Ettan DALTsix Electrophoresis System
Contents
1. volume reguired for ml final T 12 14 16 18 20 Acrylamide stock 82 95 109 123 136 1 5 M Tris Cl pH 8 8 52 5 52 5 52 5 52 5 53 Water 55 41 27 14 0 0 10 SDS 2 1 2 1 2 1 2 1 2 1 Glycerol 18 18 18 18 18 10 APS 1 05 1 05 1 05 1 05 1 05 10 TEMED 0 09 0 08 0 07 0 06 0 06 For six 1 5 mm gels Light solution 275 ml volume required for ml final T 8 10 12 14 16 Acrylamide stock 73 92 110 128 147 1 5 M Tris Cl pH 8 8 69 69 69 69 69 Water 127 109 91 73 54 10 SDS 2 75 2 75 2 75 2 75 2 15 Glycerol 0 0 0 0 0 10 APS 2 75 2 75 2 75 2 75 2 75 10 TEMED 0 59 0 47 0 39 0 34 0 29 Heavy solution 275 ml volume required for ml final T 12 14 16 18 20 Acrylamide stock 110 128 147 165 183 1 5 M Tris Cl pH 8 8 69 69 69 69 69 Water 73 55 37 18 0 10 SDS 2 75 2 75 2 75 2 75 2 75 Glycerol 19 19 19 19 19 10 APS 14 14 14 14 14 10 TEMED 0 13 0 11 0 10 0 09 0 08 36 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 10 Troubleshooting Troubleshooting 10 10 1 Electrical and mechanical Symptom Possible causes Possible solutions No current at start of run Ensure that the unit contains enough buffer to contact the upper electrode Insufficient volume of buffer in upper reservoir Buffer not circulating Pump is not primed Pump is off Turn pump off and on to purge air bubbles Turn on pump by plugging in the connector in the mains power outlet Pump i2. 1 400 mA 1 100 W 18 1130 02 ultiTemp III Thermostatic Circulator 230 V 8 1102 78 ultiTemp ll Thermostatic Circulator 115 V 8 1102 77 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD www gelifesciences com GE imagination at work and GE monogram are trademarks of General Electric Companu Ettan Immobiline IPGphor Multiphor and MultiTemp are trademarks of GE Healthcare companies GE Healthca re Bio Sciences AB 2006 2007 General Electric Company All rights reserved Bj rkgatan 30 First published Oct 2006 75184 U ppsala All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare Sweden which supplies them A copy of these terms and conditions is available on request Contact your local GE Health care representative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd 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 Asia Pacific Tel 85 65 62751830 Fax 85 65 62751829 e Australasia Tel 61 2 8820 8299 Fax 61 2 8820 8200 Austria Tel 01 57606 1613 Fax 01 57606 1614 Belgium Tel 0800 73 890 Fax 02 416 8206 e Canada Tel 1 800 463 5800 Fax 1
3. 11 5 Maintenance Polyurethane PUR e Electrical and electronic components When taking Ettan Spot Handling Workstation out of service these different materials must be separated and recycled according to local regulations This symbol indicates that the waste of electrical and electronic eguipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your eguipment 11 5 Maintenance Check the following before each run on Ettan DALTsix e all parts are clean see Section 11 2 e all parts are intact and free of cracks and other damage If a part is damaged replace the part or entire instrument before use see Section 11 3 e the power cord and high voltage cable is intact and free of cracks and other damage If the cable is damaged replace it before use e there is no leakage from parts containing buffer or cooling water Moist or liquid must be removed from the equipment before use e the circulation pump spins and that there is circulation of liquid 42 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 12 Customer service information 12 1 Technical service and repair GE Healthcare offers complete technical support for all our products If you have any questions about how to Customer service information 12 use this product or would like to arrange to repair
4. 11 Immediately pipette the water saturated n butanol onto each gel Avoid wetting adjacent plastic surfaces of the caster with n butanol 12 Allow the gradient gels to polymerize for 2 hours before disassembling the caster Gradient gel polymerization should proceed from the top down If the peristaltic pump outlet tubing was connected directly to the caster it should not be removed until polymerization is complete Do not leave the water saturated n butanol o the gels overnight as long term exposure of acrylic to butanol will damage the caster ME Sa a connector D OD channel o slide stop open closed Fig 6 2 Open and closed positions of slide valve Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 23 6 Casting gradient gels 6 3 Pouring gel solutions for gradient gels 24 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Unloading the gel caster 7 7 Unloading the gel caster protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for f WARNING When using hazardous chemicals take all suitable protective measures such as wearing safe operation and maintenance of the system 1 Make sure the caster is either near a sink or on a tray so that any liquid leaking out can be contained 2 Remove the front of the gel caster by loosening and removing the black knobbed screws and spring clamps 3 Carefully unload
5. 800 567 1008 Central East amp South East Europe Tel 43 1 972 720 Fax 43 1 972 722 750 e Denmark Tel 45 70 25 24 50 Fax 45 45 16 2424 Eire Tel 1 800 709992 Fax 44 1494 542010 e Finland amp Baltics Tel 358 9 512 3940 Fax 358 9 512 39439 e France Tel 01 69 35 67 00 Fax 01 69 41 98 77 Germany Tel 0800 9080 711 Fax 0800 9080 712 Greater China Tel 852 2100 6300 Fax 852 2100 6338 e Italy Tel 02 26001 320 Fax 02 26001 399 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Korea Tel 82 2 6201 3700 Fax 82 2 6201 3803 e Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 e Middle East amp Africa Tel 30 210 96 00 687 Fax 30 210 96 00 693 Netherlands Tel 0800 82 82 82 1 Fax 0800 82 82 82 4 e Norway Tel 47 815 65 777Fax 47 815 65 666 e Portugal Tel 21 417 7035 Fax 21 417 3184 e Russia amp other CLS amp N I S Tel 7 495 956 5177 Fax 7 495 956 5176 e Spain Tel 902 11 72 65 Fax 935 94 49 65 e Sweden Tel 018 612 1900 Fax 018 612 1910e Switzerland Tel 0848 8028 10 Fax 0848 8028 11e UK Tel 0800 515 313 Fax 0800 616 927e USA Tel 1 800 526 3593 Fax 1 877 295 8102 imagination at work 80 6492 49 AD 03 2007
6. Replacementaf compORBNIS us RR SEE EES ER Re RE RR LR Es EE GE EE ee TES RECUCW ole EE EE EE Ee ee ee TIS MOIS ONCE EE RE GE AEG 12 Customer service information uue sesse sesse se se ee ee ee ee ee Se Re Ge ee ee ee ee ee 43 12 4 TEChNICGl SenvicesGMG repair SR EE tect enti ee Ge delta otal sort ee eh 43 12 2 rderindipformatION Ee ER EE RE EE Ge ee ed es RE Ek RE EG EE ke Es EE 43 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD System function and description 1 1 System function and description 1 1 Electrophoresis System function and description In 2 D electrophoresis proteins are separated according to isoelectric point by isoelectric focusing most reliably on Immobiline DryStrip immobilized pH gradient IPG gel strips using the IPGphor or Multiphor II IEF Systems The second dimension electrophoresis separates the proteins on the basis of their molecular mass using sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE The Ettan DALTsix Electrophoresis System is designed to handle large second dimension gels in a simple efficient and reproducible manner 1 1 1 Electrophoresis Unit The Ettan DALTsix Electrophoresis Unit accommodates up to six 25 5 x 20 5 cm slab gels either 1 mm or 1 5 mm thick in a common tank under identical conditions A sample focused in an IPG strip is placed on the cathodic up
7. and about 10 50 ng of each component for silver staining Seal the IPG strip in place For each IPG strip melt an aliquot of agarose sealing solution in a heating block or boiling water bath Tip an ideal time to carry out this step is during IPG strip equilibration Allow the agarose to cool slightly and slowly pipette the solution across the length of the IPG strip taking care not to introduce or trap bubbles It will flow down between the glass plate and the support film and seal the IPG strip in place see Fig 8 2 Agarose should also be used to seal any gap between the side of the gel and a side spacer Allow a minimum of 1 min for the agarose to cool and solidify we Fig 8 2 Adding agarose overlay Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Electrophoresis 8 8 3 Preparing the electrophoresis unit Note This Section describes the procedure for preparing the electrophoresis unit using the upper buffer chamber UBC shown in Fig 8 3 If another UBC is used follow instructions supplied together with that UBC Fig 8 3 Upper chamber to be used for this instruction The Ettan DALTsix electrophoresis unit requires a total volume of about 5 7 of electrophoresis buffer to fill both the UBC and the lower buffer chamber LBC For lab cast Laemmli gels the LBC requires approximately 4 5 of 1x SDS electrophoresis buffer while the UBC requires 1 2 of buffer with a higher concentration Prepare the
8. ee ane a 11 AE elf del OE EE EE N EE Ee 12 2 3 Emergency procedures ER RO De E A EEE Ee ee Ge De RE Ee 13 3 SpecHiEatiOns ie ee GE WE Ge ee 15 3 1 vElectrophoresis UR ie ee ee De SG RS Ee ED ES EG 15 3 2 GOL Casten irer terine a e a E A EE EE E E A S 16 4 Preparing the Gel Caster es Ee ee teas A ed cco AE EDE 17 5 Casting homogeneous gels sihisee SE Ke ie ER ERG Ke 19 6 Casting gradient gels 6 1 Preparation for gradient gel casting 6 2 Gradient GAStingsSetUpP se dts EER es EER AE EE EG GER A Ge Ee ee he 6 3 Pouring gel solutions for gradient gels ee RARR ee 22 7 Unloading the gel caster EG eatin ee ees eee aes 25 8 Electrophoresis ii ie Ge ee oe eine Sandals 27 8 1 Placement of the electrophoresis Unit us seg Gr Ee ES Re ER ek ede ae ee Es es skede 27 8 2 Preparing second dimension GEIS ee eene 27 8 2 1 Equilibration and loading 27 8 3 Preparing the electrophoresis unit wi 29 84 Recommended running conditions sl 8 5 Unloading the gels and cleaning the UNI 31 9 RECIPES ee ee re Ge ee Ge ee Re ee oat 33 10 Troubleshooting 10 1 Electrical and mechanical 10 2 Geledsting taeda iad iced theres Riad ee heh lal Oa ei oe ele oe Sle hhh hoe clad ae due 103Preseastgel8 RE RE sedis GE GR EE EE ee ES 10 4Stained gels GE ES ER GR EE Ge EE E GE ER AE GEE GE SS EE 11 Care and maintenance 11 1 Unpacking inventory and set up 112 EleanINndis ss Ee ee eg EE a Ge EE De ad 113
9. electrophoresis unit WARNING Handle the gels with care The edges of the glass plates of the gels are sharp PP Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 29 8 Electrophoresis 8 3 Preparing the electrophoresis unit 30 1 Fill the electrophoresis unit with 4 5 liters of 1x electrophoresis buffer or diluted anode buffer when using Ettan DALTsix Pre cast gels and turn the pump on Fig 8 4 A Insert the anode assembly into the tank so that the circulation ports are properly aligned The anode assembly is keyed so that it may only be inserted in one orientation and the bottom edge of the assembly should fit into the slot in the bottom of the tank Fig 8 4 B Turn on the MultiTemp Ill adjust the temperature to the desired setting Insert the prepared gels into the unit Fill any unused slots with blank cassette inserts Wet the UBC sealings with 1x electrophoresis buffer or 0 1 SDS immerse the sealings in solution or spray the sealings of the UBC using a plant sprayer and apply an even pressure on the UBC to carefully slide it over the gel cassettes Fill the upper cathode chamber with 2x electrophoresis buffer or diluted cathode buffer to a level between the fill lines about 1 2 liters Fill the LBC with 1x electrophoresis buffer or water when using Ettan DALTsix Pre cast gels to the same level as the upper chamber Fig 8 4 D Place the safety lid on the unit and be
10. it please contact your local GE Healthcare representative for more information Note Only use spare parts that GE Healthcare recommends for safety reasons The warranty is not valid if parts that are not recommended by GE Healthcare are used 12 2 Ordering information Product code no Ettan DALTsix Electrophoresis Unit 100 V 80 6494 77 110 120 V 80 6485 08 220 240 V 80 6485 27 Replacement Lid 80 6490 40 Replacement Upper Buffer Chamber 80 6490 78 Replacement Anode Assembly Cassette Carrier 80 6491 35 Ettan DALTsix and DALT II Cassettes Pre Cast Gel Cassette 80 6466 65 Gel Casting Cassette 1 mm 80 6466 84 Gel Casting Cassette 1 5 mm 80 6488 69 Blank Cassette Insert 80 6467 03 Cassette Removal Tool 2 pkg 80 6474 82 Ettan DALT II Pre Cast Gels Pre Cast Gel 12 5 6 pkg 17 6002 36 Buffer Kit one run of 12 gels 17 6002 50 Ettan DALTsix Gel Caster Complete with Separator Sheets 7 pcs and Filler Sheets 6 pcs 80 6485 46 Rubber Insert 80 6493 44 Small Black Knobbed Screws 80 6493 63 Ettan DALTsix Gradient Maker 80 6487 36 Ettan DALTtwelve Gel Caster Complete with Separator Sheets 16 pcs and Filler Sheets 6 pcs 80 6467 22 Separator Sheets 16 pkg 80 6467 41 Filler Sheets 6 pkg 80 6467 60 Black Knobbed Screws 4 pkg 80 6437 58 Funnel Sponge 80 6474 06 Acrylic Feed Tube 80 6437 20 Foam Sealing Gasket 80 6023 76 Silicon Tubing Set two pieces pkg 9 mm o d 178 mm long and 12 5 mm o d 16 mm l
11. the sealing gasket is compressed evenly by the faceplate and forms a tight seal with the caster Do not overtighten the screws If casting using the filling channel in the back plate of the caster be sure to plug the barbed fitting in the faceplate If casting using the displacement method attach the tubing from the peristaltic pump to the barbed fitting filler sheets filling channel separating sheets faceplate and gel cassette sealing gasket cassette hinge spring clamp 6 filler port and cap clamp screw Fig 4 1 Gel caster with parts labelled Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 17 4 Preparing the Gel Caster 18 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 5 Casting homogeneous gels 5 Casting homogeneous gels AN AN WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING Acrylamide is a neurotoxin Never pipette by mouth and always wear protective gloves when working with acrylamide solutions IPG strips or surfaces that come into contact with acrylamide solutions Be sure the entire gel casting system is clean dry and free from any polymerized acrylamide Prepare a sufficient volume of gel overlay solution
12. G strips or surfaces that come into contact with acrylamide solutions final conc amount Acrylamide MW 71 08 30 900 g Bis N N methylenebisacrylamide MW 154 17 0 8 24g Distilled or deionized water to 3 000 ml May need filtration Weigh acrylamide and bis under a hood avoid contact with dust Filter and store at 4 C 1 5 M Tris Cl pH 8 8 final conc amount Tris MW 121 14 15M 545 g 6 M HCI to pH 8 8 about 150 ml Distilled or deionized water to 3 000 ml Adjust to pH 8 8 and store at 4 C 10 w v SDS final conc amount Sodium dodecylsulfate MW 288 38 10 10g Distilled or deionized water up to 100 ml Store at room temperature 10 w v Ammonium persulfate final conc amount Ammonium persulfate MW 71 08 10 2g Distilled or deionized water up to 20 ml Prepare fresh 10 v v TEMED final conc amount TEMED MW 116 2 10 0 5 ml Distilled or deionized water 4 5 ml Prepare fresh 33 9 Recipes 34 Displacing solution 0 375 M Tris Cl pH 8 8 30 v v glycerol bromophenol blue 200 ml amount Tris Cl 1 5 M pH 8 8 50 ml Glycerol 60 ml Bromophenol blue 2 mg Distilled or deionized water 90 ml Should be made fresh stored solution may support microbial growth Water saturated butanol amount n i or t butanol 50 ml Distilled or deionized water 10 ml Combine in a bottle and shake Use the top phase to overlay gels Store at room tem
13. GE Healthcare Ettan DALTsIx Electrophoresis System User Manual Ettan Important user information All users must read this entire manual to fully understand the safe use of Ettan DALTsix Electrophoresis System Ettan DALTsix Electrophoresis System is intended for laboratory use only not for clinical or in vitro use or for diagnostic purposes WARNING to avoid personal injury Do not proceed until all stated conditions A The WARNING sign highlights instructions that must be followed are clearly understood and met WARNING High Voltage The High Voltage sign highlights an instruction that must be strictly followed in order to avoid contact with lethal high voltage A Be sure not to proceed until the instructions are clearly understood and all stated conditions are met 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 Health
14. NING The safety lid must be firmly in place before power can be applied WARNING Always turn off the power to the gels before opening the safety lid WARNING Only connect MultiTemp Ill or a circulating water bath with earthed water pipe and low pressure water circulation Do not connect the heat exchanger to a water tap or any other water source with unregulated pressure that may exceed 82 kPa 12 PSI WARNING Check electrical cables for damage and the electrophoresis unit for damage or leakage before use If damage and or leakage is present do NOT use the equipment There is a risk for high voltage injuries WARNING If electrical cables are damaged and or any of the buffer chambers has a leakage do NOT use the equipment There is a risk for high voltage injuries WARNING The power cord should be easily accessible to enable emergency disconnection of the current gt e PPP PP Ettan DALTSIX Electrophoresis System User Manual 80 6492 49 Edition AD 11 2 Safety information 2 2 Cautions WARNING Never connect the Ettan DALTsix Electrophoresis System to higher voltage current or effect than specified for that system WARNING Always disconnect the power cord before draining the tank or before servicing the Ettan DALTsix Electrophoresis System WARNING Use care when lifting and moving the electrophoresis unit It is best to move the unit when
15. buffer Decrease power voltage or current Vertical protein streaks IPG strip not properly placed on gel surface Make sure IPG strip contacts the gel surface uniformly along its entire length Avoid gouging the surface of the separating gel Include iodoacetamide equilibration step for IPG strip Gels cast simultaneously are different sizes Gradient gels uneven layering Spots skewed or distorted Allow the solution to settle or reach equilibrium before applying the overlay Apply equal amounts of overlay solution to each gel Apply overlay as quickly as possible Add sucrose 15 w v or glycerol 25 v v to the high percent monomer solution Add a very small amount of bromophenol blue to the high percent monomer solution to track gradient formation Excessive bromophenol blue will inhibit polymerization Gels run too fast uneven migration Run at a lower power setting Use a two step program start at a low power setting until the proteins enter the gel then increase the power for the remainder of the run Uneven gel surface Overlay the running gel with water saturated butanol before polymerization begins to avoid forming an uneven gel surface Uneven gel polymerization or gradient formation Heavy background after silver staining Unusually slow or fast run Use reagents of the highest purity preferably electrophoresis grade Use deionized double distilled water Check for leaks All plates
16. care 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 Recycling and must be collected separately Please contact an authorized representative of the manufacturer for information concerning EE the decommissioning of equipment This symbol indicates that the waste of electrical and electronic R equipment must not be disposed as unsorted municipal waste Safety standards This product meets the requirement of the Low Voltage Directive LVD 73 23 EEC through the following harmonized standards e EN61010 1 e EC 61010 1 e CAN CSA C22 2 No 61010 1 e UL61010 1 EMC This device meets the requirements of the EMC Directive 89 336 EEC through the following harmonized standard e EN 55014 Content 1 System function and description sesse se se see ee ee ee ee ee ee ee ee ee ee 7 1 1 Electrophoresis System function and description ee 7 Dias Eleetrophoresis UNIE ME EE EE EE ER GE AK Ee EE EE EE EE Ee 7 1 1 2 Gel COStE Riss ae EE SE GE Ge GE E E SR GE GE ER Se STER GR ES DE ee 7 1 13 Gel Easting Cassettes ER ESE N Ee E ES REGEER GEE REEN 8 1 14 Gradient Maken EE Re Se ee GR ee GE eg ED N 9 1 2 vHeatexehangeF EG RE ER EE EE RR GE RR SE EE Ee Ee 9 2 Safety information ee EG ER Rue ES Ke KS Re ee ee Dee ee Ek N 11 2 1 Warning S as EE EE EE Ge GE ee GE cS
17. cting valve between the reservoir heavy and mixing light chambers and clamp off the exit line 5 Prepare a sufficient volume of gel overlay solution water saturated n butanol You need 1 0 ml of overlay for each 1 mm cassette and 1 5 ml for each 1 5 mm cassette 6 Make up 200 ml of displacing solution 7 Make up the gel acrylamide solutions from the stock mixes but do not add the 10 ammonium persulfate APS and 10 N N N N tetramethylethylenediamine TEMED See Section 6 3 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 21 6 Casting gradient gels 6 3 Pouring gel solutions for gradient gels reservoir back connector channel chamber mixing front chamber outlet connector Fig 6 1 DALTsix gradient maker 6 3 Pouring gel solutions for gradient gels 1 Prepare the gel caster as described in Section 4 and place identifying gel labels inside each cassette 2 When you are ready to cast the gels add the APS and TEMED and mix each gel solution thoroughly Vary the amount of TEMED added to control the rate of polymerization Once these reagents are added polymerization begins You have about 10 min to cast the gradient before the gels begin to solidify at the top Work rapidly and carefully 3 Close the slide valve out on side of white slide stop button Fig 6 1 If the outlet tubing is not controlled by a pump clamp it off near the gradient former Add the required volume of t
18. distortions can result in turn in disturbances in the second dimension Vertical gap in the 2 D pattern Bubble between IPG strip and top surface of second dimension gel Ensure that no bubbles are trapped between the IPG strip and the top surface of second dimension gel Vertical streaking Spots are vertically doubled or Incorrectly prepared equilibration solution Prepare equilibration solution according to instructions Poor transfer of protein from IPG strip to second dimension gel Insufficient equilibration IPG strip is not placed properly Use low power for sample entry phase Extend entry phase if necessary Extend equilibration time Ensure that the plastic backing of the IPG strip is against the twinned glass plate of the second dimension cassette Poor representation of higher molecular weight proteins Incorrectly prepared equilibration solution Prepare equilibration solution according to instructions Poor transfer of protein from IPG strip to second Use low power for sample entry phase Extend entry phase dimension gel 38 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD if necessary Troubleshooting 10 10 4 Stained gels Symptom Possible solution Protein spots are diffuse or broader than usual Use only highest quality reagents Make sure that polymerization is complete Check equilibration time of IPG strips Too l
19. ect only to coolant sources with regulated pressure Fig 1 5 To connect the coolant tubing lines to the tank remove the lid and the anode rack from the tank and lay the tank fill label front side down Guide the free ends of the coolant tubes through the holes in the tank base and slip them onto the plastic connector fittings then pull the collector collar onto the tubing to lock it in place The end of the tubing can be lubricated with glycerol or a mix of glycerol water to ease the fit Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 9 1 System function and description 1 2 Heat exchanger 10 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Safety information 2 2 Safety information e This system is designed for indoor use only e To avoid risk of injury operation and user maintenance should be performed by properly trained personnel only and in accordance with the instructions e Any equipment connected to the system should meet the requirements of the EN 61 010 1 or other international safety standard Within EU connected equipment must be CE labelled 21 Warnings WARNING Connect the instrument to a properly grounded electrical outlet WARNING Use EPS 601 as power supply for Ettan DALTsix Electrophoresis System If another power supply is used it must have similar specifications as EPS 601 such as floating output and leakage current detection WAR
20. ed as a unit and can be stood upright to dry The cassettes can be cleaned in an automatic dishwasher Cassettes are 27 x 22 cm and produce a gel about 25 5 x 20 5 cm A 1 0 mm thick gel has a volume of approximately 52 ml anda 1 5 mm thick gel has a volume of approximately 78 ml Fig 1 3 Gel Casting Cassettes 8 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD System function and description 1 11 4 Gradient Maker The Ettan DALTsix Gradient Maker is designed for producing linear gradients of aqueous solutions ranging in volume from 200 2 000 ml The gradient maker can be used to form convex and concave exponential gradients with the addition of a one holed rubber stopper a piece of rigid tubing and a piece of flexible tubing Fig 1 4 Ettan DALTsix Gradient Maker 12 Heat exchanger The heat exchanger is built into the base of the separation tank The white alumina ceramic heat exchange surface is fragile and the user should avoid dropping anything directly on surface When connected to a circulator bath coolant passes through a serpentine chamber beneath the ceramic plate The thin ceramic permits rapid heat exchange between the coolant and the buffer in the electrophoresis tank The ceramic plate is attached with silicone rubber adhesive The heat exchanger tubing connectors are 13 mm o d The heat exchanger is rated to a maximum of 0 8 atmospheres above ambient 12 psig Do not connect it to a water tap Conn
21. empty WARNING The casting unit when filled with glass plates and gel solutions is very heavy Use caution when trying to move or lift the caster WARNING Fire hazard never use flammable liquids in the electrophoresis tank WARNING The protection provided by the equipment may be impaired if this equipment is used in a manner not specified by the manufacturer WARNING Only accessories and parts approved or supplied by GE Healthcare may be used for operating maintaining and servicing this product Pe Pe P P 22 Cautions CAUTION Rinse and flush the tank and pumping system with distilled or deionized water before and after use CAUTION Do not run the circulation pump if the electrophoresis tank is empty CAUTION Do not operate with buffer temperature above 40 C All plastic parts are rated for 40 C continuous duty 12 Ettan DALTSIX Electrophoresis System User Manual 80 6492 49 Edition AD Safety information 2 CAUTION Turn the buffer circulation pump on during electrophoresis to minimize uneven heating even if not connected to a thermostatted circulator CAUTION Connect the heat exchanger to an external thermostatted circulating bath Overheating will cause irreparable damage to the unit CAUTION Do not autoclave or boil this unit or any of its parts 2 3 Emergency procedures Emergency shutdo
22. esired speed to avoid introducing any air bubbles For displacement casting pump the gel solution into the caster until it is about 7 11 cm below the final desired gel height Stop the flow of acrylamide and transfer the feed tube to the container holding the displacement solution Restart the pump to displace the gel solution in the tubing and the V trough at the bottom of the caster The remaining acrylamide solution is forced into the cassettes to the final gel height When the final desired gel height is reached stop the pump and clamp off the tubing near the barbed fitting at the bottom of the caster For filling channel casting pour the gel solution into the channel until the solution reaches the final desired height about 1 cm below the top edge of the short plate The amount of gel solution required in either case will be 550 ml for six 1 5 mm gels and 425 ml for six 1 mm gels Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 19 5 Casting homogeneous gels 9 Immediately pipette the water saturated n butanol onto each gel Avoid wetting adjacent plastic surfaces of the caster with n butanol OR Spray do not pipette 0 1 w v SDS solution on the edges of the cassettes for example with a plant sprayer so the gels are covered By doing this curved edges on the gels are prevented 10 Allow the homogeneous gels to polymerize for at least one hour before disassembling the caster If the peristaltic pump
23. gin electrophoresis A B Il MAX fill line MIN fill line fill line ettes LBC start with cas Fig 8 4 Filling the buffer chambers Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Electrophoresis 8 8 4 Recommended running conditions Run conditions Day run Applicable to 1 mm thick 12 PAA gel and Laemmli Buffer system Set the MultiTemp temperature to 25 C Step mA gel Voltage V W gel Time h min 1 102 80 1 1 00 2 401 500 13 4 30 6 002 1 For 1 5 mm gels increase the current by 50 2 Continue the electrophoresis until the bromophenol blue reaches the end of the gel Run Conditions Overnight Set the MultiTemp temperature to 30 C Step mA gel Voltage V W gel Time h 1 10 80 1 1 2 121 150 2 15 172 1 For 1 5 mm gels increase the current by 50 2 Continue the electrophoresis until the bromophenol blue reaches the end of the gel 8 5 Unloading the gels and cleaning the unit N WARNING The HIGH VOLTAGE and power switch MUST be turned off before removing the safety lid unloading the gels drain and cleaning the unit 1 Carefully pull the UBC upward using the handles at each end 2 Asthe UBC is pulled upward make sure that the cassettes remain in the anode assembly 3 Take out the anode assembly 4 Remove the gel cassettes from the anode assembly 5 Open the gel cassettes using a Wonder Wedge 80 6127 88 to
24. he final solution to the reservoir back chamber 4 Carefully open the slide valve valve in on button side and allow just enough solution to flow through the connector channel to fill it to the edge of the mixing chamber then close the valve Be sure no large bubbles remain to obstruct flow through the channel 5 Add the required volume of the light solution to the mixing chamber and start the magnetic stirrer 6 Simultaneously open the outlet tubing if clamped off and start the pump Adjust the pump rate so that the solution is not forced up in a fountain that mixes with the overlying solution 7 Immediately open the connecting valve between the mixing and the reservoir chamber 8 Watch the gradient solution enter the caster It is important that no bubbles disturb the gradient watch the delivery carefully 9 Just as the last of the gradient mix is pumped out of the mixing chamber add 200 ml of displacement solution to the mixing chamber and pump it through until almost all of the gradient mix has entered the cassettes It is convenient to include a dye in the displacement solution to visually track the boundary between the gradient mix and the displacement solution 10 When the gels have reached the desired height and before air is introduced stop the peristaltic pump It is important that no air bubbles disturb the gradient 22 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Casting gradient gels 6
25. ild detergent Clean the Gel Casting Cassettes and Pre cast Gel Cassettes with a dilute solution of a laboratory cleanser such as RBS 35 from Pierce Chemical Company Rinse the cassettes thoroughly with distilled or deionized water e Do not autoclave or heat any part above 40 C e Do not expose the unit or its parts to organic solvents including gt 20 ethanol e If using radioactive reagents decontaminate the unit with a cleaning agent such as CONTRAD 70 or Decon 90 from Decon Laboratories Inc Note Local security regulations must be followed when handling radioactive reagents 11 3 Replacement of components N WARNING Never use the eguipment if any part cracked or broken Risk for electric shock If any of the components becomes cracked or broken it should be replaced The upper buffer chamber UBC should be replaced if any of the rubber flaps become cut or torn To prevent damage to the flaps ensure that all sharp edges on glass plates are smoothed and exercise care when employing the cassette removal tool With normal use the UBC ribs may become bowed upward this bowing should not affect the performance of the seal 11 4 Recycling Ettan Spot Handling Workstation contains the following materials e Stainless steel e Aluminium e Glass e Plastic Acetal POM Polyethylene Terephthalate Polyester PETP Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 41 11 Care and maintenance
26. ith the heavier high concentration solution sinking below the light solution This generates a downward gradient of increasing acrylamide gel percentage 6 2 Gradient casting setup WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING Acrylamide is a neurotoxin Never pipette by mouth and always wear protective gloves when working with acrylamide solutions IPG strips or surfaces that come into contact with acrylamide solutions 1 Be sure the entire gel casting system is clean dry and free from any polymerized acrylamide Remove the rubber insert from the base of the V shaped feed channel of the caster The caster should be placed on a level bench to ensure that the gels and gradients are even and level 2 Adda magnetic stir bar of the appropriate dimensions 20 30 mm long to the mixing chamber and place the unit on a magnetic stirrer If volumes will be less than half the capacity of the unit an identical stir bar should be placed in the reservoir chamber as well to balance the displacement and prevent backflow into the reservoir when the chambers are first connected 3 Connect tubing to the outlet connector and pump and adjust pump speed if used Connect the tubing to the gel casting unit 4 Close the conne
27. lean surface short glass plate side up 8 Using forceps remove the equilibrated IPG strip from the equilibration solution and rinse with fresh SDS electrophoresis buffer Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 27 8 Electrophoresis 8 2 Preparing second dimension gels 28 10 11 Holding one end of the IPG strip with forceps carefully draw it across the exposed top part of the long gel plate until the strip is completely on the glass plate and centered Using a thin plastic spatula ruler or spacer push against the plastic backing of the IPG strip not the gel itself and slide the strip between the two glass plates and down into contact with the surface of the slab gel The strip should just rest on the surface of the gel Avoid trapping air bubbles between strip and the slab gel or piercing the second dimension gel with the strip By convention the acidic or pointed end of the IPG strip is on the left The gel face of the strip should not touch the opposite glass plate See Fig 8 1 Fig 8 1 IPG strip are loaded onto cassette and slid into place Apply molecular weight marker proteins optional Apply the markers to a sample application piece in a volume of 15 20 ul then cover the piece with 50 ul of agarose sealing solution Pick up the application piece with forceps and place next to one end of the IPG strip The markers should contain 0 2 1 0 ug of each component for Coomassie blue staining
28. livered from GE Healthcare except for alterations described in the User Manual e connected to other CE labelled GE Healthcare modules or other products as recommended Compliance with safety standards e 1EC61010 1 e N61010 1 e UL61010 1 e CAN CSA C22 2 No 61010 1 15 3 Specifications 3 2 Gel Caster 16 32 Gel Caster Gel capacity Acrylamide solution volume total Dimensions h x w x d Weight empty 1 0 mm Gel Casting Cassette Cassette dimensions w x h x d Slab gel dimensions w x h x d 1 5 mm Gel Casting Cassette Cassette dimensions w x h x d Slab gel dimensions w x h x d Ettan DALTsix Gradient Maker Volume Weight 6 gels 1 0 mm or 1 5 mm thick 425 ml for 1 0 mm thick gels 550 ml for 1 5 mm thick gels 27x37x8cm 3 6 kg 27 6 x 21 7 x 0 70 cm 25 5 x 20 5 x 0 10 cm 27 6 x 21 7 x 0 75 cm 25 5 x 20 5 x 0 15 cm 1000 ml 0 73 kg Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 4 Preparing the Gel Caster 4 Preparing the Gel Caster Set up the gel caster near a sink in a tray or on a drainboard so that any liquid that overflows spills or drains out of the unit during pouring or disassembly can be easily contained The Ettan DALTsix Gel Caster accommodates up to six 1 mm or 1 5 mm gel cassettes with separator sheets 0 5 mm between them If you are not planning to cast a full set of gels use the blank cassette inserts purchased separately with separator
29. ns will migrate slower above pH 8 8 Protein spots are at both extremes but not in center The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the full range of proteins Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 39 10 Troubleshooting 10 4 Stained gels 40 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Care and maintenance 11 11 Care and maintenance 11 1 Unpacking inventory and set up Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit 11 2 Cleaning WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system For day to day operation of the unit the cleaning procedure outlined in unit operation is sufficient thoroughly rinsing the electrophoresis tank with distilled or deionized water If desired the unit can be periodically cleaned with a dilute solution of a m
30. ong 80 6437 39 Replacement Tilt Leg with Nylon Screw 80 6474 25 Replacement Face Plate 80 6474 44 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 43 12 Customer service information 12 2 Ordering information 44 Product code no Accessories 2 D Electrophoresis Using Immobilized pH Gradients 80 6429 60 Cassette Rack 2 pkg 80 6467 98 Equilibration Tubes 12 pkg 80 6467 79 Stainless Steel Staining Tray Set 80 6468 17 GelSeal 1 4 oz tube 80 6421 43 Roller 80 1106 79 Fluorescent Rulers 2 pkg 80 6223 83 Wonder Wedge 80 6127 88 PlusOne Electrophoresis Chemicals and Reagents Urea 500 g 7 1319 01 Dithiothreitol DTT 1 g 7 1318 01 Bromophenol Blue 10 g 7 1329 01 Glycerol 87 1 7 1325 01 Acrylamide IEF acrylic acid lt 0 002 1 kg 17 1300 02 Acrylamide IEF 40 solution 1 7 1301 01 N N Methylene bisacrylamide 25 g 7 1304 01 N N Methylene bisacrylamide 2 solution 1 7 1306 01 Agarose NA 10g 7 0422 01 N N N N tetramethylethylenediamine TEMED 25 ml 7 1312 01 Ammonium Persulfate APS 25 g 17 1311 01 Tris 500 g 7 1321 01 Glycine 500 g 7 1323 01 Sodium Dodecylsulfate SDS 100 g 7 1313 01 Silver Staining Kit Protein 7 1150 01 Molecular Weight Markers W Range 2512 16949 2 mg vial 1 vial 80 1129 83 W Range 14400 94000 575 mg vial 10 vials 7 0446 01 W Range 53000 212000 175 mg vial 10 vials 7 0615 01 Companion Products EPS 601 Power Supply 6 600 V
31. ong can lead to diffusion and too short can lead to incomplete equilibration Make sure the IPG strip rests on the slab gel surface without damaging it Problems with first dimension see troubleshooting guides for IPGphor or Multiphor units or 2 D Electrophoresis Principles and Methods Protein spots are poorly resolved Allow gel to polymerize completely Begin electrophoresis as soon as the IPG strips are loaded to prevent diffusion of low molecular weight proteins Running too fast Reduce the power current or voltage Reduce the temperature setting Problems with the first dimension Smeared or comet shaped spots Check pH of cathode buffer Should be between 8 3 and 8 8 Make sure that 2x Laemmli buffer is used in the upper cathode chamber Buffer or SDS depleted Protein spots are near the buffer front Buffer depleted Check pH of upper cathode buffer Should be below pH 8 3 8 8 Be sure that 2x Laemmli buffer is being used in the upper cathode chamber Pore size of the gel is too large Increase the T Proteins degraded during sample preparation Add protease inhibitors during sample preparation Check the pH of the 4x gel buffer It should be pH 8 8 Proteins will migrate faster below pH 8 8 Protein spots have not entered the gel when buffer front has reached the bottom of the gel The gel pore size is too small Decrease the T Check the pH of the 4x gel buffer It should be pH 8 8 Protei
32. outlet tubing was connected directly to the caster it should not be removed until polymerization is complete Do not leave the water saturated n butanol on the gels overnight as long term exposure of acrylic to butanol will damage the caster 20 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Casting gradient gels 6 6 Casting gradient gels gt gt 6 1 Preparation for gradient gel casting Successful gradient gel casting requires planning timing and practice A full cast with the Ettan DALTsix Gel Caster requires 420 550 ml of acrylamide stock Polymerization begins as soon as TEMED and APS are added to the acrylamide stock At this point there is no time to adjust the gradient maker or the cassettes and separators in the gel caster To familiarize yourself with the gel caster and gradient maker before casting gels you should set up the unit and carry out a practice gradient cast substituting water for the appropriate volume of light solution and a mixture of glycerol and water for the appropriate volume of heavy solution A pore gradient gel results from mixing varying proportions of two solutions of different acrylamide concentrations and densities a light solution and a heavy solution The heavy gel solution contains glycerol and a higher concentration of acrylamide During the gradient pouring procedure the mixing ratio of high concentration acrylamide solution to low concentration solution gradually changes w
33. per surface of a slab gel and sealed in place with agarose Up to six gel cassettes are inserted into the electrophoresis unit and any unused slots are filled with blank cassette inserts The Upper Buffer Chamber Buffer Seal UBC is pushed down over the cassettes holding the gel cassettes in slots flanked by a double rubber gasket Use EPS 601 Power Supply for power supply If any other power supply is used it must have similar specifications as the EPS 601 Power Supply such as a floating output leakage current detection and be capable of handling up to 600 V 400 mA or 100 W A pump mounted under the lower buffer chamber circulates the buffer pumping it up into the chamber on the right hand side between the cassettes down the left side and over the internal heat exchanger before returning to the pump The pump starts when it is plugged in For temperature control the heat exchanger located in the bottom of the unit must be connected to a MultiTemp Ill or similar circulating water bath that have earthed water pipe and low pressure water circulation 1 1 2 Gel Caster The Ettan DALTsix Gel Caster holds up to six 1 mm or 1 5 mm gel cassettes with separator sheets for casting homogenous or gradient gels When desired fewer gels can be cast at one time by using blank cassette inserts to occupy any unneeded volume The removable faceplate and separator sheets simplify loading and unloading the casting unit The groove in the back of the cas
34. perature indefinitely Gel storage solution 0 375 M Tris Cl pH 8 8 0 1 w v SDS 2 0 final conc amount Tris Cl 1 5 M pH 8 8 0 375 M 500 ml 10 w v SDS 0 1 w v 20 ml Distilled or deionized water to 2 000 ml Store at 4 C 10x SDS electrophoresis buffer 250 mM Tris 1 92 M glycine 1 0 w v SDS approximate pH 8 3 20 I final conc amount Tris MW 121 14 250 mM 605 0 g Glycine MW 75 07 1 92M 2882 0 g SDS MW 288 38 1 0 w v 200 0 g Distilled or deionized water to 201 Do not adjust the pH of this solution SDS equilibration buffer 50 mM Tris Cl pH 8 8 6 M urea 30 v v glycerol 2 w v SDS bromophenol blue 200 ml final conc amount Tris Cl 1 5 M pH 8 8 50 mM 6 7 ml Urea MW 60 06 6M 72 07 9 Glycerol 87 v v MW 92 09 30 v v 69 ml SDS MW 288 38 2 w v 409g Bromophenol blue trace A few grains Distilled or deionized water to 200 ml Store at 20 C This is a stock solution Add DTT or iodoacetamide before using Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Recipes 9 Agarose sealing solution 25 mM Tris 192 mM glycine 0 1 w v SDS bromophenol blue 0 5 w v agarose 25 ml final conc amount 1x SDS electrophoresis buffer see above 25 ml Agarose NA or M 125mg Bromophenol blue trace A few grains Combine all ingredients in a 250 ml Erlenmeyer flask Swirl to disperse On a low setting heat in a microwave oven until the agarose is com
35. pletely melted about 1 min Do not allow the solution to boil over Allow the agarose to cool slightly before using Do not adjust pH Homogeneous gel solutions 600 ml volume required for ml Final 10 12 5 15 Acrylamide stock 200 250 300 1 5 M Tris Cl pH 8 8 150 150 150 Water 237 187 137 10 SDS 6 00 6 00 6 00 10 APS 6 00 6 00 6 00 10 TEMED 1 03 0 83 0 69 450 ml volume required for ml final T 10 12 5 15 Acrylamide stock 150 188 225 1 5 M Tris Cl pH 8 8 113 113 113 Water 178 140 103 10 SDS 45 45 45 10 APS 45 45 45 10 TEMED 0 77 0 62 0 52 Note The amounts of TEMED 0 025 0 09 v v and APS 0 1 w v suggested here are based on our experience You may want to changes these volumes for your laboratory because of differences in temperature and reagent quality Perform a small scale test before using a new composition to check that your solution polymerizes in about 10 min The gel recipes are based on Laemmli U K Nature 227 680 685 1970 Gradient gel solutions For six 1 mm gels Light solution 210 ml volume required for ml final T 8 10 12 14 16 Acrylamide stock 55 68 82 95 109 1 5 M Tris Cl pH 8 8 52 5 52 5 52 5 525 52 5 Water 98 85 71 58 44 10 SDS 2 1 2 1 2 1 2 1 2 1 Glycerol 0 0 0 0 0 10 APS 2 1 2 1 2 1 2 1 2 1 10 TEMED 0 45 0 36 0 30 0 25 0 22 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 35 9 Recipes Heavy solution 210 ml
36. r gel is damaged Bubbles between the gel and the glass plate Use the roller to remove any bubbles or excess liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Interfering substances in the first dimension There is an unfilled gap between the gel and one Contaminants in the sample can cause distortions or swollen regions in the IPG strip following IEF Modify sample preparation to limit these contaminants See 2 D Electrophoresis Using Immobilized pH Gradients Principles and Methods 80 6429 60 When sealing the IPG strip into place ensure that some of dye front on one side of the gel of the spacers the agarose sealing solution flows down any gap that may exist between the gel and spacer Distortion in the 2 D pattern Bubbles between the gel and the glass plate Liquid between the gel and the glass plate Use the roller to remove any bubbles or excess liquid between the gel and the glass plate Ensure that no visible bubbles remain and that the gel adheres firmly to the glass and resists movement Interfering substances in the first dimension Contaminants in the sample can cause distortions or swollen regions in the IPG strip following IEF These
37. required buffers as described below Note Do not overfill the upper and lower chambers by using more buffer than required Electrophoresis buffer for Ettan DALTsix with lab cast Laemmli gels Gel thickness Anodic buffer Cathodic buffer LBC vol I UBC vol I 1 0 mm gels 1xSDS 45 2x SDS 1 2 electrophoresis buffer electrophoresis buffer 1 5 mm gels 1xSDS 43 3x SDS 1 2 electrophoresis buffer electrophoresis buffer For the DALT Pre cast Gels and Buffer Kit only half of the 100x anode lower buffer is used for each run the slightly reduced buffer concentration should not affect the run conditions To prepare the anode buffer dilute half the contents of the 100x anode buffer into 4 5 of water For the cathodic buffer a single bottle of 10x cathode upper buffer should be diluted to a final volume of 1 2 with deionized water and the full amount added to the UBC Please consult the user instructions for the DALT Pre cast Gels for detailed technical information regarding the assembly of gels in their cassettes WARNING Check electrical cables for damage and the electrophoresis unit for damage or leakage before use If damage and or leakage is present do NOT use the equipment There is a risk for high voltage injuries WARNING The high voltage current MUST always be disconnected when the safety lid of the electrophoresis unit is taken off The high voltage current MUST never be switched on unless the safety lid is on the
38. s broken Service calll 10 2 Gel casting Symptom Possible solutions Gel caster leaks Apply a light film of GelSeal compound to the foam gasket on the front plate before clamping and casting Check the foam gasket for cracks or nicks and replace if necessary If the stack is too thick the front plate may not seat firmly against the gasket Remove one or more of the filler sheets until the gasket seals Incomplete gel polymerization Use only recent stocks of the highest quality reagents If the dry ammonium persulfate does not crackle when water is added to it replace with fresh reagent Use fresh ammonium persulfate Solutions of extreme pH may not polymerize Degas the monomer solution Oxygen inhibits polymerization Increase both ammonium persulfate and TEMED by 30 50 Adjust the gel solution temperature to a minimum of 20 C Gel is too soft too brittle or white Check and adjust crosslinker concentration Standard SDS gels should have a crosslinker concentration of 2 6 C g bis x 100 g monomer g bis Make up fresh acrylamide stock solution Gel exhibits swirls Dye front curves up smiles If gel polymerized too fast lt 10 min reduce the concentration of catalyst APS and TEMED by 25 If gel polymerized too slowly gt 50 min increase the concentration of catalyst APS and TEMED by 50 Make up fresh acrylamide stock solution Check circulation of the buffer Pre chill the
39. separate the plates on the side opposite the hinge 6 For lab cast gels run an edge of the Wonder Wedge down each side of the cassette along the spacer and carefully lift the gel out of the cassette For pre cast gels lift the gel out by grasping the GelBond backing 7 Tiltthe entire electrophoresis unit on the side of the unit do NOT tilt to front or back to empty the buffer from the LBC into a sink or a waste container See Section 8 1 for instructions of how to place the unit for safe use N WARNING Ettan DALTsix electrophoresis unit is heavy especially when filled with buffer Handle the unit with care to avoid personal damage 8 Rinse all the components with distilled or deionized water Flush the pump with distilled or deionized water by filling the unit and turning the pump on Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 31 8 Electrophoresis 8 5 Unloading the gels and cleaning the unit 32 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 9 Recipes Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Recipes 9 WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Acrylamide stock 30 8 T WARNING Acrylamide is a neurotoxin Never pipette by mouth and always wear protective gloves when working with acrylamide solutions IP
40. sheets between them to occupy the extra space Gel labels for easy indexing of gels and samples can be placed in the cassettes at any time during the assembly of the caster Check that the caster is level Remove the faceplate and lay the caster on its back If casting gels by the displacement method remove the triangular wedge in the V shaped base Fill the gel caster starting with a separator sheet against the back wall The separator sheets make it easier to remove the cassettes from the unit after polymerization Fill the caster by alternating cassettes with separator sheets The rubber hinge should be on the left side of the caster with the matching ends of the cassette down End with a separator sheet then use the thicker filler sheets 1 0 mm to bring the level of the stack of cassettes and spacers even with the edge of the caster Remove the gray foam seal from the groove in the faceplate and lubricate it with a light coating of GelSeal compound to help ensure a liquid tight seal Place the gasket back in the groove on the faceplate Avoid stretching the gasket by seating it from the ends first working toward the middle Turn two black knobbed screws into the two threaded holes across the bottom until they are well engaged two to three full turns Carefully place the faceplate onto the caster with the bottom slots resting on their respective screws Clamp both sides of the faceplate with six spring clips and tighten the screws Be sure
41. spacers and gaskets must be clean dry and free of grease Check the UBC It should be free of nicks or tears Check the pH of the buffer If the pH is wrong make fresh buffer do not back titrate Check recipes gel concentrations and buffer dilutions For example do not use Tris HCl in place of Tris base for the electrophoresis buffer Discard older acrylamide solutions and use only reagents of highest quality Only use freshly deionized urea of highest quality Adjust power current or voltage Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 37 10 Troubleshooting 10 3 Pre cast gels Symptom 10 3 Pre cast gels Possible causes Possible solutions Second dimension electrophoresis proceeds slowly with high current One of the slots in the upper buffer chamber is open The UBC is damaged Anodic buffer has mixed with cathodic buffer from overfilling of either the cathodic or the anodic reservoir All 6 slots in the UBC should be occupied by either a gel cassette or a blank cassette Carefully fill both buffer chambers to the same level Ensure that the level of the anode lower buffer does not come above level of the buffer in the UBC when the electrophoresis unit is fully loaded Dye front is irregular Pronounced downward curving of the The top surface of the gel has been damaged during application of the IPG strip Take care during application of the IPG strip that neithe
42. ter provides the channel through which the gel solution is poured into the caster unless gradients are being the cast by displacement through the lower tubing fitting Ettan DALTsix System components e Six slot vertical slab electrophoresis unit e Gel caster e Gel casting cassettes e Gradient maker Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 7 1 System function and description 1 1 Electrophoresis System function and description GES a Lid with leads a Safety interlock Upper buffer chamber Anode assembly cassette carrier Lower buffer chamber Ceramic heat exchager Buffer circulation pump Fig 1 1 Exploded view of Ettan DALTsix Electrophoresis Unit Fig 1 2 Ettan DALTsix Gel Caster for casting 1 0 and 1 5 mm large format gels 1 1 3 Gel Casting Cassettes The Ettan DALTsix Electrophoresis System uses the same cassettes as the Ettan DALTtwelve Electrophoresis System see Fig 1 3 The gel casting cassettes are pre assembled Two glass plates are joined along one edge by a hinge strip of silicone rubber and the vinyl side spacers 1 0 mm or 1 5 mm thick are glued in place To complete assembly close the two plates like a book and press the plates together over the length of the spacer Gels are removed by opening the book after the run and carefully lifting out the gel slab Care must be taken to ensure that the gel does not adhere to the spacers and tear during removal The cassette is clean
43. the cassettes from the unit by pulling forward on the separator sheets 4 Rinse the top surface of each gel with distilled water to remove the n butanol and any unpolymerized acrylamide Remove the separator sheet if still attached and rinse the glass cassettes with water to remove any acrylamide adhering to the glass plates 5 Examine the gels for polymerization defects and discard any unsatisfactory gels 6 Store the acceptable gels in an airtight container at 4 C with a small amount of gel storage solution to keep the gels from drying out 7 Rinse the gel caster and all tubing with mild detergent then rinse thoroughly with deionized water Clean the separator and spacer sheets with a mild detergent and rinse with deionized water Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 25 7 Unloading the gel caster 26 Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD Electrophoresis 8 8 Electrophoresis 8 1 Placement of the electrophoresis unit The Ettan DALTsix electrophoresis unit should be placed close to a sink for easy rinsing and draining The tubing leading to and from the heat exchanger should be connected to a circulating water bath such as the MultiTemp III the heat exchanger should not be connected to a water tap or any other coolant supply that lacks pressure regulation An EPS 601 or equivalent power supply should be placed conveniently near the electrophoresis unit in a dry place wi
44. thout risk for liquid exposure 8 2 Preparing second dimension gels WARNING When using hazardous chemicals take all suitable protective measures such as wearing N protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system 8 2 1 Eduilibration and loading For a detailed description of the components of the SDS eguilibration solution and the eguilibration process please consult 2 D Electrophoresis Using Immobilized pH Gradients 80 6429 60 1 Prepare SDS equilibration buffer Just prior to use add DTT to the buffer to a concentration of 1 w v 2 Place the IPG strips in individual tubes with the support film toward the wall 3 Add 10 15 ml of the DTT containing solution to each tube Typically two 18 cm strips can be equilibrated with 10 ml of buffer or two 24 cm strips can be equilibrated with 15 ml of buffer 4 Incubate the strips for 10 15 min with gentle agitation Do not over equilibrate as proteins can diffuse out of the strip during this step 5 Second equilibration Prepare SDS equilibration buffer with iodoacetamide added to 2 5 w v and repeat steps 3 and 4 6 Before equilibration is completed prepare the gel cassettes for loading by rinsing the top of the gel with deionized water and draining Before loading the IPG strips make sure that the gel surface and plates are dry 7 Lay the prepared gel flat on a c
45. water saturated n butanol You need 1 0 ml of overlay for each 1 mm cassette and 1 5 ml for overlay for each 1 5 mm cassette Mixing 1 mm and 1 5 mm cassettes in one casting is not advised Note If using glass plates in stead of cassettes ensure that the plates are properly aligned If casting using the displacement method make up 200 ml of displacing solution 0 375 M Tris Cl pH 8 8 30 v v glycerol bromophenol blue For a full 6 gel 1 5 mm cassette set make up 600 ml of acrylamide gel stock solution without ammonium persulfate APS or N N N N tetramethylethylenediamine TEMED For a full 6 gel 1 mm cassette set make up 450 ml of acrylamide gel stock solution as above This amount of gel solution will provide you with sufficient volume to cast gels using either the filling channel or a peristaltic pump Note For best result the acrylamide gel stock solution should be 8 C when APS and TEMED is added Assemble the gel caster as described in the proceeding section the caster should be placed on a level bench or on a leveling table so that gel tops are level Add the appropriate volumes of APS and TEMED only when ready to pour the gels not before Once these two components are added polymerization begins and the gel solution should be completely poured within 10 min Pour the gel solution into the filling channel If displacement casting is being used the flow rate on the peristaltic pump should be increased slowly to the d
46. wn In a situation where there is a risk of injury turn off the current by e disconnecting the external power supply cable from its power outlet e disconnecting the Ettan DALTsix from its power outlet Power failure routine In the event of a power failure the run is interrupted in an undefined state Ettan DALTSIX Electrophoresis System User Manual 80 6492 49 Edition AD 13 2 Safety information 2 3 Emergency procedures 14 Ettan DALTSIX Electrophoresis System User Manual 80 6492 49 Edition AD Specifications 3 3 Specifications 3 1 Electrophoresis Unit Maximum voltage high voltage connection Maximum current high voltage connection Maximum power high voltage connection Gel capacity Electrophoresis buffer volume Dimensions h x w x d Weight empty Weight filled Maximum temperature Environmental operating conditions Installation category Pollution degree 100 V model 115 V model 230 V model Compliance with standards Ettan DALTsix Electrophoresis System User Manual 80 6492 49 Edition AD 600 V 400 MA 100 W 6 gels 5 51 40 3 x 54 2 x 16 0 cm 11 1 kg 16 8 kg 40 C Indoor use 4 40 C Humidity up to 90 Altitude to 2000 m II 2 100 V 50 60 Hz 8 W 80649477 110 120 V 60 Hz 8 W 80648508 220 240 V 50 60 Hz 7 W 80648527 The declaration of conformity is valid for the instrument only if it is e used in laboratory locations e used in the same state as it was de
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