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Ettan DALTtwelve System - GE Healthcare Life Sciences

1. WARNING Always disconnect the power supply cord from the control unit before disconnecting the cables and cords between the separation unit and the control unit WARNING Take care when handling glass sheets to avoid injury caused by sharp edges e Connect the instrument to a properly grounded electrical outlet e The safety lid must be firmly in place and the pump valve set to circulate before power can be applied e Stop the run before opening the safety lid e Rinse and flush the tank and pumping system with distilled or deionized water before and after use e Always disconnect the power cord before maintenance or repair e Do not run the circulation pump if the separation unit is empty e Do not operate with buffer temperature above 50 C All plastic parts are rated for 50 C continuous duty e Turn the pump on during electrophoresis to minimize heating Overheating will cause irreparable damage to the unit e For runs near the lower temperature limits 10 to 15 C it may be necessary to operate the unit in a cold room especially in laboratories where the ambient temperature is above 25 C e Do not autoclave or boil this unit or any of its parts e Make sure there are no loose parts or debris inside the equipment e Use care when lifting and moving the separation unit It is best to move the unit when empty e When filled with glass plates and gel solutions the casting unit is very heavy Use caution when moving o
2. amount TrisCl 1 5 M pH 8 8 25 ml Glycerol 50 ml Bromophenol blue 2 mg Distilled or deionized water 25ml Should be made fresh stored solution may support microbial growth Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 39 10 Recipes 40 Water saturated butanol amount nor t butanol 50 ml Distilled or deionized water 5 ml Combine in a bottle and shake Use the top phase to overlay gels Store at room temperature indefinitely Gel storage solution 0 375 M TrisCl pH 8 8 0 1 w v SDS 2 I final conc amount TrisCl 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 10 SDS electrophoresis buffer 250 mM Tris 1 92 M glycine 1 w v SDS approximate pH 8 3 10 I final conc amount Tris MW 121 14 250 mM 303 g Glycine MW 75 07 1 92 M 1440 9 SDS MW 288 38 1 w v 100g Distilled or deionized water to 101 Do not adjust the pH of this solution SDS equilibration buffer 50 mM TrisCl pH 8 8 6 M urea 30 v v glycerol 2 w v SDS bromophenol blue 200 ml final conc amount TrisCl 1 5 M pH 8 8 50 mM 6 7 ml Urea MW 60 06 6M 72 079 Glycerol 87 v v MW 92 09 30 v v 69 ml SDS MW 288 38 2 w v 40g 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 Sealing solution 0 25 m
3. edge providing a surface for sliding the IPG strip into position lt Fig 8 2 Pre cast gel cassette The buffer in the gel is part of a unique buffer system that gives longer shelf life and shorter run times than the conventional Laemmli Tris glycine system while retaining the protein capacity and robustness of that system Separations performed using Ettan DALT Gel 12 5 are similar to those seen with a 12 5 Laemmli gel The associated buffer kit contains all the reagents necessary for a single run of up to 12 Ettan DALT Gel 12 5 gels in the Ettan DALTtwelve Electrophoresis Unit 8 3 Preparing the Ettan DALTtwelve Electrophoresis Unit WARNING Never overfill the vessel or spill liquid on the units due to the risk of electrical shock Always make sure the equipment is disconnected from supply voltage when filling the tanks Ensure that the valve on the Ettan DALT Electrophoresis Unit is set to circulate Fill the tank to the 7 5 I fill line with distilled or deionized water Add the entire contents 75 ml of the bottle of 100 anode solution from the buffer kit Avoid pouring the 100x anode solution on the buffer seal tubing by spreading it slightly with one hand while pouring the solution see Fig 8 3 Turn on the pump to mix Set the unit to the desired temperature 25 C is recommended 32 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Electrophoresis on pre cast gels 8 Fig 8 3 Addition of 10
4. If the problem persists Service calll Make sure that the lid is completely closed Make sure that the pump valve is turned completely to circulate Service call Apply alight film of GelSeal compound to the foam gasket before 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 Use only recent stocks of the highest quality reagents If the dry ammonium persulphate does not crackle when water is added to it replace with fresh reagent Use fresh ammonium persulphate Solutions of extreme pH may not polymerize Degas the monomer solution Oxygen inhibits polymerization Increase both ammonium persulphate and TEMED by 30 to 50 Adjust the gel solution temperature to a minimum of 20 C 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 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 buffer Decrease power voltage or current Ettan DALTtwelve System User Ma
5. 13 Customer service information 13 1 Technical service and repair GE Healthcare 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 GE Healthcare Support IMPORTANT Request a copy of the GE Healthcare Health and Safety Declaration form before returning the item No items can be accepted for servicing or return unless this form is properly completed Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 49 13 Customer service information 50 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Ordering information 14 14 Ordering information Product Code number Ettan DALTtwelve Electrophoresis Unit and Power Supply Control Unit 110 to 120 V 80 6466 46 220 to 240 V 80 6466 27 Replacement Lid 80 6473 11 Replacement Buffer Seal 80 6473 30 Buffer Seal Removal Tool 2 pkg 80 6474 63 Replacement Baffle Plate 80 6473 49 Replacement Flow Guide 80 6473 68 Replacement Anode Plate 80 6473 87 Ettan DALT Cassettes Pre cast Gel Cassette 80 6466 65 Gel Casting Cassette 1 0 mm 80 6466 84 Gel Casting Cassette 1 5 mm 80 6488 69 Blank Cassette Insert 80 6466 03 Cassette Removal Tool 2 pkg 80 6474 82 Ettan DALT Pre cast Gels Pre cast Gel 12 5 6 pkg 17 6002 36 Buffer Kit one run of 12 gels 17 6002 50 Ettan DALTtwelve Gel Caster Complete with Separator Sheets 16 pcs and Filler Sheets
6. Methylene bisacrylamide 2 solution 1 17 1306 01 Agarose IEF 10 g 17 0468 01 N N N N tetramethylethylenediamine TEMED 25 ml 17 1312 01 Ammonium Persulphate APS 25 g 7 1311 01 Tris 500 g 17 1321 01 Glycine 500 g 17 1323 01 Sodium Dodecylsulphate SDS 100 g 17 1313 0 Silver Staining Kit Protein 17 1150 01 Molecular Weight Markers MW Range 2512 to 16949 2 mg vial 1 vial 80 1129 83 MW Range 14400 to 94000 575 mg vial 10 vials 17 0446 01 MW Range 53000 to 212000 175 mg vial 10 vials 17 0615 01 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC For local office contact information visit GE imagination at work and GE monogram are trademarks of General Electric Company www gelifesciences com contact Ettan IPGphor and Multiphor are trademarks of GE Healthcare companies All third party trademarks are the property of their respective owners GE Healthcare Bio Sciences AB Bjorkgata n 30 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 are available on request Contact your local GE 751 84 U ppsala Healthcare representative for the most current information Sweden 2006 2009 General Electric Company All rights reserved First published 2006 www gelifesciences com GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buc
7. all the cables between the control unit and separation unit are properly connected The high voltage connector on the control unit has a voltage of 600 volts DC High voltage indicator High voltage connector 600 VDC Signal and power cable connector Fig 1 2 Front of Power Supply Control Unit Air vents Mains switch Power supply inlet Fig 1 3 Rear of Power Supply Control Unit 6 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Ettan DALTtwelve System 1 1 1 3 Electrical connections The power supply control unit provides the separator with 600 VDC from the upper connector The lower connector provides power and control to the separator See Fig 1 2 On the rear side of the power supply control unit there is a power inlet to which the power supply cord is connected and an RS232 port See Fig 1 3 1 1 4 Gel Caster The Ettan DALTtwelve Gel Caster holds up to fourteen 1 0 mm or thirteen 1 5 mm gel cassettes with separator sheets for casting homogenous or gradient gels see Fig 1 4 Fewer gels can be cast at one time by using blank cassette inserts to occupy unneeded volume The removable faceplate and separator sheets simplify loading and unloading of cassettes from the unit The unit also has removable tilt support legs that allow the caster to be tipped back for more convenient loading of gel casting cassettes Fig 1 4 Gel Caster 1 1 5 Gel Casting Cassettes The gel casting cassettes
8. 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 cleaned 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 and a 1 5 mm thick gel has a volume of approximately 78 ml Fig 1 5 Gel Casting Cassette Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 7 1 Ettan DALTtwelve System gt gt gt gt 1 2 Important information IMPORTANT Ettan DALTtwelve System is intended for laboratory use only not for clinical or in vitro use or for diagnostic purposes 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 In case of an emergency situation the mains plug must always be accessible and easy to disconnect
9. concentrations and buffer dilutions For example do not use TrisCl in place of Tris base for the electrophoresis buffer Discard older acrylamide solutions and use only reagents of the highest quality Only use freshly deionized urea of the highest quality Adjust power current or voltage Ensure that all 12 slots in the buffer seal are occupied Do not pour more than the suggested volume 7 5 into the lower reservoir Ensure that the level of the anode lower buffer does not come above buffer seal when the separation unit is fully loaded Remove any excess anode buffer from the upper reservoir Ensure that the level of cathode buffer is not above the air vents in the upper reservoir Take care during application of the IPG strip that neither gel is damaged 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 Contaminants in the sample can cause dimension There is an unfilled gap between the gel and one of the spacers 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 the agarose sealing solution flows down any ga
10. in the proper position see Fig 8 6 Fig 8 6 Initial placement of the gel Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 33 8 Electrophoresis on pre cast gels 8 34 Move the gel and allow it to fall against the glass so that the edges of the gel are against not overlapping the side spacers and so that the bottom anodic edge of the gel is flush within 1 mm of the bottom anodic edge of the glass plate The protruding side support film margins but not the gel should rest on top of the side spacers see Fig 8 7 Fig 8 7 Final placement of the gel Use the roller to press out any bubbles and excess buffer from between the gel and the glass Press firmly against the plastic support film with the roller and roll over the entire gel see Fig 8 8 After rolling the gel should adhere firmly to the glass and resist further movement Fig 8 8 Removing air bubbles Close the cassette and snap the plastic frame to the glass plate see Figs 8 9 and 8 10 Fig 8 9 Closing the cassette l I Fig 8 10 Snapping the cassette Repeat the procedure for each second dimension gel to be run Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Electrophoresis on pre cast gels 8 8 4 Applying the IPG strip 1 Leave the loaded gel cassette lying flat on the bench top with the glass plate down and the plastic frame up 2 Rinse the IPG strip Pour some of the diluted 1 cathodic b
11. than 1 cm below the top of the cassettes you can add up to 25 ml more displacing solution to the balance chamber The gradient is now in hydrostatic equilibrium in the unit ready to polymerize see Fig 4 5 water q id displacing solution b c Fig 4 5 Finished cast Immediately pipette gel overlay solution water saturated n butanol onto each gel Apply 1 ml of overlay for each 1 0 mm cassette and 1 5 ml for each 1 5 mm cassette Allow gels to polymerize at least 2 h Quickly reopen clamp c and restart the pump to empty the gradient maker of any excess polymerizing acrylamide Collect the excess in a waste container Dispose of unpolymerized acrylamide according to applicable safety guidelines Rinse the gradient maker well to prevent polymerization within the tubing lines Place the feed tube in a larger waste vessel or a sink drain Pour a liter of water into each chamber of the gradient maker and open all clamps Start the pump to flush the system Flush another 2 of water through the gradient maker and tubing Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Polymerization 5 5 Polymerization Allow nongradient gels to polymerize for at least 1 h allow gradient gels 2 h to polymerize Gradient gel polymerization should proceed from the top down You can observe this through the front and sides of the caster The level of the dense displacing solution falls farther
12. the gels begin to solidify at the top Work rapidly and carefully Pour the light solution into the right side of the gradient maker the chamber that is narrower at the bottom Light in right Fill the tubing between the light and heavy chambers with light solution Carefully open the clamp on the light chamber exit tube a and then very slowly open the heavy chamber exit tube clamp b Allow light solution to fill the tube coming from the light chamber all the way to the Y connector and back up to the point at which the heavy tube enters the heavy chamber Fill the entire tube with light solution no bubbles but do not allow light solution into the heavy chamber itself 4 3 Priming the gradient marker with light solution light solution I I Fig 4 3 Priming the gradient marker with light solution Close both clamps again All three clamps should now be closed Add the heavy solution to the heavy left chamber the chamber that is wider at the bottom until the liquid level reaches a point about 2 cm below the level of light solution in the adjacent chamber Load the balance chamber on the side of the caster with 75 ml of displacing solution see Fig 4 4 The feed tube should be seated securely in the grommet seal to prevent leakage of displacing solution into the caster light solution heavy displacing so
13. the parameters 7 1 Controller interface in programming mode Fig 7 1 Controller interface in programming mode Sample program Twelve gels electrophoresed at 180 W constant power and 25 C with a 5 W gel entry phase The second phase is extended longer than required to ensure that the dye front runs off the gel Step 1 Const Watt 60 W Time 1 00 45 hrs Pump Auto Temperature 25 C Step 2 Const Watt 180 W Time 2 04 00 hrs Pump Auto Temperature 25 C Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 25 7 Electrophoresis 26 Titer Run Type determines the method of power regulation The options for run type are constant power constant current constant voltage and crossover mode In crossover mode the voltage and current limits for the run are set manually instead of using the instrument defaults of 600 V and 1 000 mA As the run progresses the power supply operates in the mode that is limiting Timer controls the duration of the electrophoresis run The options are continuous run timed step timed and hold and volt hours Up to three timed steps of up to 100 h each can be programmed using the timed step mode With timed step mode all the steps must have the same run type Pump controls buffer circulation through the separation unit The options for the pump are on off and auto Auto mode activates the pump only when power is applied Temperature controls the cooling or heating of the buffer in t
14. the separation unit through the buffer seal slots flanked by rubber gaskets Any unused slots are filled with the blank cassette inserts The buffer seal is an effective current and liquid barrier The platinum wire cathode is attached to the underside of the lid and the platinum wire anode is in the lower tank Safety interlocks prevent the application of power to the separation unit unless the lid is closed properly and the pump valve is in the circulate position The lid is easily removed for cleaning by sliding it off its hinges A pump located in the lower chassis of the unit circulates the buffer pumping it up into the main chamber on the left side between the cassettes down the right side and over the internal heat exchanger before returning to the pump The default setting for the pump is auto the pump will come on only when a run is started Turning the lever at the back of the unit from circulate to drain drains the tank The temperature is controlled by Peltier modules attached to the heat exchanger beneath the tank Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 5 1 Ettan DALTtwelve System 1 1 2 Power Supply Control Unit The Ettan DALTtwelve System is controlled from the Power Supply Control Unit see Fig 1 2 The unit supplies a maximum power output of 200 W with a maximum of 600 V or 1 A The temperature control range is 10 C to 50 C WARNING The equipment may not be plugged into the mains power outlet unless
15. 0 anode to the separation unit solution 8 3 1 Inserting the Ettan DALT Gel 12 5 into the pre cast gel cassette 1 Open the gel package Cut around the gel on two sides at about 1 cm from the edge to avoid cutting the gel or the support film Remove the gel from the package The gel is cast onto a plastic support film and does not cover the film entirely The gel is covered with a protective plastic sheet Markings on the protective sheet indicate the orientation of the gel and the direction of electrophoresis The bottom or anodic edge of the gel is flush with the edge of the support film The support film protrudes approximately 15 mm beyond the top or cathodic edge of the gel and approximately 5 mm at either side 2 Open an Ettan DALT Pre cast Gel Cassette and place it on the bench top with the hinge down see Fig 8 4 Fig 8 4 Pre cast gel cassette open 3 Pipette 2 to 4 ml of gel buffer onto the centre of the glass plate see Fig 8 5 Fig 8 5 Gel buffer 4 Remove the protective plastic sheet from the gel Handling the gel only by the side support film margins hold it gel side down over the glass plate Ensure that it is oriented with the cathodic edge of the gel toward the cathodic edge of the cassette Flex the centre of the gel downward slightly and lower it toward the glass plate so that the middle of the gel contacts the puddle of gel buffer The gel buffer will lubricate the gel and allow it to be moved and placed
16. 10 l min 200 W 600 V DC 1 00A 10 C to 50 C 6 35 kg 33 x 26x 19 cm Indoor use 4 C to 40 C Humidity up to 90 Altitude to 2000 m ll 2 110 to 120 VAC 50 to 60 Hz 220 to 240 VAC 50 to 60 Hz 650 W EN61010 1 IEC61010 1 UL61010 1 CSA C22 2 1010 1 CE Certified EN61326 Gel capacity Acrylamide solution volume total Dimensions h x w x d Weight 1 0 mm Gel Casting Cassette 14 for 1 0 mm thick gel 13 for 1 5 mm thick gel 950 ml for 1 0 mm thick gel 1200 ml for 1 5 mm thick gel 26x 21 x 36 cm empty 5 kg loaded 19 kg Cassette dimensions h x w x d Slab gel dimensions h x w x d 1 5 mm Gel Casting Cassette 21 7 x 27 6 x 0 70 cm 20 5 x 25 5 x 0 10 cm Cassette dimensions h x w x d Slab gel dimensions h x w x d Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 21 7 x 27 6 x 0 75 cm 20 5 x 25 5 x 0 15 cm 1 Ettan DALTtwelve System 10 Gradient Maker Dimensions h x w x d Maximum gradient volume Weight Peristaltic Pump for Gradient Maker 54x 19x 18 cm 21 5 4 kg Supply voltage Max Power Consumption Max input current 115 V Max input current 230 V Weight Dimensions h x d x w Environmental operating conditions Installation category Pollution degree Product certifications Complies with EMC standard IP class 100 to 118 VAC 50 to 60 Hz 220 to 237 VAC 50 to 60 Hz 37W 1 5 A SC output 0 9 A SC output 4 1 kg 13 5 x 18
17. 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 Triangular 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 long 80 6437 39 Replacement Tilt Leg with Nylon Screw 80 6474 25 Replacement Faceplate 80 6474 44 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 51 14 Ordering information 52 Product Code number DALT Gradient Maker with Peristaltic Pump 115 V 80 6067 65 230 V 80 6067 84 Gradient Maker Gasket Divider 80 6068 41 Gasket Adjuster Rod 80 6068 60 Knobs 4 pkg 80 6437 58 Plastic Feed Tubing 1 8 m 80 6068 03 Bow Tie Mixer Kit 80 6068 22 Acrylic Feed Tube 80 6437 20 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 Plate Separation Tool 80 6127 88 PlusOne Electrophoresis Chemicals and Reagents Urea 500 g 17 1319 01 Dithiothreitol DTT 1 g 17 1318 01 Bromophenol Blue 10 g 17 1329 01 Glycerol 87 1 17 1325 01 Acrylamide IEF acrylic acid lt 0 002 1 kg 7 1300 02 Acrylamide IEF 40 solution 1 17 1301 01 N N Methylene bisacrylamide 25 g 7 1304 01 N N
18. APS 45 45 45 45 45 10 TEMED 0 96 0 77 0 64 0 55 0 48 Heavy solution 450 ml volume required for ml final T 12 14 16 18 20 Acrylamide stock 180 210 240 270 300 1 5 MTrisCl pH 8 8 113 113 113 113 113 Water 120 90 60 30 0 10 SDS 45 45 45 45 45 Glycerol 31 31 31 31 31 10 APS 2 3 2 3 2 3 2 3 2 3 10 TEMED 0 21 0 18 0 16 0 14 0 13 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 41 10 Recipes 42 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 11 Troubleshooting Troubleshooting 11 Symptom Possible causes Possible solutions Electrical and mechanical No current at start of run Insufficient volume of buffer in Ensure that the unit contains enough buffer to upper reservoir contact the upper electrode Buffer not circulating Pump is not primed Turn circulation valve to drain to fill with buffer Pump is off or set to auto Pump is broken Display on the control unit blank Unit is not turned on Unit is not plugged in Display is broken Control unit display malfunctions Open circuit warning Safety interlocks not engaged Gel casting Gel caster leaks Incomplete gel polymerization Gel is too soft too brittle or white Gel exhibits swirls Dye front curves up smiles then back to circulate On control unit set pump to on Service call Turn unit on at power switch in back Plug in unit Service call Turn the AC power switch off for a few seconds then on again
19. Care and maintenance DOV ACICOIAG Aa sa cemtca tse taagases tung eects NN SNR AA tenancy 47 12 2 Replacingiinternal COmPONeNtS scecden ccteatssiaciiondda E LE A AREER AAA R 47 13 Customer service information 13 1 Te hnical service and re PO 222 sasse nissan sned Ne River 49 14 Ordering information Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Ettan DALTtwelve System 1 1 Ettan DALTtwelve System In 2 D electrophoresis proteins are separated according to isoelectric point by isoelectric focusing most reliably on immobilized pH gradient IPG strips using the IPGphor IEF System The second dimension electrophoresis separates the proteins on the basis of their molecular mass using sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS PAGE The Ettan DALTtwelve System is designed to handle multiple large second dimension gels in a simple efficient and reproducible manner 1 1 Ettan DALTtwelve System components e 12 slot vertical slab separation unit e 200 W Power Supply Control Unit e gel caster e gel casting cassettes e gradient maker with peristaltic pump 1 1 1 Separation Unit Fig 1 1 Ettan DALTtwelve Separation Unit The Ettan DALTtwelve Separation Unit accommodates up to twelve 25 5 x 20 5 cm slab gels for separation under identical conditions A sample focused in an IPG strip is placed on the cathodic surface of a slab gel and sealed with agarose Up to 12 gels are inserted into
20. DS equilibration buffer with iodoacetamide added to 2 5 w v and equilibrate the strips with this solution as in steps 3 to 4 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 P p if Fig 7 3 IPG strip being positioned on cassette Fig 7 4 IPG strip begin seated against slab gel Lay the prepared gel flat on a clean surface Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 27 7 Electrophoresis 28 10 11 Using forceps remove the equilibrated IPG strip from the equilibration solution and rinse with fresh SDS electrophoresis buffer Holding one end of the IPG strip with forceps carefully draw it across the long gel plate until the strip is completely on the glass plate and centered see Fig 7 3 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 7 4 IPG strip begin seated against slab gel The strip should just rest on the surface of the gel Avoid trapping air bubbles between strip and the slab gel and avoid 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 t
21. GE Healthcare Ettan DALTtwelve System Second dimension gel electrophoresis User Manual Important user information All users must read this entire manual to fully understand the safe use of Ettan DALTtwelve system Important Ettan DALTtwelve system is intended for research use only and should not be used in any clinical or in vitro procedures for diagnostic purposes Safety notices This manual contains warnings and cautions concerning the safe use of the product See definitions below WARNING The WARNING sign highlights instructions that must be followed to avoid personal injury Do not proceed until all stated conditions are clearly understood and met CAUTION WARNING The system must be connected to a grounded power supply socket WARNING If liquid is spilled on the equipment the power supply shall be disconnected immediately WARNING The system must not be opened by the user It contains high voltage circuits that can give a lethal electrical shock 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 Certification This product meets all requirements of applicable CE directives A copy of the corresponding Declaration of Co
22. H 8 8 Proteins will migrate more slowly above pH 8 8 Protein spots are at both extremes but The molecular weight range of the sample not in center requires an acrylamide concentration gradient to resolve the full range of proteins Vertical protein streaks IPG strip not properly placed on Make sure IPG strip uniformly contacts the gel gel surface surface along its entire length Avoid gouging the surface of the separating gel Spots skewed or distorted Gels run too fast uneven Run at a lower power setting Use a two step migration 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 Use reagents of the highest purity preferably electrophoresis grade Use deionized double distilled water Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 45 11 Troubleshooting 46 Symptom Possible causes Possible solutions Distortion in the 2 D pattern Vertical gap in the 2 D pattern Vertical streaking Spots are vertically doubled or twinned Poor representation of higher molecular weight proteins Bubbles between the gel and the glass plate Liquid between the gel and the glass plate Use the roller t
23. M Tris 192 mM glycine 0 1 w v SDS bromophenol blue 0 5 w v agarose 25 ml final conc amount SDS electrophoresis buffer see above 25 ml Agarose IEF NA or M 125 mg 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 completely melted about 1 min Do not allow the solution to boil over Allow the agarose to cool slightly before using Do not adjust pH Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 10 1 Homogeneous gel solutions Recipes 10 900 ml volume required for ml final T 10 12 5 15 Acrylamide stock 300 375 450 1 5 MTrisCl pH 8 8 225 225 225 Water 356 281 206 10 SDS 9 9 9 10 APS 9 9 9 10 TEMED 1 54 1 24 1 03 Note The amounts of TEMED 0 025 to 0 09 v v and APS 0 1 w v suggested here are based on our experience You may want to change 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 10 2 Gradient gel solutions Light solution 450 ml volume required for ml final T 8 10 12 14 16 Acrylamide stock 120 150 180 210 240 1 5 MTrisCl pH 8 8 113 113 113 113 113 Water 207 178 148 118 88 10 SDS 45 45 45 45 45 10
24. as the gels contract upon polymerization Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 21 5 Polymerization 22 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Unloading the gel caster 6 6 Unloading the gel caster 1 Make sure the caster is either near a sink or on a tray so that any liquid leaking out can be contained WARNING When handling the equipment manually the power shall be switched off If there is a risk that liquid may be splashed or spilled on the equipment then the power supply cord must be disconnected while handling the equipment 2 Remove the front of the gel caster by loosening and removing the black knobbed screws 3 Carefully unload 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 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 good 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 DALTtwelve Syste
25. bromophenol blue The buffer system in this gel and buffer kit is covered by United States Patent 6 090 252 and others Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 31 8 Electrophoresis on pre cast gels 8 2 Description of the system The Ettan DALT Gel 12 5 gel is a pre cast polyacrylamide gel for the second dimension of large format 2 D electrophoresis It is bound to a plastic support film which provides ease of handling and dimensional stability The gel is intended for use in the Ettan DALTtwelve System The gel is inserted into a cassette that allows it to be run in a vertical mode with liquid buffers The gel is used with the Ettan DALT Buffer Kit that includes concentrated buffers for running the gels gel buffer for seating the gel in the pre cast gel cassette and sealing solution for attaching the IPG strip to the top of the slab gel rn es _ Pet Fig 8 1 Ettan DALTtwelve Gel 12 5 and buffer kit components The pre cast gel cassette holds the Ettan DALT gel vertically in the Ettan DALT Electrophoresis Unit It consists of a glass plate with spacers glued to the vertical edges and connected to a rigid plastic frame along one edge by a flexible hinge The gel is placed against the glass plate between the spacers When the cassette is closed and snapped together the frame presses the gel evenly against the glass plate The glass plate extends 5 mm higher than the plastic frame at the cathodic
26. d almost touching the left wall at the height determined by the volume of water 4 To make a funnel for introducing heavy solution into the left side bend the remaining top of the divider to the right 5 Tighten the faceplate screws and close the pinch clamps on the outflow tubing at the bottom of the gradient maker before adding water or gel solutions Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 15 4 Casting gradient gels 16 Using water in place of gel solution put half of the required volume in the right chamber and the other half in the left chamber Adjust the angle of the gradient divider so that the level of liquid in the heavy or left chamber is about 2 cm below the level of liquid in the light chamber You can use tape or wax pencil on the outside of the gradient maker to mark the angle of the gradient divider Open the pinch clamps to remove the water or pour the water out of the top of the gradient maker Repeat this procedure whenever the required volume of acrylamide solution changes as a result of changing the number of gels you are casting 4 33 Calibrating the peristaltic pump Install the pump head and tubing on the pump controller as directed in the User Manual supplied with the pump Calibrate the pump flow rate before the first use and after every 10 to 20 uses to ensure proper flow rates This calibration procedure assumes a desired flow rate of 335 ml min for 14 gels Example You wa
27. e 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 Configure the gradient divider for the number of gels you are casting If necessary calibrate the gradient pump flow rate See Calibrating the peristaltic pump on page 9 3 Besure that the faceplate screws on the gradient maker are fingertight and all the gradient maker lines are clamped off There are three clamps one coming from each chamber and one after the bow tie mixer Close all three 4 Prepare a sufficient volume of gel overlay solution water saturated n butanol You need 1 ml of overlay for each 1 0 mm cassette and 1 5 ml for each 1 5 mm cassette 5 Make up 100 ml of displacing solution See Section 10 2 6 Make up the gel acrylamide solutions from the stock mixes but do not add the 10 ammonium persulphate APS and 10 N N N N tetramethylethylenediamine TEMED See Section 10 2 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Casting gradient gels 4 4 6 Pouring gel solutions for gradient gels 1 Prepare the gel caster as described on page 6 placing gel labels in each cassette 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
28. e contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Care and maintenance 12 12 Care and maintenance 12 1 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 WARNING During all maintenance the equipment shall be disconnected from the mains supply voltage For day to day operation of the unit the cleaning procedure outlined in unit operation thoroughly rinsing the separation tank with distilled or deionized water is sufficient If desired the internal components of the separation unit can be removed for a more thorough cleaning see below The unit can also be periodically cleaned with a dilute solution of a mild 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 DONOT autoclave or heat any part above 50 C e DO NOT expose the unit or its parts to organic solvents including gt 20 ethanol e f using radioactive reagents decontaminate the unit with a cleaning agent such as CONTRAD 70 or Decon 90 from D
29. e electrophoresis buffer from the tank to expose 2 to 3 cm of the gel cassettes This will ease removing the remaining cassettes When the first cassette either blank or gel is removed a sucking sound will be heard as air is drawn into the lower chamber For runs of six or fewer gels alternating gel cassettes with blank cassettes also eases the removal of the gel cassettes at the end of a run Leave enough of the electrophoresis buffer in the tank to act as a lubricant between the glass cassettes and the buffer seal There are two methods for removing the first cassettes from the unit using a the cassette removal tool or b the hands A Carefully insert the cassette removal tool between the cassette and the buffer seal with the folded tip facing the cassette until the tip is beneath the bottom edge of the cassette Verify that the tool is caught on the bottom edge of the cassette then lift it out slowly with the tool Fig 7 6 Fig 7 6 Using the cassette removal tools B By hand apply upward pressure alternately to each side of the cassette gradually shifting it up until you can grasp it and remove it Fig 7 7 Fig 7 7 Removing cassettes by hand 2 Open the cassettes using a Wonder Wedge 80 6127 88 and carefully transfer the gels to a staining tray 80 6468 17 for example Take care to ensure that the gel does not adhere to the spacers Note Viny gloves are less sticky than latex gloves and make it easier to handle larg
30. e gels Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 29 7 Electrophoresis 30 When all of the gels and blank cassette inserts have been removed drain the buffer by turning the pump valve to drain with the pump on Emptying will take about 1 min After the buffer has been removed pour 3 to 4 I of distilled or deionized water into the unit and allow it to drain Rinse the unit with 5 to 7 of distilled or deionized water in circulate mode empty again and repeat if necessary Remove the lid from the unit by sliding it to the left and rinse it with distilled or deionized water Slide the lid back on its hinges before using the unit again In most cases thorough rinsing is all the cleaning that is necessary If a more thorough cleaning is required see Section 12 for a detailed description of the removal of all the internal components Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 8 Electrophoresis on pre cast gels 8 Electrophoresis on pre cast gels Ettan DALT Gel 12 5 is a pre cast polyacrylamide gel for the second dimension of two dimensional electrophoresis The gel is cast onto a plastic support film 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 System together with the Ettan DALT Buffer Kit The gel is formulated for long shelflife and when used with the buffer kit generate
31. eavy feed tube emamna pump Fig 4 2 Pump connected to gradient maker A gradient gel results from using two gel solutions of different acrylamide concentrations and densities a light solution and a heavy solution The heavy gel solution contains glycerol During the gradient pouring procedure the mixing ratio of heavy solution to light solution gradually increases with the heavier solution underlaying the light solution This generates a downward gradient of increasing gel percentage To ensure balanced flow when the gradient maker is filled with equal volumes on each side of the divider the height of the heavy gel solution in the gradient maker should be 1 to 2 cm less than the height of the light solution Under these conditions the two solutions are in hydrostatic equilibrium see Section 4 2 Hydrostatic equilibrium can also be achieved by using equal masses instead of volumes of the heavy and light solutions Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 17 4 Casting gradient gels 18 4 5 Gradient casting setup 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 of any polymerized acrylamide Place the white sponge in th
32. econ Laboratories Inc 12 2 Replacing internal components To remove the anode plate or any of the other internal components follow the directions below 1 Insert the buffer seal removal tools through the fifth or sixth slot with the wider end underneath the tubes Turn the tools 90 and place as close to the end blocks as possible Carefully pull upward on the buffer seal until it is removed from the unit see Fig 12 1 Fig 12 1 Using the buffer seal removal tools 2 Slide the two baffle plates upward and out of the unit 3 The two flow guides are removed by pulling out the retaining pins and lifting the blocks out Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 47 12 Care and maintenance 48 buffer plate baffle plate K i flow guide X anode plate Fig 12 2 Internal components 4 The anode plate then can be removed by unscrewing the flathead nylon screw and lifting the plate out To reassemble the unit replace the anode plate and screw making sure that the sealing sleeve is in place Spread a small amount of GelSeal compound on the plug with a swab applicator to prevent corrosion Replace the flow guides and baffle plates Put a light film of GelSeal compound on the gasket of the buffer seal and reinsert it into the unit using even pressure Make sure that it is fully seated before using the unit Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Customer service information 13
33. f the cooling system capacity at that temperature Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 37 9 Recommended running conditions 38 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 10 Recipes Recipes 10 WARNING Acrylamide is a neurotoxin Always use mechanical pipettes and wear gloves when working with acrylamide solutions Note Always adhere to local and national regulations when handling chemical substances Acrylamide stock 30 8 T 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 in a hood avoid contact with dust Filter and store at 4 C 1 5 M TrisCl pH 8 8 final conc amount Tris MW 121 14 1 5 M 545g 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 dodecylsulphate MW 288 38 10 10g Distilled or deionized water to 100 ml Store at room temperature 10 w v Ammonium persulphate final conc amount Ammonium persulphate MW 71 08 10 2g Distilled or deionized water to 20 ml Prepare fresh 10 v v TEMED final conc amount TEMED MW 116 2 10 0 5 ml Distilled or deionized water 45 ml Prepare fresh Displacing solution 0 375 M TrisCl pH 8 8 50 v v glycerol bromophenol blue 100 ml
34. he separation unit The temperature range is from 10 C to 50 C On the display cooling is indicated by and heating is indicated by The pump must be on to properly cool or heat buffer in the tank The lower temperature limit is a function of the ambient temperature and the power reading To reach the lower temperature limit 10 C or in laboratory environments where the ambient temperature is elevated it may be necessary to place the unit in a cold box or cold room 7 3 Preparing the electrophoresis unit for lab cast gels The Ettan DALTtwelve Electrophoresis Unit requires a total volume of about 10 of electrophoresis buffers to fill both the upper and lower chambers For lab cast Laemmli gels the lower buffer chamber is filled with 7 5 of 1 x SDS electrophoresis buffer while the upper buffer chamber is filled with 2 5 of 2 x SDS electrophoresis buffer With the full volume of the lower buffer in the unit the liquid buffer serves as a lubricant for inserting the glass cassettes through the buffer seal Lubrication of the cassettes and the rubber surfaces of the buffer seal is vital when loading the unit Forcing dry cassettes through the slots can severely damage the seal The electrophoresis buffer can be made within the tank using the internal circulating pump to mix the solution WARNING Do not overfill the tank Spillage can cause shortcircuiting of internal circuits and the breakdown of electrical isolation which increases the risk
35. he unit if they are wet Distilled or deionized water from a squirt bottle can be used to wet the cassettes and inserts as they are being loaded into the unit When all 12 slots are filled the buffer level should be slightly below the level of the buffer seal gaskets Fig 8 14 Loading gels into the separation unit 2 Dilute the cathode buffer to working strength by adding both bottles of 10x cathode buffer total volume 250 ml to 2 25 of distilled or deionized water 3 Pour the diluted 1 cathode buffer into the top of the tank to the fill line Some of this buffer may drip through the gasket and mix with the lower anode buffer during the run but this will not affect performance or results 4 Program the desired run parameters into the control unit close the lid of the electrophoresis tank and press start stop to begin electrophoresis 8 6 Detection The Ettan DALT Gel 12 5 gel can be stained or visualized with a variety of commonly used techniques including Coomassie Blue and silver staining When using the PlusOne Silver Staining Kit Protein a modified staining protocol should be used Prepare the staining reagents 250 ml per gel as indicated in the kit instructions with the following exceptions e Prepare twice the fixing solution called for 500 ml per gel rather than 250 ml e Prepare the developing solution with twice the formaldehyde called for 100 I per 250 ml of developing solution rather than 50 Stain the gel
36. is about 1 to 2 cm below the final desired gel height Stop the flow of acrylamide and remove the feed tube from the balance chamber grommet Once the feed tube is removed the dense displacing solution flows down the connecting tube filling the V well and sloped trough at the bottom of the caster The remaining acrylamide solution is forced into the cassettes to the final gel height The amount of gel solution required will be 800 to 850 ml for gels 1 0 mm thick and 1150 ml for 1 5 mm thick gels 11 Immediately pipette 1 ml of water saturated butanol onto each gel If you are using a peristaltic pump to pour the gels rinse the gel solution from the pump before it begins to polymerize 12 Allow the homogeneous gels to polymerize for at least 1 h before disassembling the caster 14 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Casting gradient gels 4 4 Casting gradient gels 4 1 Preparing for gradient gel casting Successful gradient gel casting requires planning timing and practice A full cast with the Ettan DALTtwelve Gel Caster requires approximately 0 9 to 1 2 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 divider or the cassettes and separators in the gel caster To familiarize yourself with the gel caster and gradient maker before casting gels we recommend setting up the unit and configuring the gradient di
37. kinghamshire 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 imagination at work 80 6476 53 AC 04 2009
38. lution solution L E J i i aV i wo O a Fig 4 4 Properly filled caster and gradient marker Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 19 4 Casting gradient gels 10 11 12 13 14 15 16 17 20 Open the clamp below the mixer c to start flow to the gel caster via the peristaltic pump Carefully open the clamp on the light chamber exit tube a and turn on the pump to bring a small amount of solution into the caster Light solution should begin to flow through the feed tube and mixer toward the caster At this point a small amount of light solution can enter the caster When the light solution level in the gradient maker falls to about 1 cm above the level of the heavy solution open the heavy chamber exit tube clamp b Watch the gradient enter the caster When the caster is filled to about 1 to 2 cm below the final desired gel height or the gradient maker is empty turn off the pump and close the feed tube clamp c Stop the pump before air enters the feed tube Pull the feed tube out of the balance chamber grommet Place the end in a waste container to collect the excess polymerizing acrylamide As soon as the feed tube is removed the dense blue displacing solution flows down the connecting tube to the unit It should completely fill the V well and the sloped trough at the bottom of the caster If the V well is not completely filled and the level of gel in the cassettes is more
39. m User Manual 80 6476 53 Edition AC 23 6 Unloading the gel caster 24 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Electrophoresis 7 7 Electrophoresis 7 1 Installing the power supply control unit WARNING Never connect the power supply cable before all other cables are connected and have been checked for damage The unit should be placed close to a sink for easy rinsing and draining It must be placed on astable support such as a bench or sturdy table The ventilation openings must not be blocked and the power switch and outlet must be easily accessible A length of rubber or vinyl tubing sufficiently long to reach a sink should be attached to the drain port on the back of the unit before use The unit should not be placed on bench paper or any other material that might be pulled in by the air intake fans as any hindrance to air circulation will reduce the cooling capacity 7 2 Programming the power supply control unit The control unit has four programmable parameters Run Type Timer Pump and Temperature When the unit is turned on the default settings of constant power continuous run auto pump and 25 C are shown on the display The set read button toggles the controller between the set and read modes The start stop button starts and stops the execution of the programmed electrophoresis run The lt and gt buttons move the cursor between run parameters and the A and W buttons change the settings of
40. ne TEMED For a full 13 gel 1 5 mm cassette set make up 1 2 I of acrylamide gel stock solution as above This amount of gel solution will provide you with sufficient volume to cast gels using either a funnel or a peristaltic pump Assemble the gel caster as described in the preceding section the caster should be placed on a level bench or on a leveling table so that gel tops are level The white triangular sponge may be omitted Connect the feed tube to either a funnel held in a ring stand above the top of the gel caster about 30 cm or a peristaltic pump Insert the other end of the feed tube into the grommet in the bottom of the balance chamber see Fig 3 1 Fig 3 1 Gel caster in use Fill the balance chamber with 100 ml of the displacing solution 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 funnel taking care to avoid introducing any air bubbles into the feed tube If a peristaltic pump is being used the flow rate should be increased slowly to the desired speed to avoid introducing any air bubbles into the feed tube Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 13 3 Casting homogeneous gels 10 Use the peristaltic pump to pump the gel solution into the caster until it
41. nformity 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 Recycling WARNING Only use mains cables delivered or approved by GE Healthcare WARNING Do not block the rear panel of the system The mains power switch must always be easy to access gt gt gt b gt 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 This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning HENNE the decommissioning of equipment This is a Class A product In a domestic environment it might cause radio interference in which case the user might be required to take appropriate measures WARNING All repairs should be done by personnel authorized by GE Healthcare Do not
42. nt to cast a full tank of gels and determine that the flow rate is 500 ml min when you set the flow rate at 3 For a flow rate of 335 ml min 335 500 3 2 the appropriate flow rate setting to deliver 335 ml min 1 Place the inlet side of the tubing in a beaker that contains 1 of water Place the outlet side of the tubing in a 1 graduated cylinder Set the flow speed at 3 on the dial and start the pump Stop the flow of liquid after exactly 2 min Measure the water in the outlet cylinder and divide by 2 to determine the flow in ml min To determine the appropriate flow setting divide the desired flow rate by the flow rate in step 5 multiply this result by the flow speed used in step 3 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Casting gradient gels 4 4 4 Casting gradient gels The gradient maker is a simple unit with two chambers that are defined by an adjustable silicone rubber gasket clamped between two acrylic plates You can modify the shape of the gel gradient by adjusting the movable divider Solutions flow out of the two chambers in proportion to the relative widths at the surface of the liquid join at the Y connector and then are thoroughly mixed in an in line bow tie mixer that has no moving parts Three pinch clamps control the flow at the exits from the light a and heavy b chambers and after the mixer c to control flow into the peristaltic pump Fig 4 2 light chamber h
43. nual 80 6476 53 Edition AC 43 11 Troubleshooting Symptom Possible causes Possible solutions Gel casting Gels cast simultaneously are different Allow the solution to settle or reach sizes equilibrium before adding the overlay Gradient gels uneven layering Unusually slow or fast run Pre cast gels Second dimension separation proceeds All of the slots in the buffer seal slowly with high current Dye front is irregular Pronounced downward curving of the dye front on one side of the gel are not occupied by either gel cassettes or blank cassettes Anodic buffer has mixed with cathodic buffer from overfilling of either the cathodic or the anodic reservoir The top surface of the gel has been damaged during application of the IPG strip Bubbles or liquid between the gel and the glass plate Add equal amounts of overlay solution to each gel Add overlay as quickly as possible Add sucrose 15 w v or glycerol 25 v v to the highpercentage monomer solution Add a very small amount of bromophenol blue to the highpercentage monomer solution to track gradient formation Note Excessive bromophenol blue will inhibit polymerization Check for leaks All plates spacers and gaskets must be clean dry and free of grease Make sure buffer is at the fill level and not covering the vent holes Check the pH of the buffer If the pH is exceeded make fresh buffer do not back titrate Check recipes gel
44. o prevent the rubber tubing from sticking to the cassettes Once the buffer level reaches the sealing tubes the gels should slide in easily Note Forcing cassettes through the rubber tubes of the buffer seal without sufficient lubrication can damage the buffer seal Fig 7 5 Adding agarose sealing solution Fill any unused slots with the blank cassette inserts When the last cassette is put into place buffer will be pushed out of the lower tank into the upper tank via the two air vents at the corners of the sealing assembly The final level of electrophoresis buffer in the upper tank should not be above the openings for the air vents Recommendation For electrophoresis runs of six or fewer gels it is helpful to alternate gel cassettes with blank cassette inserts Alternating cassettes will make it considerably easier to remove the cassettes from Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Electrophoresis 7 the unit following the run The blank cassette inserts are easily removed first leaving a larger gap that makes it easier to grasp and remove the gel cassettes 7 6 Unloading and cleaning the separation unit CAUTION During all cleaning and draining operations the unit shall be disconnected from the mains power outlet Any liquid on the equipment must be dried off before connecting the power 1 After the run has been completed remove one or more of the blank cassette inserts or gels and drain enough of th
45. o 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 Contaminants in the sample can cause dimension Bubble between IPG strip and top surface of second dimension gel Incorrectly prepared equilibration solution Poor transfer of protein from IPG strip to second dimension gel nsufficient equilibration PG strip is not placed properly ncorrectly prepared equilibration solution Poor transfer of protein from IPG strip to second dimension gel distortions or swollen regions in the IPG strip following IEF These distortions can result in disturbances in the second dimension Ensure that no bubbles are trapped between the IPG strip and the top surface of the second dimension gel Prepare equilibration solution according to instructions Use low power for sample entry phase Extend entry phase if necessary Prolong equilibration time Ensure that the plastic backing of the IPG strip is against the glass plate of the second dimension cassette Prepare equilibration solution according to instructions Use low power for sample entry phase Extend entry phase if necessary 11 1 Recycling This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Pleas
46. of electrical shock 1 Before filling the tank turn the pump valve to circulate see Fig 7 2 y A Fig 7 2 Circulation valve Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Electrophoresis 7 Fill the electrophoresis tank to the 7 5 I fill line with 1x SDS electrophoresis buffer Turn on the pump control unit and turn the pump on If the pump fails to begin circulating buffer immediately the pump must be primed turn the pump valve to drain then back to circulate while the pump is on On the control unit adjust the temperature to the desired setting 7 4 Preparing second dimension gels equilibration and loading For a detailed description of the components of the SDS equilibration solution and the equilibration process please consult 2 D Electrophoresis Using Immobilized pH Gradients 80 6429 60 WARINING Take care when handling glass sheets You risk cutting yourself on sharp edges 1 7 Prepare SDS equilibration buffer Just prior to use add DTT to the buffer to a concentration of 1 w v Place the IPG strips in individual tubes with the support film toward the wall Add 5 to 10 ml of the DTT containing solution to each tube Typically two 18 cm strips can be equilibrated with 10 ml of buffer Incubate the strips for 10 to 15 min with gentle agitation Do not overequilibrate as proteins can diffuse out of the strip during this step Second equilibration optional Prepare S
47. open any covers or replace any parts unless specifically stated in the instructions Contents 10 Ettan DALTtwelve System 1 1 Ettan DALTtwelve System components PEs 1 1 1 Separation Unit wees 205 1 1 2 Power Supply Control Unit 6 KTS Electrical GONNE GC H NS zeguan aaee ea a iat ea AAE RERE 7 E E EO A AA AOA AA E A A A SS Ta 7 11 5 Gel Casting CASSETES inienn aa a a a a a s 7 12 J IMPOFLANtINFOFMALION a sinstshettannerta cs aaa EE E E E E EA 8 T3 Specifi Catonis asec cassiihenhatds daa heal dasa G isda cea ck ea bel nol ble al addi ol 9 Preparing the gel caster Casting homogeneous gels Casting gradient gels 4 1 Preparing for gradient gel Tasting isisascescdiecalncadiaisadiecadhssledaicanthanwuislluldgeiahdasldcaidantas 15 4 2 Configuring the gradient divider m m 15 43 Calibrating the Peristaltic PUM Di irseresnaeneinean anaana 16 AAs CASUAG OTO ONE NE GES ies nn terse NTA ANDREA RR NS aerial on 16 4 5 Gradient CASING SetU D a aeaa e a ar lancet best aa aeae eao s ea ar oaeoi Eiei 17 4 6 Pouring gel solutions for gradient Gels csssssssssssssssssessesesecececcesssssssssssssssssssusuusssssssssceeeseeceeeeeeesesesssssssssssssssssasessees 18 Polymerization Unloading the gel caster Electrophoresis 7 1 Jastalling the power supply CcONntrolUnitesssute cscs E A NE 25 7 2 Programming the power supply control unit o eeeesssesssssssesssessessssesemsssseeessssseesesseeseesssseseuuss
48. ouch the opposite glass plate Apply molecular weight marker proteins optional Apply the markers to a sample application piece in a volume of 15 to 201 then cover the piece with 50 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 to 1 0 mg of each component for Coomassie blue staining or about 10 to 50 mg 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 bubbles It will flow down between the glass plate and the support film and seal the IPG strip in place see Fig 7 5 Agarose should also be used to seal any gap between the side of the gel and aside spacer Allow a minimum of 1 min for the agarose to cool and solidify 7 5 Loading the separation unit WARNING Switch the equipment off before opening the covers to avoid exposure to high voltage Once the electrophoresis tank has reached the desired temperature and the gels are ready carefully slide the gels one by one into the tank Until the buffer reaches the bottom of the rubber sealing tubes the cassettes should be lubricated with buffer or water t
49. p should not touch the plastic support film Fig 8 12 Seating the IPG strip against the gel 4 Seal the IPG strip in place For each IPG strip melt an aliquot of sealing solution from the buffer kit in a 100 C heating block or boiling water bath Tip An ideal time to carry out this step is during IPG strip equilibration Allow the solution to cool slightly then slowly pipette the solution across the length of the IPG strip taking care not to introduce bubbles It will flow down between the glass plate and the support film and seal the IPG strip in place see Fig 8 13 There may be a gap of up to 2 mm between the edge of the gel and the side spacer Any gap should be plugged by allowing some of the sealing solution to flow down the gap Allow a minimum of 1 min for the agarose to cool and solidify Fig 8 13 Sealing the IPG strip with agarose 5 Repeat the procedure for each second dimension gel to be run Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 35 8 Electrophoresis on pre cast gels 36 8 5 Inserting gels into the separation unit WARNING Disconnect the unit from the supply voltage when inserting gels 1 When the lower tank buffer has reached the desired temperature insert the loaded gel cassettes with the IPG strips in place see Fig 8 14 Push blank cassette inserts into any unoccupied slots Load the unit from back to front Gel cassettes and blank cassette inserts slide much more easily into t
50. p that may exist between the gel and the spacer 44 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Troubleshooting 11 Symptom Possible causes Possible solutions Stained gels Protein spots are diffuse or broader than Use only the highest quality reagents usual Make sure that polymerization is complete Check equilibration time of IPG strips Too long 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 Problems with first dimension see troubleshooting guides for IPG phor 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 Protein spots are near the buffer front 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 The gel pore size is too small Decrease the when buffer front has reached the T bottom of the gel Check the pH of the 4x gel buffer It should be p
51. r ifting the caster e If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Ettan DALTtwelve System 1 e Only accessories and parts approved or supplied by GE Healthcare may be used for operating maintaining and servicing this product WARNING To avoid risk of electrical shock ensure that all electrode contacts are clean and dry and there is no excess liquid on the upper rim of the separation unit Inspect the cables for damage before use of the instrument and contact Technical Support if any damage is apparent 1 3 Specifications Ettan DALTtwelve Electrophoresis Unit Gel capacity Electrophoresis buffer volume Dimensions h x w x d Weight Maximum temperature Environmental operating conditions Buffer circulation pump rate Ettan DALTtwelve Power Supply Control Unit Maximum output power Maximum output voltage Maximum output current Temperature control range Weight Dimensions h x w x d Environmental operating conditions Installation category Pollution degree Supply voltage Max Power Consumption Product and safety certifications Complies with EMC standard Ettan DALTtwelve Gel Caster 12 gels 101 54 x 46 x 51 cm lid closed 74 x 46 x 51 cm lid open 24 5 kg 50 C Indoor use 4 C to 40 C Humidity up to 90 Altitude to 2000 m
52. s a discontinuous buffer system yielding rapid runs with sharp reproducible results Performance and capacity of this gel and buffer system are similar to the widely used Laemmli Tris glycine buffer system These instructions describe how to use Ettan DALT Gel 12 5 together with the Ettan DALT Buffer Kit for the second dimension of 2 D electrophoresis 8 1 Ettan DALT Gel 12 5 and Ettan DALT Buffer Kit 8 1 1 Package contents Each gel package contains six gels and instructions The buffer kit contains four bottles of buffer and 12 tubes of sealing solution The solutions are sufficient for a single run of up to 12 gels Product Quantity Product number Ettan DALT Gel 12 5 6 17 6002 36 Instructions 1 71 5019 56 Ettan DALT Buffer Kit enough to run 12 gels 17 6002 50 Anode buffer 1 bottle 75 ml Cathode buffer 2 bottles 2 x 125 ml Gel buffer 1 bottle 60 ml Sealing solution 12 tubes 12 x 1 ml Technical data Gel composition Separation range Gel dimensions Buffer in gel Gel backing Shelf life Storage 100 anode buffer 10x cathode buffer Gel buffer Sealing solution T 12 5 C 3 12 125 acrylamide 0 375 bisacrylamide Mr 12000 to 120000 255 x 196 x 1 mm Piperidinopropionamide PPA Polyester film 265 x 211 mm 6 months 4 C to 8 C 5 M diethanolamine DEA 5 M acetic acid 0 25 M Tris 1 92 M glycine 1 w v SDS Piperidinopropionamide PPA Gel buffer with 0 5 agarose and 0 002
53. s according to the following protocol Fixing 2 x 60 min Sensitization 60 min Water wash 5x8min Silver 60 min Wash 4x1min Developing 10 mint Stop 60 min Wash 2x30 min Preserve 40 min The first fixing step may be prolonged up to 3 days if desired for the sake of convenience Approximate time this step may be visually monitored The gels should be transferred to stop solution when the spots have reached the desired intensity and before the background staining becomes too dark Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 9 Recommended running conditions Recommended running conditions 9 Approximate run Gels Step Power w gel Duration h min Day runs set temperature to 25 C 1mm thick gels 1 5 0 30 lab cast and precast 2 17 max 180 4 00 1 5 mm thick gels 1 5 0 30 2 17 max 180 6 00 Overnight runst set temperature to 30 C and power supply to continuous run 1 0 mm thick gels 1 5 mm thick gels 1 5 17 00 17 00 The time shown is approximate Stop electrophoresis when the dye front is 1 mm from the bottom of the gel For the best possible resolution faster separation times should be used Use the faster lt 6 h protocols instead Note The run times provided should only be used as guidelines or estimates Decreasing the number of gels per run allows increased watts per gel which reduces run times The maximum power specified for each temperature is the limit o
54. seeeassseseunssseseeset 25 7 3 Preparing the electrophoresis Unit for lab cast gels mms 26 7 4 Preparing second dimension gels equilibration and loading 27 75 Loading thesepdration UN Ite save en nA EEA A EN E i SS ANA AMRA ARS YE 28 7 6 Unloading and cleaning the separation unit o cccccsssssssssssssssssssssssssssessssesseseeesssssssssssssssmsssssssessesseseeseseeessssses 29 Electrophoresis on pre cast gels 8 1 Ettan DALT Gel 12 5 and Ettan DALT Buffer Kit oc cccccssssssssscccecceccessessnsssuununsssssssseseeseseeseeceeseessssssssssssssaneeete 31 Bill Packag contents ds sciscizcencsacciescavonsci aa nsec acca scones chee NAN OOR NE 31 8 2 Description f the SYSTENN ss sd serves eA A Mn SNR NORR A EESE 32 8 3 Preparing the Ettan DALTtwelve Electrophoresis Unit m lt cosewosossssmsmssomsmesssmsmsssmsmsmssmsmsmmusmsn 32 8 3 1 Inserting the Ettan DALT Gel 12 5 into the pre cast gel cassette m ee cmweememmwsswssmsmeemsmsmm 33 84 Applying the POSED E EAEE AA E O ERA ER 35 8 5 Inserting gels into the separation unit sme 36 86 Detection oerien ne iiit i aE Banden EAE RAAR E TEA a E ah aes 36 Recommended running conditions Recipes 10 2 HOMOGENEOUS gelsol tioNSsonee E EEE E R EA a a canes 41 10 2 Gradi nt gelsolytioN Sess a aa a a a a eaheaatee 41 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 3 11 Troubleshooting TTT RE CY GUNG 2232 iae cet iia i es cn ha ee E eae eae GE dot Sea Ue ee 46 12
55. t is compressed evenly by the faceplate and forms a tight seal with the caster Do not overtighten the screws see Fig 2 3 T gt Fig 2 3 Gel caster filled and level Insert the end of the rigid plastic feed tube supplied with the gel caster into the rubber grommet in the floor of the hydrostatic balance chamber on the side of the caster The feed tube must be snug so that there is no leakage from the balance chamber into the caster The feed tube should be connected with flexible tubing directly to either a peristaltic pump or a funnel Fig 3 1 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Casting homogeneous gels 3 3 Casting homogeneous gels 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 of any polymerized acrylamide Prepare a sufficient volume of gel overlay solution water saturated n butanol You need 1 ml of overlay for each 1 0 mm cassette and 1 5 ml for each 1 5 mm cassette Make up 100 ml of displacing solution 0 375 M TrisCl pH 8 8 50 v v glycerol bromophenol blue For a full 14 gel 1 0 mm cassette set make up 0 9 I of acrylamide gel stock solution without adding the 10 w v ammonium persulphate APS or 10 v v N N N N tetramethylethylenediami
56. that the caster is level Remove the faceplate and tip the caster back so that it rests on the removable tilt support legs If casting gradient gels place the triangular sponge in the base of the V shaped feed channel otherwise it may be omitted Fig 2 2 Fig 2 2 Gel caster with gel cassette tipped back Ettan DALTtwelve System User Manual 80 6476 53 Edition AC 11 2 Preparing the gel caster 12 Start filling the gel caster by placing a separator sheet against the back wall to make it easier to remove the last cassette 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 offset end of the cassette up End with a separator sheet then use the thicker spacer sheets to bring the level of the stack even with the edge of the caster Lubricate the foam gasket with a light coating of GelSeal compound to help ensure a liquid tight seal Place the gasket in the groove on the faceplate Avoid stretching the gasket by seating it from the ends toward the middle Turn four black knobbed screws into the four 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 Screw the four remaining black knobbed screws into the holes at the sides of the faceplate and tighten all eight evenly Be sure the sealing gaske
57. uffer into a 100 ml graduated cylinder or similar vessel Using forceps remove the equilibrated IPG strip from the equilibration solution and dip it into the cathodic buffer in the cylinder This step lubricates the IPG strip and washes off any particulate material that may be precipitated on the surface of the IPG strip 3 Place the IPG strip on the top cathodic surface of the gel Holding one end of the IPG strip carefully draw it across the shelf formed by the extension of the glass plate beyond the plastic frame until the strip is completely on the glass plate and centered gel face upward see Fig 8 11 M INF Fig 8 11 Placing of the IPG strip Push the IPG strip into place Using a thin spatula or ruler push against the plastic backing of the IPG strip to slide it a short distance into the gap between the glass plate and the support film and plastic frame Be sure to push against the backing of the IPG strip not the gel itself see Fig 8 12 Place the cassette upright in the cassette rack with the glass plate forward Continue to slide the IPG strip down until it contacts the surface of the second dimension gel The strip should just rest on the surface of the gel Avoid trapping bubbles between the strip and the slab gel and avoid piercing the second dimension gel with the strip Note the orientation of the IPG strip relative to the gel conventionally the acidic pointed end of the IPG strip points to the left The gel face of the stri
58. vider as described below Follow the gradient pouring procedure substituting water for the appropriate volume of light solution and a mixture of glycerol and water for the appropriate volume of heavy solution When the angle of the gradient divider is adjusted correctly and you are comfortable with the gel casting procedure clean all parts of the caster and gradient maker including sponges separator sheets and filler blocks with a solution of mild detergent followed by a deionized water rinse 4 2 Configuring the gradient divider You can adjust the shape of the gradient divider to suit the number of gels to be cast and the shape of the gradient you want When casting a full tank of gels the angle of the adjustable divider to the floor of the gradient maker should be about 40 see Fig 4 1 For fewer gels decrease the divider angle Whatever angle you use a straight divider gives a linear gradient divider pinch clamps Fig 4 1 Configuration of the gradient maker 1 Determine the amount of gel solution needed to cast the desired number of gels 2 Loosen the faceplate screws on the empty gradient maker to adjust the gasket angle Refer to the divider configuration in Fig 4 1 as a guideline for the angle you should use Pull the divider slightly to help move it into position Use the red adjuster rod provided with the gradient maker to push the gradient divider down 3 Fora linear gradient straighten the divider with its upper en
59. x 22 cm Indoor use 0 C to 40 C Humidity 10 to 90 2 EN61010 1 UL508 CSA C22 2 No 14 IEC 61010 EN61326 IP22 Ettan DALTtwelve System User Manual 80 6476 53 Edition AC Preparing the gel caster 2 2 Preparing the gel caster Unpack the equipment and take care when moving the equipment into position Use the handles to lift and position the units on a level and stable surface IMPORTANT Make sure that the supply cord and the power outlet are easily accessible and that no ventilation openings in the enclosure are blocked in any way WARNING Make sure that there are no loose parts or debris inside the equipment Set up the gel caster near a sink in a tray or on a drainboard so that any liquid that may overflow from the unit or drain out of it during pouring or disassembly can be easily contained The Ettan DALTtwelve Gel Caster see Fig 2 1 can accommodate up to fourteen 1 0 mm or thirteen 1 5 mm gel cassettes with separator sheets between them If you are not planning to cast a full set of gels use the blank cassette inserts with separator sheets in between provided with the separation unit to occupy the extra space faceplace feed tube hydrostatic balance chamber grommet knobbed r tl Screw support leg Fig 2 1 Exploded view of caster Gellabels for easy indexing of gels and samples can be placed in the cassettes at any time during the assembly of the caster 1 Check

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