DIGE Gel and DIGE Buffer Kit - GE Healthcare Life Sciences
Contents
1. IPG strip Interfering substances Contaminants in the sample can in the first dimension Distortion inthe Interfering substances 2 D pattern in the first dimension cause distortions or swollen regions in the IPG strip following IEF These distortions can result in disturbances in the second dimension Modify sample preparation to limit these contaminants Refer to 2 D Electrophoresis Using Immobilized pH Gradients Principles and Methods code no 80 6429 60 for suggestions Contaminants in the sample can cause distortions or swollen regions in the IPG strip following IEF These distortions can result in turn in disturbances in the second dimension Modify sample preparation to limit these contaminants Refer to 2 D Electrophoresis Using Immobilized pH Gradients Principles and Methods code no 80 6429 60 for suggestions DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 15 Troubleshooting 4 Symptom Possible cause Remedy Vertical gapin Bubble between IPG Ensure that no bubbles are trapped the 2 D pattern strip and top surface between the IPG strip and the top of second dimension surface of second dimension gel gel Vertical streaking Incorrectly prepared Prepare equilibration solution equilibration solution according to instructions Poor transfer of Employ a low power or current protein from IPG strip sample entry phase in the second to second gel dimension electrophoresis run Pr2. is intended for use in the Ettan DALTtwelve and Ettan DALTsix electrophoresis units The gel is used together with the DIGE Buffer Kit which includes concentrated buffers for running the gel and Sealing Solution for attaching the IPG strip to the top of the slab gel Ettan DALTtwelve and DALTsix units are electrophoresis instruments designed for the second dimension of large format 2 D electrophoresis using either 24 cm or 18 cm IPG strips The electrophoresis units can accommodate up to 12 gels or up to 6 gels respectively either precast or lab cast The buffer system used in the gel gives longer shelf life than the conventional Laemmli Tris glycine system while retaining the capacity and robustness of that system Separations performed using the DIGE Gel are similar to those seen with a 12 5 Laemmli gel The DIGE Buffer Kit contains all the reagents necessary for a single run of up to 12 DIGE Gels in the Ettan DALTtwelve electrophoresis unit and two runs of up to 6 gels in the Ettan DALTsix electrophoresis unit see Fig 2 1 e s 9 Lehre la 7 SS N Fig 2 1 DIGE Gel and DIGE Buffer Kit DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 6 Instructions for use 3 3 Instructions for use WARNING Always wear gloves protective clothing and eye protection when handling DIGE gels DIGE Buffer IPG strips gel cassettes or any other equipment these items will come into contact with Note Always
3. level of the anode buffer does not come above the sealing assembly when the electrophoresis unit is fully loaded If excess anode buffer is in the upper reservoir it should be removed with a pipette Ensure that the level of cathode buffer does not come above the air vents in the corners of the upper reservoir Lack of mixing between upper and lower reservoirs can be verified by adding bromophenol blue dye to the lower reservoir prior to loading the unit with gels Several drops of 1 w v bromophenol blue will impart sufficient color to the anode buffer DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 14 Troubleshooting 4 Symptom Possible cause Remedy Second One of the slots inthe All six slots in the upper buffer dimension upper buffer chamber chamber must be occupied by either a separation is open gel cassette or a blank cassette proceeds slowly with high current Ettan DALTsix only Upper buffer chamber Change upper buffer chamber is damaged Anodic buffer has Ensure that the level of the anode mixed with cathodic lower buffer does not come above buffer from overfilling the level of the buffer in the upper of either the cathodic buffer chamber when the reservoir or the anodic electrophoresis unit is fully loaded reservoir Dye front is The top surface of the Take care during application of the IPG irregular gel has been damaged strip that the gel is not damaged during application of the
4. run 12 gels 28 9374 52 Contains Anode Buffer 2 bottles 2 x 125 ml Cathode Buffer 4 bottles 4 x 125 ml Sealing Solution 12 tubes 12 x 1 ml DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB T7 Technical information 5 5 2 Technical specifications DIGE Gel Gel composition Separation range Gel dimensions Buffer in gel Gel cassette Shelf life Storage T 12 5 C 3 12 125 acrylamide 0 375 bisacrylamide 12 120 kDa 255 x 196 x 1mm Special buffer based on piperidinopropionamide PPA Low fluorescent glass 12 months 4 C to 8 C DIGE Buffer Kit Anode Buffer 2 bottles Cathode Buffer 4 bottles Sealing Solution Shelf life Storage Special buffer based on piperidinopropionamide PPA 0 25 M Tris 1 92 M glycine 1 w v SDS Gel Buffer with 0 5 agarose and 0 002 bromophenol blue Estimated 12 months 4 C to 8 C DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 18 5 3 Recommended equipment accessories Technical information 5 and reagents Designation Code No Ettan DALTtwelve Separation Unit and 80 6466 27 Power Supply Control Unit 230 VAC Ettan DALTtwelve Separation Unit and 80 6466 46 Power Supply Control Unit 115 VAC Ettan DALTsix Electrophoresis Unit including buffer 80 6485 27 circulation pump and Peltier cooling 230 VAC Ettan DALTsix Electrophoresis Unit including buffer 80 6485 08 circulation pump and Pel
5. trademarks are the property of their respective owners 2009 General Electric Company All rights reserved First published Oct 2008 CyDue This product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent number 5 268 486 and equivalent patents in the US and other countries 2 D Fluorescence Difference Gel Electrophoresis 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE technology is covered by US patent numbers 6 043 025 6 127 134 and 6 426 190 and equivalent patents and patent applications in other countries and exclusively licensed from Carnegie Mellon University CyDye this product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent numbers 5 569 587 5 627 027 and equivalent patents in other countries The purchase of CyDye DIGE Fluors includes a limited license to use the CyDye DIGE Fluors for internal research and development but not for any commercial purposes A license to use the CyDye DIGE Fluors for commercial purposes is subject to a separate license agreement with GE Healthcare All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare Europe GmbH Munzinger Stra
6. use the highest quality reagents and the purest water available Note Do not store opened bottles of DIGE Buffer 3 1 Storage of gels Store the gels horizontally in the original packaging at 4 C to 8 C 3 2 Preparing samples and first dimension IEF For instructions in preparing samples for 2 D electrophoresis and running first dimension IEF see 2 D Electrophoresis Principles and Methods code no 80 6429 60 AC and Ettan DIGE System User Manual 18 1173 17 AB 3 3 Preparing Ettan DALTtwelve electrophoresis unit Instructions for using Ettan DALTtwelve Electrophoresis Unit can be found in the instrument s User Manual 1 Ina separate container dilute the concentrated cathode buffer included in the DIGE Buffer Kit to working strength by adding four bottles of DIGE cathode buffer total volume 500 ml and fill up with distilled or deionized water to a total volume of 2 25 l 2 Ensure that the valve on the separation unit is set to circulate Add the entire contents of two bottles of DIGE anode buffer stock solution included in the DIGE Buffer Kit into the tank see Fig 3 1 Rinse the bottles with distilled or deionized water and pour it into the tank Fill the tank to the 7 5 fill line with distilled or deionized water in this way washing the DIGE anode DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 7 Instructions for use 3 buffer from the buffer seal Fig 3 1 Add the anode buffer Note A
7. GE Healthcare DIGE Gel and DIGE Buffer Kit Precast polyacrylamide gels and buffers for 2 dimensional electrophoresis User Manual Important user information All users must read this entire manual to fully understand the safe use of DIGE Gel and DIGE Buffer Kit WARNING that must be followed to avoid personal injury Do not proceed until all stated conditions are clearly understood and met f The WARNING sign highlights instructions CAUTION The CAUTION sign highlights instructions that must be followed to avoid damage to the product or other equipment Do not proceed until all stated conditions are met and clearly understood Note The Note sign is used to indicate information important for trouble free and optimal use of the product CE Certifying This product meets all requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a stand alone unit or connected to other CE marked GE Healthcare instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from GE Healthcare except for alterations described in this manual Contents DEO CUCU ON poe cyeateetse ee eeirorie nt even eee eee ee 5 Description of the system esesessesssse
8. ation To reduce vertical streaking in the second dimension it is necessary to apply two equilibration steps The first step saturates the IPG strip with the SDS system and the second step blocks the protein thiol groups The equilibration solution contains buffer urea glycerol reductant SDS and dye Prepare equilibration solution Prepare SDS equilibration buffer below This is a stock solution Just prior to use add 50 mg DTT per 10 ml SDS equilibration buffer 0 5 w v SDS equilibration buffer Final concentration Amount 1 5 M Tris Cl pH 8 8 50 mM 6 7 ml Urea FW 60 06 6M 72 07 g Glycerol 87 v v 30 v v 69 ml SDS FW 288 38 2 w v 40g Bromophenol blue 0 001 w v 2mg Distilled or deionized water to 200 ml Store in 40 ml aliquots at 20 C Equilibration Place the IPG strips in individual tubes with the support film toward the tube wall Add 10 ml DTT containing solution to each tube Place the tubes on a rocker and equilibrate for 15 min Note When using CyDye DIGE saturation dyes Labeling Kit for scarce samples repeat the first equilibration with DTT containing SDS equilibration solution for another 15 min Second equilibration A second equilibration is performed with an iodoacetamide solution instead of DTT Prepare a solution of 450 mg iodoacetamide per 10 ml of SDS equilibration buffer 4 5 w v Decant the first equilibration solution and add the same volume of iodoacetamide containing equ
9. gaskets If the buffer level is too low add distilled or deionized water to the lower buffer chamber If excess anode buffer is in the upper reservoir remove it with a pipette Pour the diluted cathode buffer into the tank to the fill line some of this buffer may drip through the gasket and mix with the anode buffer during the run but this will not affect performance or results DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 10 Instructions for use 3 3 8 Inserting gel cassettes into Ettan DALTsix When the electrophoresis buffer has reached the desired temperature insert the loaded gel cassettes with the IPG strips in place 1 Wet the UBC sealings with cathode buffer or 0 1 SDS immerse the sealings in solution or spray the sealings of the UBC using a plant sprayer and carefully slide the UBC over the gel cassettes Note Do not move the UBC repeatedly up and down as this will reduce the sealing effect 2 Ina separate container add 2 x 125 ml 2 bottles of cathode buffer Rinse the bottles and fill up with distilled or deionized water to 1 2 3 Fill the UBC with 1 2 liters of diluted cathode buffer and use a funnel to adjust the buffer level in the lower buffer chamber to the same height as in the UBC by adding water or diluted anode buffer 3 9 Recommended running conditions Ettan DALTtwelve Program the Ettan DALTtwelve electrophoresis unit to deliver the following protocol Constant power Temperat
10. ilibration solution to each tube Place the tubes on a rocker and equilibrate for an additional 15 min Note The subsequent steps of electrophoresis unit preparation insertion of gels and melting of the Sealing Solution can be performed while the IPG strips are equilibrating DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 9 Instructions for use 3 3 6 Applying Immobiline DryStrip gels Take out the gels from the refrigerator and keep gels at room temperature Open the gel package and remove the gel The DIGE gels come in glass cassettes and are ready to be used Allow the gels to reach room temperature before use For more information about the application of strips see Ettan DIGE System User Manual 18 1173 17 Edition AB 3 7 Inserting gel cassettes into Ettan DALTtwelve When the electrophoresis buffer has reached the desired temperature insert the loaded gel cassettes with the IPG strips in place Note Gel Cassettes and Blank Cassette Inserts slide much more easily into the unit if they are wet Wetting the cassette with some cathode buffer using a soaked kleenex or alternatively distilled or deionized water from a squirt bottle can be used to wet the cassettes and Blank Cassette Inserts as they are being loaded into the unit 1 Fit Blank Cassette Inserts into any unoccupied slots 2 Load the unit from back to front 3 Whenall 12 slots are occupied the buffer level should be slightly below the level of the
11. mension of 2 dimensional 2 D electrophoresis The gel is cast in a low fluorescent glass cassette that is compatible with 2 D DIGE analysis The gel size is 255 x 196 x 1 mm The gel is a homogeneous 12 5 polyacrylamide gel cross linked with bisacrylamide It is intended to be used in the Ettan DALTtwelve and Ettan DALTsix electrophoresis units together with the DIGE Buffer Kit The gel is formulated for long shelf life and when used with the DIGE Buffer Kit generates a discontinuous buffer system offering rapid runs with sharp reproducible results The performance and capacity of this gel and buffer system are similar to the widely used Laemmli Tris glycine buffer system This User Manual describes how to use DIGE Gel together with the DIGE Buffer Kit for the second dimension of 2 D electrophoresis Note The gels are not bound to the glass and are intended to be kept in the glass cassettes through electrophoresis and scanning For preparative gels and consecutive spot picking we recommend the backing supported DALT gels 17 6002 36 or the Ettan Spot Picker Nonbacked Gel Kit 11 0002 93 see Section 3 13 DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 5 Description of the system 2 2 Description of the system DIGE Gel is a precast polyacrylamide gel for the second dimension of large format 2 D electrophoresis It is cast in a low fluorescent glass cassette which is compatible with 2 D DIGE analysis The gel
12. olong entry phase if necessary nsufficient Prolong equilibration time equilibration PG strip was not Equilibrate IPG strip in two steps 1st equilibrated with step with DTT 0 5 and 2nd step odoacetamide ina with iodoacetamide 4 5 second equilibration according to instructions on page 9 step Spots are PG strip is not placed Ensure that the plastic backing of vertically dou properly the IPG strip is against the glass bled or plate of the second dimension twinned cassette Poor Incorrectly prepared Prepare equilibration solution representation of equilibration solution according to instructions higher molecular weight proteins Poor transfer of protein Employ a low power or current from IPG strip to sample entry phase in the second second dimension gel dimension electrophoresis run Prolong entry phase if necessary Bubbles lagging These bubbles do not affect the after the front result DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 16 Technical information 5 5 Technical information 5 1 Package contents DIGE Gel Each gel package contains three gels Designation No per pack Code No DIGE Gel 3 28 9374 51 DIGE Buffer Kit Each kit contains 6 bottles of buffer 12 tubes of sealing solution and the Short Instruction The solutions are sufficient for a single run of up to 12 gels or two DALTsix runs Designation No per pack Code No DIGE Buffer Kit Sufficient to
13. s units For information regarding unloading gels from the electrophoresis units please refer to the respective instrument User Manuals The gels should not be removed from the cassettes 3 12 Detection Scan the gels as soon as possible after the second dimension SDS PAGE is finished in order to minimize protein diffusion Typbhoon Variable Mode Imager or Ettan DIGE Imager are recommended for scanning DIGE second dimension SDS PAGE gels Store the gels in a refrigerator in a closed container and keep the gels moist Allow the gels to reach room temperature before scanning Keep the gels in the glass cassettes throughout scanning DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 12 Instructions for use 3 3 13 Further analysis of protein spots For preparative gels and consecutive spot picking the backing supported DALT gels 17 6002 36 and the DALT Buffer Kit 17 6002 50 can be used CyDye DIGE fluor minimal dye Cy 5 can be included for matching and detection purposes Alternatively the non supported DIGE gels can be used with the Ettan Spot Picker Nonbacked Gel Kit 11 0002 93 DIGE gels can be removed from the glass cassettes for staining or Western Blotting To open the cassette use a spatula to remove the glue at one side of the cassette Open carefully to avoid damaging the gel Adding small amounts ml of water or buffer on top of the gel will aid the opening process With the gel positioned on one glass pla
14. sse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 28 9460 89 AB 03 2009
15. ssessssesessesessesesseseesesses 6 Instructions for USe eseneseesosoorossosessosessesssrosssrossesessesesessssee 7 31 Stotage OF gelsin aa a N RTE ER EAN 7 3 2 Preparing samples and first dimension IEF ecscccscsssssssssssseeeessecsee 7 3 3 Preparing Ettan DALTtwelve electrophoresis unit cscs 7 3 4 Preparing Ettan DALTsix electrophoresis unit nccccccccsssssssssseeeeesseee 8 3 5 Equilibrating ImmMobiline DryStrip GelS ccsseseccssssscccccsssssssssssseeeeeses 9 3 6 Applying Immobiline DryStrip gels 10 3 7 Inserting gel cassettes into Ettan DALTtwelve vices 10 3 8 Inserting gel cassettes into Ettan DALTSIX vcs 11 3 9 Recommended running conditions Ettan DALTtwelve 11 3 10 Recommended running conditions Ettan DALTSIX 11 3 11 Unloading gels from electrophoresis units ecccccsccccsssssssssssseeees 12 BAZ s Detectie e T AE AR 12 3 13 Further analysis of Protein SPOts eccccccccssssssssssseeessssscsscsssssssssseeeees 13 Troubleshooting ssiiissoisirossisiresstoseoreseseocirecreesittssieseseoserssie 14 Technical information seseesssesssessssessesesesssseesessesessessosesesee 17 SL Package CONTENTS nesine A AA 17 5 2 TechAICASpECIfCAtiON Sieniniai a aE a 18 5 3 Recommended equipment accessories and reagents 19 DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB Introduction 1 1 Introduction DIGE Gel is a precast polyacrylamide gel for the second di
16. te the DIGE gel can be easily moved to a tray for post staining e g using Deep Purple Please note that the gel is not attached to a glass plate DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 13 4 Troubleshooting Troubleshooting 4 This section concerns troubleshooting problems that have their origin in the second dimension separations using DIGE Gel For a more comprehensive guide to troubleshooting problems with 2 D electrophoresis see 2 D Electrophoresis Using Immobilized pH Gradients Principles and Methods 80 6429 60 Symptom Possible cause Remedy No current at start of run Insufficient volume of buffer in upper reservoir Ensure that the upper reservoir contains enough buffer to contact the upper electrode Buffer not circulating Ettan DALTsix only Pump is not primed Pump is off Pump is broken Turn pump off and on to purge air bubbles Turn pump on Call for service Second dimension separation proceeds slowly with high current Ettan DALT twelve only All the slots in the sealing assembly are not occupied by either gel cassettes or Blank Cassette Inserts Anodic buffer has mixed with cathodic buffer from overfilling of either the cathodic reservoir or the anodic reservoir Ensure that all 12 slots in the sealing assembly are occupied Do not pour more than the suggested volume 7 5 I into the lower reservoir Ensure that the
17. tier cooling 115 VAC ultiTemp IIl Thermostatic Circulator 115 V 18 1102 77 ultiTemp IIl Thermostatic Circulator 230 V 18 1102 78 EPS 601 Power Supply 18 1130 02 DALT Blank Cassette Insert 80 6467 03 Cassette Rack 80 6467 98 PlusOne Urea 17 1319 01 PlusOne Tris 17 1321 01 PlusOne Glycerol 17 1325 01 PlusOne Dithiothreitol 17 1318 01 PlusOne Sodium Dodecylsulfate 17 1313 01 PlusOne Bromophenol Blue 17 1329 01 Equilibration Tube Set 12 pk 80 6467 79 Immobiline DryStrip Dry polyacrylamide gels 0 5 mm T 4 C 3 after rehydration cast on plastic backing 12 pk pH interval Code No 18 cm strip Code No 24 cm strip 3 5 4 5 17 6002 38 3 7 NL 7 6002 43 4 7 17 1233 01 17 6002 46 6 9 17 6001 88 17 6002 47 6 11 17 6001 97 3 10 17 1234 01 17 6002 44 3 10 NL 17 1235 01 17 6002 45 3 5 6 NL 17 6003 56 17 6003 57 5 3 6 5 17 6003 61 17 6003 62 6 2 7 5 17 6003 66 17 6003 67 7 11 NL 17 6003 71 17 6003 72 3 11 NL 17 6003 76 17 6003 77 DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 19 For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com proteomics imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company Cy CyDye Deep Purple Ettan Immobiline MultiTemp and Typhoon are trademarks of GE Healthcare companies All third party
18. ure 22 C Phase Power W gel Duration 1 1W 1 hour 2 17 W max 180W Until the bromophenol dye front reaches the bottom of the gel approximately 4 5 hours for a full set of 12 gels For overnight runs the power is set to 1 W gel and the recommended tempera ture is 15 C Alternatively set the temperature to 22 C Run for 1 hour and then increase to 1 5 W gel 3 10 Recommended running conditions Ettan DALTsix The maximum rated electrical input for the electrophoresis unit is 400 mA 100 W and 600 V For overnight runs we recommend to set the temperature to 15 C Alternatively set the temperature to 22 C For further run conditions see the DIGE Gels and DIGE Buffer Kit Short Instructions 28 9460 86 in the DIGE Buffer Kit DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB led Instructions for use 3 Run conditions Day run Set the MultiTemp temperature to 22 C Step mA gel Voltage V W gel Time hours mins 1 10 80 1 1 0 2 50 500 17 4 00 5 00 The maximum rated input power for the electrophoresis unit is 100 W Continue the electrophoresis until the bromophenol blue reaches the end of the gel Run Conditions Overnight Set the MultiTemp temperature to 15 C Step mA gel Voltage V W gel Time hours mins 1 12 150 1 5 15 00 17 00 Continue the electrophoresis until the bromophenol blue reaches the end of the gel 3 11 Unloading gels from electrophoresi
19. void pouring the DIGE anode buffer onto the tubing by spreading the tubing elements apart using one hand while pouring the solution with the other hand 3 Switch the separation unit on 4 Turn the pump on to mix set separation unit to desired temperature A temperature of 22 C is recommended for day runs and 15 C for overnight runs 3 4 Preparing Ettan DALTsix electrophoresis unit Instructions for running the Ettan DALTsix electrophoresis unit can be found in the instrument s User Manual 1 When preparing to run a gel in the DALTsix instrument insert the anode assembly in the tank and then fill the lower buffer chamber tank Add one bottle 125 ml of DIGE Anode Buffer stock solution included in the DIGE Buffer Kit into the tank Fill the electrophoresis unit to the 4 5 fill line with distilled or deionized water and turn the pump on Note Use only one bottle of the DIGE Anode lower Buffer for each DALTsix run 2 Turnon the circulation pump 3 Connect an external MultiTemp Ill thermostatic circulator and set the temperature to 22 C for a day run and to 15 C for an overnight run Equilibrate the buffer to 15 C before starting an overnight run DIGE Gel and DIGE Buffer Kit User Manual 28 9460 89 Edition AB 8 Instructions for use 3 3 5 Equilibrating Immobiline DryStrip gels The equilibration step saturates the Immobiline DryStrip gel IPG strip with the SDS buffer system required for the second dimension separ
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