower the separation chamber onto the 4 screws and seat the chamber __ Circulation ports 2 Separation chamber _ Membrane _ Alignment screws 4 Ed 5 Carefully lay the next more basic membrane into the depression on the separa tion chamber and lower a second properly oriented separation chamber onto a E the four screws and seat it sg 1 _ Anode end gt Ss lt 5 chamber 21 Figure 11 Fluid flow diagram The protein is drawn through the sample vessel draw tube by peristaltic pump action on the peri staltic tubing The sample then enters the lower port of the separation chamber and circulates back into the sample vessel by way of the sample vessel tubing 22 6 Repeat step 5 until all membranes are installed in sequence The last membrane should be the most basic 7 After all separation chambers are in place seat the cathode end chamber onto the 4 screws and fit the 4 black knobs onto the screws tightening them until the assembly is secure Finger tighten the knobs making sure to tighten each one equally Create a fluid path for each chamber of the separation module Each chamber used between 1 and 8 requires a separate fluid circuit that runs from the sample vess
54. te all stock solu tions to final concentration with double distilled water Final conc acrylamide g bisacrylamide g dd H 0 30 T 4 C 57 6 2 4 to 200 ml 30 T 5 C 57 0 3 0 to 200 ml 30 T 6 C 56 4 3 6 to 200 ml 30 T 8 C 55 2 4 8 to 200 ml 30 T 10 C 54 0 6 0 to 200 ml Cast the membranes Important 2 Always wear gloves when handling membranes 3 both for your protection and to avoid contaminat ing the membranes Handle the membranes as 4 little and as gently as pos sible to avoid damaging them 5 6 Important Prepare and cast only one membrane solution at a time 7 in order to prevent prema ture polymerization Figure 9 8 Membrane casting cham bers 9 18 Prepare deionize filter and degas enough acrylamide bis monomer stock solu tion for a minimum of 10 ml final diluted volume for each membrane pH value Recipes are listed in Table 3 Remove acrylamido buffers from 4 C storage and allow to warm to room tem perature for 30 minutes Cover the work area with absorbent paper Label one membrane mould block 2 casting wells each 1 mm x 4 7 cm dia with glass cover plates for each pH mem brane formulation Always make two membranes and use the best one Coat the clean dry cover plates for each mold block with 1 ml of Repel Silane solution and allow to dry several minutes Repel Silane 2 dichlorodimethyl silane in 1 1 1 chloro trichloroethane prevents membrane adhesion to the cov
55. tension Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser a Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsabilit Amersham Biosciences garantit Putilisateur que le produit livr a subi avec succ s tous les essais pr vus pour s assur er qu il est conforme aux sp cifications et normes en vigueur La garantie incluse dans les conditions de livraison n est valable que si le produit a t install et utilis conform ment aux instructions fournies par Amersham Biosciences La soci t Amersham Biosciences ne sera en aucun cas responsable de tout dommage caus directement ou indirecte ment par toute utilisation incorrecte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives commerciales tout emp chement d utilisation et tout autre risques ayant un rapport avec Putilisation du produit mais sans aucune limitation quant a la nature de ces dommages Copyright 1998 Amersham Biosciences Tous droits r serv s La reproduction le stockage dans un syst me de r cup ration d informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publication en totalit ou en partie sont s
56. the pH The buffering capability and ionic strength of a membrane can be specified by using the appropriate range of buffers The buffering capacity of any membrane with acrylamido buffers is several thousand times higher than the protein solution in terms of buffering and titrant molarity These properties cause the membranes to isolate the protein in the appropriate chamber and keep the protein isoelectric Table 2 Acrylamido buffer characteristics Note Use utmost care to follow all procedures precisely and to avoid all contamination Proper membrane prepara tion is essential to ensure effective and reproducible protein separations Table 3 Recipes for 30 T x C Acrylamide bisacrylamide stock solution 17 Listed below are all commercially available acrylamido buffers and each buffer s type pK value and molecular mass Mr relative to H Acrylamido buffer type pK M 2 Acrylamido 2 methylpropane sulfonic acid acid titrant 1 0 207 2 Acrylamidoglycolic acid acid 3 1 145 N Acryloylglycine acid 3 6 129 4 Acrylamidobutyric acid acid 4 6 157 2 Morpholinoethylacrylamide base 6 2 184 3 Morpholinopropylacrylamide base 7 0 198 N N Dimethylaminoethylacrylamide base 8 5 142 N N Dimethylaminopropylacrylamide base 9 3 156 N N Diethylaminopropylacrylamide base 10 3 184 N N N Triethylaminoethylacrylamide base titrant gt 12 198 Available as Immobilines from Amersham Biosciences or as acrylamido buffers from Fluka Biochemi
57. tic acid Lysine Citric acid Arginine y amino butyric acid Histidine Formic acid ddH20 may be used for certain neutral or acidic proteins NaOH lt 2mM can be used but may result in pH shock for certain proteins Adjust the pH of the dilute acid or base to within 1 to 2 pH units of the membrane pH at each electrode Avoid titrating with strong acids and bases Then add the same con centration of solubilizing agent used in the other sample vessels Fill the sample vessel in the cathode chamber circuit with catholyte buffer and the sample vessel in the anode chamber circuit with anolyte buffer Load the sample Small to medium quantities If the IsoPrime is intended to separate moderate to heavily contaminated proteins use one of two strategies to purify the sample Fill all sample vessels with the solution used to solubilize the protein except the sample vessel that feeds into the chamber delimited by membranes with pH values slightly above and slightly below the pl of the target protein Fill this remaining sample vessel with the protein solution Under electrophoretic separation contaminants or other isoforms will move out of the chamber containing the target protein and into adjacent chambers leaving a purified product Or load the protein into one of the adjacent chambers delimited by membranes with pHs slightly above or slightly below the pI of the protein to be purified Under elec trophoretic separation the protein of interest
58. trictement interdits sans autorisation pr alable crite de la soci t Wichtige Benutzerinformationen F r ein vollst ndiges Verst ndnis und eine sichere Handhabung dieses Produktes ist es notwendig da der Benutzer dieses Handbuch vollst ndig durchliest A Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Deutsch Ein Ausrufezeichen in einem gleichseitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und Wartungsanweisungen in der dem Ger t beiliegenden Dokumentation hinweisen Ein Blitzsymbol in einem gleichseitigen Dreieck soll den Benutzer auf die Gefahr anliegender Hochspannungen hinweisen Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and Haftung Amersham Biosciences garantiert da das gelieferte Produkt sorgf ltig auf die Einhaltung der ver ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gew hrleistungsanspr che gelten nur dann wenn das Produkt gem f den von Amersham Biosciences gelieferten Anweisungen installiert und benutzt wurde Amersham Biosciences bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbufen Einkommensverluste entgangene Gesch ftsabschl sse Verlust
59. valently incorporate acrylamido buffers to fix a series of isoelectric pH points that define steps in a pH gradient The peristaltic pump circu lates the material to be separated from sample vessels through the separation cham bers where electrofocusing takes place Proteins move by electrophoresis through the isoelectric membranes until they reach a chamber bounded by membranes with pHs which bracket the pI of the protein With appropriately selected membrane pHs each chamber and the sample vessel it is connected to will contain an isoelectrically homo geneous fraction at the end of the run Important safety information This instrument and its components are for in vitro research use only Warning Operation of this instrument requires potentially hazardous high volt age It should be used only by trained persons Turn off and disconnect any high voltage power supply before opening the safety enclosure lid The high voltage safety interlock is not to be used as the primary high voltage switch for the instru ment Warning If liquid is observed to have spilled or overflowed during a separation run turn off the high voltage power supply and the AC mains power to the entire instrument before opening the safety enclosure lid Disconnect any instrument from the AC mains before cleaning or servicing Acrylamide is a neurotoxin Wear gloves and work under a fume hood when han dling a Caution Refer to the manual 2 1

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