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Hoefer DALT System - GE Healthcare Life Sciences

1. 80 6431 50 Hoefer DALT System DALT Electrophoresis Tank DALT Gradient Maker DALT Multiple Gel Caster DALT Blotting Kit User Manual Rev A 12 98 Amersham Biosciences Important User Information E Please read this entire manual to fully understand the safe English and effective use of this product user to the presence of important operating and maintenance instructions in the literature accompanying the instrument The exclamation mark within an equilateral triangle is intended to alert the If you have any comments on this manual please send them to us at Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences guarantees that the product delivered has been thoroughly tested to assure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Amersham Biosciences Amersham Biosciences shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business oppor tunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product O 1998 Amersham Bioscienc
2. Installation category Pollution degree Buffer circulation pump rate 115 V 230 V Product certifications DALT Multiple Gel Caster Gel Capacity 1 0 mm thick 1 5 mm thick Acrylamide solution volume total Dimensions h x w x d Weight DALT Gel Cassettes Glass cassette dimensions w x h Slab gel dimensions 10 gels 20 liters 22 liters lid closed 38 x 48x 33cm 15 x 19 x 13 in lid open 50 x 48 x 33 cm 20x 19 x 13 in 16 8 kg 200 W 500 V DC 1000 mA 30 C Indoor use 4 40 C Humidity up to 90 Altitude up to 2000 m Il 2 20 liters min 50 60 Hz 2 4 180 W 50 60 Hz 1 0 83 A EN61010 1 UL3101 1 CSA C22 2 1010 1 CE 25 gels 23 gels 1 800 ml 29 x 340 25 cm 33 5 kg 25 5 x 20 4 cm 1 0 mm x 23 4 x 19 5 cm 1 5 mm x 23 4 x 19 5 cm Hoefer DALT System Function and Description Hoefer DALT System DALT Gradient Maker Dimensions h x w x d 54x 19x 18cm Maximum gradient volume 2 000 ml Weight 5 4 kg Peristaltic Pump for Gradient Maker 115 V 37 W 1 5A 230 V 37 W 0 9A Weight 4 1 kg 9 Ibs Dimensions h x w x d 13 5 x 18 x 22cm Environmental operating conditions Indoor use O 40 C Humidity 10 90 Altitude up to 2000 m Installation category Il Pollution degree 2 Product certifications EN61010 1 UL508 cUL 115 V IEC 1010 230 V CE DALT Blotting Kit Capacity 5 gels Rack dimensions h x w x d 28 x 23 x 26 cm Weight 3 2 kg This declarati
3. Note that the light mix is always assumed to have a lower acrylamide concentration than the heavy mix the gradient increases in T from top to bottom These solutions can be degassed though this is not usually necessary Light Solution 1000 ml Amounts shown in ml Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 233 267 300 333 367 400 433 467 500 533 567 600 633 1 5M Tris Cl 250 250 250 250 250 250 250 250 250 250 250 250 250 pH 8 8 Water 494 461 428 395 362 329 295 262 229 196 162 129 96 10 SDS 10 10 10 10 10 10 10 10 10 10 10 10 10 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 TEMED 245 2 14 1 90 1 71 1 55 143 1 32 122 1 14 1 07 1 01 0 95 0 90 Heavy Solution 1000 ml Amounts shown in ml Final T 8 9 10 11 12 13 14 15 16 17 18 19 20 Acryl Stock 267 300 333 367 400 433 467 500 533 567 600 633 667 1 5MTris Cl 250 250 250 250 250 250 250 250 250 250 250 250 250 pH 8 8 Water 400 366 333 300 267 233 200 167 133 100 67 33 0 10 SDS 10 10 10 10 10 10 10 10 10 10 10 10 10 Glycerol 68 68 68 68 68 68 68 68 68 68 68 68 68 10 APS 5 00 5 00 5 00 5 00 5 00 5 00 5 00 5 00 5 00 5 00 5 00 5 00 5 00 10 TEMED 0 71 0 64 0 57 0 52 0 47 0 44 0 41 0 38 0 35 0 34 0 32 0 30 0 28 Amersham Biosciences 49 Gradient Gel Solutions Hoefer DALT System For 25 gels 1 0 mm 750 ml each Heavy and Light by volume in ml Ligh
4. 1 5 M Tris Cl 41 57 10 Ammonium persulfate 41 concentration 56 in gradients 49 in homogeneous gels 46 in gradient preparation 19 10 SDS 41 10 TEMED 42 concentration in gradients 49 in homogeneous gels 46 A acrylamide in IPG strips 27 ordering 62 precautions for use 14 18 stock 41 active cooling 34 agarose sealing solution 28 43 balance chamber 13 banana plug corrosion 37 blotting kit 34 to 38 description 7 bow tie mixer 18 bromophenol blue in heavy solution 56 C calibrating peristaltic pump 17 casting gels gradient 18 to 21 non gradient 14 configuring the gradient divider 16 cooling bath 11 temperature 24 34 creatine kinase charge standards 45 crosslinker concentration 56 culture tubes screw cap 11 Amersham Biosciences 63 DALT System components 3 to 7 specifications 9 unpacking 7 degassing solutions 11 displacing solution 42 recommended volume 14 20 dithiothreitol in equilibration 27 E electroendosmosis minimizing 27 electrophoresis buffer 56 conditions 32 electrophoresis tank description 3 loading 24 EPS 2A200 11 EPS 601 11 equilibration buffer 27 ethylene glycol precaution 24 F flow rate calibration 17 G gel cassettes cleaning 33 description 5 loading 31 unloading 33 gel casting unit description 5 preparation 12 unloading 23 gel labels 54 placement 13 geloverlay 14 19 42 application 22 gel storage 23 42 gels gradient 18t021
5. 10g Water up to 100 ml Store at room temperature 10 Ammonium Persulfate Final Conc Amount Ammonium persulfate MW 228 2 10 2g Water up to 20 ml Make fresh 4l Recipes 42 Hoefer DALT System 10 TEMED Final Conc Amount VA MW 116 2 10 O5m Water 4 5 ml Prepare fresh in glass vessel Displacing Solution 0 375 M Tris Cl pH 8 8 50 glycerol bromophenol blue 200 ml Amount Tis CI 1 5M pH8 8 DOM Glycerol 100 ml Bromophenol blue 2 mg Water 50 ml Prepare fresh Stored solution may support microbial growth Water saturated n Butanol Amount n butanol 50 ml Double distilled 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 Tris Cl pH 8 8 0 1 SDS 2 0 liters Final Conc Amount 15MTisC pH8 amp 8 500mI 10 SDS 0 1 20 ml Double distilled H20 to 2000 ml Store at 4 C Amersham Biosciences Hoefer DALT System Amersham Biosciences Recipes SDS Electrophoresis Buffer 25 mM Tris 192 mM glycine 0 1 SDS approximate pH 8 3 20 liters Final Conc Amount Tris MW 121 14 25 mM 60 5 g Glycine MW 75 07 192 mM 288 0 g SDS FW 288 38 0 1 w v 20 0 g Double distilled H20 to 20 liters Do not adjust the pH of this solution SDS Equilibration Buffer 50 mM Tris HCI pH 8 8 6 M urea 30 glycerol 2 SDS bromophenol blue 200 ml Final Conc Amount Tris Cl pH 8 8
6. MI 11 I e pil Iud r I 5 Make a note a label on the tank lid when you change the buffer Although it is possible to use each tank of buffer for 2 3 runs by mixing the center and cathode left chambers between runs it is better to change the buffer before every run Fresh buffer ensures more consistent results When a tank is not going to be used for several days siphon out the buffer rinse the tank with water and allow the tank to sit empty This prevents growth of bacteria and molds which can occur in used buffer Such contamination can give rise to mysterious protein bands across a whole set of gels 6 When the buffer is distributed in all chambers replace the barrier combs in the outer slots to accommodate the 20 x 25 cm cassettes Figure 15 Top view of DALT Tank with barrier Pump combs in place Outer barrier comb slot accommodates 20 x 25 cm cassettes Cassette When properly positioned the ends of the silicone rubber flaps face the outer walls of the tank Figure 15 26 Amersham Biosciences Hoefer DALT System Amersham Biosciences Loading and Running Second Dimension Gels Loading and Running Second Dimension Gels Immobiline DryStrip gels provide a pH gradient IPG that is immobilized in an acrylamide gel and supported by a plastic film backing The Hoefer DALT System can accommodate the entire length of an 18 cm IPG strip plus markers and up to 10 gels
7. Transfer Membranes The DALT Blotting kit includes 50 pieces of blotting paper For electrophoresis tank blotting Amersham Biosciences offers a complete line of transfer membranes including pure nitrocellulose supported nitrocellulose PVDF and nylon Choose the appropriate transfer membrane for your application Small Gadgets e A small plastic spatula or thin plastic ruler to insert and seat IPG gels on the tops of the slab gels between the glass plates e Plastic covered dish racks Rubbermaid or equivalent for holding the DALT gel cassettes during the loading process e microwave oven or 100 C heating block for preparing the agarose sealing solution A Wonder Wedge 80 6127 88 for prying open the gel cassettes after the DALT run e Screw cap culture tubes 25 x 200 mm for storing IPG strips at 40 C e GelSeal for lubricating the gradient gasket to assure a leakproof seal e A peristaltic pump is recommended for more rapid removal of buffer than the small siphon pump supplied with the DALT Tank The pump can be set up to deliver used buffer that is not radioactive directly to a lab drain e vacuum pump for degassing solutions e Thick latex dishwashing gloves with textured fingers 11 Preparing the Gel Caster 12 Hoefer DALT System Preparing the Gel Caster Set up the gel caster near a sink in a tray or container that can act as a catch basin for any liquid that may overflow the unit or empty out of it wh
8. 8 122 124 G rg A Postel W G nther S 1988 The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 9 531 546 G rg A 1993 Two dimensional electrophoresis with immobilized pH gradients current state Biochem Soc Trans 21 130 132 Matsudaira P 1987 Sequence from Picomole Quantities of Proteins Electroblotted into Polyvinylidene Difluoride Membranes J Biol Chem 262 10035 O Farrell PH 1975 High resolution two dimensional electrophoresis of proteins J Biol Chem 250 4007 4021 Stellway E J and Dahlberg A E 1980 Electrophoretic transfer of DNA RNA and protein onto DBM paper Nucleic Acids Res 8 299 Towbin H et al 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose procedure and some applications Proc Natl Acad Sci USA 76 4350 4353 Amersham Biosciences Hoefer DALT System Amersham Biosciences Customer Service Information Customer Service Information Technical Service and Repair Amersham Biosciences offers complete technical support for all our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Amersham Biosciences representative IMPORTANT Request a copy of the Amersham Biosciences Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this f
9. Wedge 11 Amersham Biosciences Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Hoefer Immobiline IPGphor Multiphor MultiTemp are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc All other trademarks and registered trademarks are the property of their respective companies or organizations All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Amersham Biosciences 1998 AII rights reserved Printed in the USA Amersham Biosciences
10. can be run simultaneously To prepare for second dimension electrophoresis first equilibrate the focused IPG strips in SDS buffer before loading them onto DALT slab gels IMPORTANT Prepare the second dimension vertical gel and tank buffer before IPG strip equilibration Prepare agarose sealing solution during IPG strip equilibration The exposed edges of the gels tend to dry out over a period of hours Coordinate both IPG and DALT runs so that the end of the IPG run coincides with the end of unloading the DALT casting unit Start the DALT polymerization 2 5 hours before the IPG equilibration to allow up to 2 hours for gel polymerization and 30 minutes for unloading the cassettes from the casting unit Equilibrating IPG Strips The SDS equilibration buffer contains urea glycerol SDS Tris buffer and tracking dye During equilibration the sample proteins are saturated with SDS for mobility in the second dimension gel Urea and glycerol minimize electroendosmosis effects due to the IPG strip In addition the equilibration buffer contains DTT to fully reduce the sample prior to the second dimension separation Equilibration Buffer 50 mM Tris Cl maintains strip pH in a range that allows proper stacking The pH is typically adjusted to 8 8 to compensate for the acidifying effect of the optional iodoacetamide treatment Urea 6 M together with glycerol reduces the effects of electroendosmosis by increasing the viscosity of the buffer E
11. circuler l eau dans l changeur vertical durant l lectrophor se pour minimiser l chauffement afin d viter des dommages irr parables l instrument Pour des coulages plus long on peut aussi contr ler la temp rature en refroidissant le tampon avant l utilisation et ou en utilisant l instrument dans une chambre froide e Ne pas soulever la Cuve DALT en s appuyant sur la pompe e Ne pas autoclaver ou st riliser cette unit ni aucune de ses pi ces d tach es e L unit de coulage des gels quand elle comprend les plaques de verre et la solution d acrylamide est tr s lourde Prenez toute vos pr cautions quand vous d placez ou soulevez l unit e 51 l instrument n est pas utilis en conformit avec les recommandations du fabriquant les protections de s curit qui quipent cet appareil peuvent tre rendues in fficaces e Seulement les accessoires et pi ces detach es approuv s ou fournis par Amersham Biosciences sont recommand s pour l utilisation l entretien et r paration de cet appareil Amersham Biosciences Hoefer DALT System Amersham Biosciences Specifications Hoefer DALT System Function and Description DALT Electrophoresis Tank with Buffer Circulation Pump Gel capacity 1 0 or 1 5 mm thick Electrophoresis buffer volume Blotting buffer volume Dimensions h x w x d Weight Maximum wattage Maximum voltage Maximum amperage Maximum temperature Environmental operating conditions
12. fit connectors female to fit 9 mm i d tubing Quick fit connectors male to fit 9 mm i d tubing Accessories MultiTemp III Thermostatic Circulator includes insulation tubing for cooling 115 VAC 230 VAC Hoefer EPS 2A200 Power Supply 200V 2000 mA EPS 601 Power Supply 600 V 400 mA PlusOne Electrophoresis Chemicals and Reagents Urea Dithiothreitol DTT Bromophenol Blue Glycerol 8796 Acrylamide IEF acrylic acid lt 0 002 Acrylamide IEF 40 solution N N Methylene bisacrylamide N N Methylene bisacrylamide 2 solution Agarose NA N N N N tetramethylethylenediamine TEMED Ammonium persulfate APS Tris Glycine Sodium dodecylsulfate SDS Molecular Weight Markers MW Range 2 512 16 949 2 mg vial MW Range 14 400 94 000 575 ug vial MW Range 53 000 212 000 175 ug vial 2 D Technical Manual 2 D Electrophoresis Using Immobilized pH Gradients Lo oL HE 500g lg 10g 1000 ml 1 kg 1000 ml 25g 1000 ml 10 g 25 ml 25g 500g 500 g 100g 1 vial 10 vials 10 vials Code No 80 6069 17 80 6069 55 80 6069 74 80 6069 93 80 6421 43 80 6127 88 80 6437 58 80 6115 15 80 6115 53 18 1102 77 18 1102 78 80 6406 99 18 1130 02 17 1319 01 17 1318 01 17 1329 01 17 1325 01 17 1300 02 17 1301 01 17 1304 01 17 1306 01 17 0422 01 17 1312 01 17 1311 01 17 1321 01 17 1323 01 17 1313 01 80 1129 83 17 0446 01 17 0615 01 80 6429 60 Amersham Biosciences Index Numerics
13. for your system use the pump to circulate the buffer to avoid buffer depletion at the electrodes Assemble the Transfer Cassette Note Always wear gloves 1 when handling membranes to avoid contamination with skin proteins Amersham Biosciences Pre wet nitrocellulose or nylon membranes with distilled water Pre wet PVDF or other hydrophobic membranes in methanol Then soak all membrane types in transfer buffer for 2 5 minutes If necessary equilibrate the gel in transfer buffer for 10 15 minutes Open the cassette by releasing both latch tabs along the edge opposite the hinges Place the opened cassette into a tray filled with at least 6 cm of transfer buffer Assemble the transfer stack so that molecules will migrate toward the membrane For negatively charged macromolecules such as proteins coated with SDS build the stack on the anode half of the cassette 35 The DALT Blotting Kit Note Carefully position the gel Proteins may begin to transfer immediately Once transfer has begun moving the gel will distort results or cause shadow bands on the membrane Figure 22 Transfer stack assembly Orient the stack so that the negatively charged molecules migrate toward the anode 36 Hoefer DALT System Place one 6 mm thick foam sponge on the opened submersed cassette and press gently until all air is expelled Place one sheet of blotting paper on the sponge and then place the membrane on the blot
14. make a funnel for introducing heavy solution into the left side bend the remaining top of the divider to the right Tighten the faceplate screws and close the pinch clamps on the tubing that runs out of the gradient maker before adding water or gel solutions Using water in place of gel solution put half 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 chamber is about 2 cm below the level of liquid in the light chamber You may use tape or wax pencil on the outside of the gradient maker to record the angle of the gradient divider Avoid using marker ink that can only be removed with methanol Most solvents including methanol can craze the plastic parts of the gradient maker 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 or thickness of gels you are casting Calibrating the Peristaltic Pump Install the pump head and tubing to the pump controller as directed in the manual supplied with the pump Calibrate the pump flow before the first use and after every ten to twenty uses to assure proper flow rates This calibration procedure assumes a desired flow rate of 440 ml min for a full tank of gels 2000 ml acrylamide and 325
15. membrane proteins Biochemistry 15 616 623 Berkelman T and Stenstedtt T 1998 2 D Electrophoresis Using Immobilized pH Gradients Principles and Methods Amersham Biosciences Bjellqvist B Ek K Righetti P G Gianazza E G rg A Westermeier R Postel W 1982 Isoelectric focusing in immobilized pH gradients principle methodology and some applications Biochem Biophys Methods 6 317 339 Bjevllqvist B Sanchez J C Pasquali C Ravier Paquet N Frutiger S Hughes G J Hochstrasser D 1993 Micropreparative two dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins Electropboresis 14 1375 1378 Blomberg A Blomberg L Norbeck J Fey S J Larsen P M Larsen M Roepstorff P Degand H Boutry M Posch A G rg A 1995 Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two dimensional gel electrophoresis Electrophoresis 16 1935 1945 Dunn M J Corbett J M 1996 2 dimensional polyacrylamide gel electrophoresis Meth Enzymol 271 177 203 Gershoni J M and G E Palade 1983 Protein Blotting Principles and Applications Anal Biochem 131 1 15 G rg A Postel W Weser J G nther S Strahler J R Hanash S M Somerlot L 1987 Elimination of point streaking on silver stained two dimensional gels by addition of iodoacetamide to the equilibration buffer Electrophoresis
16. non gradient 14 GelSeal 11 56 glycerol in equilibration 27 Index gradient gels casting 18t021 solutions 49 troubleshooting 56 gradient maker 16 divider configuration 16 description 6 pump assisted 18 H heat exchanger 24 homogeneous gel casting 14 solutions 46 hydrostatic balance chamber 6 13 hydrostatic equilibrium 21 immobilized pH gradient see IPG strips iodoacetamide 28 IPG strips 11 description 27 equilibration 27 loading onto slab gels 29 storage 58 L labeling gels 54 M migration direction 37 migration rate adjusting 57 MultiTemp 11 24 non gradient gels 14 solutions 46 0 overlay 22 42 57 P peristaltic pump 11 18 polymerization incomplete 56 time 14 15 19 22 power supply current leak 57 electrophoresis 11 64 Hoefer DALT System proteins contamination 26 35 migration 59 spots on gels 22 transfer from gel 34 transfer parameters 38 proteolytic degradation 58 Q Quick fit disconnect fittings 24 R run conditions electrophoresis 32 transfer 39 S SDS electrophoresis buffer 24 43 SDS equilibration buffer 43 silver stain 33 sodium dodecyl sulfate 27 specifications 9 T TEMED 19 56 Towbin buffer 44 tracking dye 28 transfer conditions 39 parameters 38 40 transfer buffer 44 notes 34 reusing 39 transfer cassette assembly 35 58 loading 37 transferring proteins 34 U unpacking the system 7 urea in equilibration 27 W Wonder
17. of the heat exchanger WARNING Do not use automotive antifreeze solution Ethylene glycol is toxic and may be fatal if ingested inhaled or absorbed through the skin Filling the Tank with SDS Electrophoresis Buffer Because the DALT Tank has its own circulating pump you can make the tank buffer within the tank itself The size and number of the cassettes used determine the amount of SDS electrophoresis buffer needed to cover the cassettes You can run any number of Amersham Biosciences Hoefer DALT System Preparing the DALT Tank for Electrophoresis gels up to the tank maximum The empty slots seal themselves and require no blank cassette Since the filled tank is too heavy to empty directly use a siphon pump to remove buffer from the tank Between runs rinse the tank with water 1 Two or three hours before the beginning of a run fill the tank with the appropriate volume of de ionized water Use tape to mark the level of the liquid on the outside of the tank for future runs Always use water near room temperature Never fill the tank with liquid at a temperature very different from the tank itself IMPORTANT Fill the tank with water before turning the pump on The pump is not self priming and can be damaged if run dry 2 Turn on the pump after the tank is filled At first the pump blows some bubbles through the circulating flutes in the tank and then establishes a vigorous circulating action in the tank If the pump does
18. of the strip should just rest on the surface of the slab gel Avoid trapping air bubbles between the plastic backing and the glass plate or cutting into the SDS gel with the strip The gel face of the strip should not touch the opposite glass plate Optional Apply size marker proteins Apply the markers to a paper IEF sample application piece in a volume of 15 20 ul For less volume cut the sample application piece proportionally Place the IEF application piece on a glass plate and pipette the marker solution onto it Apply approximately 50 ul agarose sealing solution to seal markers in the sample application pieces Pick up the application piece with forceps and apply to the top surface of the gel next to one end of the IPG strip The markers should contain 200 1000 ng of each component for Coomassie staining and about 10 50 ng of each component for silver staining Slowly moving the pipette from left to right across the gel deliver agarose sealing solution onto the IPG strip to seal it into place Carefully avoid bubbles when sealing with the agarose See Figure 17 Wait 2 5 minutes to allow the agarose to fully solidify before proceeding 29 Loading and Running Second Dimension Gels Hoefer DALT System Figure 17 Completely cover the IPG strip with agarose sealing solution 4 Keep a log of run conditions and the identification number of the DALT gel onto which each IPG strip is loaded 30 Amersham Bioscienc
19. the cassette with a minimum of mixing resulting in a smooth flat gel top surface Apply equal volumes of overlay to each gel to produce gels of consistent heights 2 Cover the top of the gel caster with plastic wrap and let the gels polymerize Polymerization Allow non gradient gels to polymerize for at least 1 hour allow gradient gels 2 hours 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 as the gels contract upon polymerization Amersham Biosciences Hoefer DALT System Amersham Biosciences Unloading the Gel Caster Unloading the Gel Caster 1 Remove the front of the gel caster The dense displacing solution will leak out into the tray or drain board beneath the casting unit Carefully unload the cassettes from the unit Pull forward on the separator sheets to easily separate gels Quickly rinse the top surface of each gel with water to remove n butanol and unpolymerized acrylamide Wash the cassette glass plates carefully with water and use a dish brush to remove any acrylamide adhering to the outer surfaces As each cassette is washed place it hinged side up in a dish rack standing in a plastic container with about 0 5 cm of tap water in the bottom The container retains the excess liquid as it drains from the surface of the gel The water in the container
20. the tank close the lid Figure 19 Center lid hinge Banana plug lead Banana plug 31 Loading and Running Second Dimension Gels Hoefer DALT System 4 Attach the electrical leads to make proper electrical contact with the power supply Migration proceeds toward the red or right chamber 5 Turn on the power supply to begin the separation Electrophoresis Conditions in the DALT Tank 1 Set the power supply to 100 125 V constant voltage for overnight runs or at 40 mA per gel for runs to be completed during the same day A set of ten 20 x 25 cm gels can be done in about 9 hours at maximum settings of 600 mA and 200 V Run times can vary widely depending on the acrylamide gradient or concentration the temperature and pH of the buffers and the number and size of cassettes Observe the progress of your first run and set future schedules accordingly 2 Run the gels until the blue tracking dye reaches the right side of the gel cassette the bottom of the gel as seen through the front of the DALT Tank The bromophenol blue tracking dye introduced into the IPG strip from the equilibration buffer or included in the agarose overlay migrates just ahead of the smallest proteins Figure 20 For consistency and reproducibility establish a specific finish line distance for the dye front It may be the actual bottom edge or a few millimeters before but when the blue line reaches that point the run is done Figure 2
21. 0 Tracking dye progresses from left to right Closed lid the DALT Tank i Red or right chamber Buffer level 32 Amersham Biosciences Hoefer DALT System Amersham Biosciences Loading and Running Second Dimension Gels Unloading the DALT Cassettes 1 When you are ready to remove the cassettes unplug the buffer circulation pump and the circulating water bath turn off the power supply and disconnect the power leads Open the DALT Tank lid only when the power supply is turned off and disconnected NOTE Wear gloves when handling gels that will be silver stained since fingerprints will show after staining Remove the cassettes carefully one at a time using both hands The cassettes are slippery because of the presence of SDS Rubber gloves may improve your grip on the glass Unload the cassettes into dish racks ten per rack that are placed in plastic containers of water to minimize gel drying Place a cassette on a flat surface and pry it open very carefully with a plastic wedge such as the Wonder Wedge Avoid metal wedges or spatulas which can easily chip the glass Put the stiff wedge end into the top comer of the cassette away from the hinge and twist it gently Take care not to chip the glass of the cassette When opening the cassette make sure that the gel adheres to one of the plates and is not sticking partly to both to avoid tearing the gel If the gel sticks to the plate with the space
22. 1 5 M solution 50 mM 6 67 ml Urea FW 60 06 6M 72 07 g Glycerol 87 v v MW 92 09 30 v v 69 ml SDS FW 288 38 2 w v 4 0g Bromophenol blue trace a few grains Double distilled H20 to 200 ml Store at 20 C This is a stock solution Add DTT or iodoacetamide before using Agarose Sealing Solution 25 mM Tris 192 mM glycine 0 1 SDS 0 5 agarose approximate pH 8 3 25 ml Final Conc Amount SDS Electrophoresis Buffer see above 25 ml Agarose 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 dissolved about 1 minute Do not allow the solution to boil over Allow the agarose to cool slightly before using Do not adjust pH The volume of sealing solution prepared from this recipe is sufficient for a complete set of second dimension DALT gels 43 44 Recipes Hoefer DALT System Towbin Buffer for DALT Blotting Tank 25 mM Tris 192 mM glycine approximate pH 8 3 22 liters optional 20 v v methanol Final Conc Amount Tris FW 121 14 25 mM 66 6 g Glycine FW 75 07 192 mM 317 1g Dissolve in 15 liters distilled water and mix in the DALT Tank itself by turning on the circulation motor Remove barrier combs Add methanol as required Alternatively mix in a carboy with a magnetic stirbar Optional Adding 0 1 SDS can improve transfer efficiency but may decr
23. 18 18 18 18 18 18 18 18 18 18 18 1096 APS 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 10 TEMED 441 3 85 342 3 08 2 79 2 57 2 38 2 20 2 05 1 93 1 82 1 71 1 62 12 gels 1 5 mm 1300 ml Amounts shown in ml Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acrylamide 257 293 330 367 403 440 447 513 550 587 623 660 697 Stock 1 5M Tris 275 275 275 275 275 275 275 275 275 275 275 275 275 CI pH 8 8 Water 544 507 471 434 398 361 325 288 252 215 179 142 105 1096 SDS 11 11 11 11 11 Ti 11 11 11 11 11 11 11 10 APS 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 10 TEMED 2 70 2 35 2 09 1 88 1 71 1 557 145 1 34 1 25 1 18 RIR 1 05 0 99 Amersham Biosciences Hoefer DALT System Gradient Gel Solutions Gradient Gel Solutions The concentrations of APS and TEMED in gradient solutions vary to assure top down polymerization We suggest for light solutions 0 025 0 09 v v TEMED and 0 1 w v APS for heavy solutions 0 0071 0 0028 v v TEMED and 0 05 w v APS The amounts of TEMED and APS suggested here are based on our experience You may want to change these volumes for your lab because of differences in temperature and reagent quality Perform a small scale test before first using a new composition to check that your light solution polymerizes in about 10 minutes and your heavy solution in about 20 minutes Vary the TEMED concentration to control the rate of polymerization
24. 2 38 10 SDS 4 4 4 4 4 4 4 4 4 4 4 4 4 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 4 4 4 4 4 4 4 4 4 4 4 4 4 10 TEMED 0 98 0 86 0 76 0 68 0 62 0 57 0 53 049 0 46 0 43 0 40 0 38 0 36 Heavy Solution Final T 8 9 10 11 12 13 14 15 16 17 18 19 20 Acryl Stock 107 120 133 147 160 173 187 200 213 227 240 253 267 1 5MTris Cl 100 100 100 100 100 100 100 100 100 100 100 100 100 pH 8 8 Water 160 147 133 120 107 93 80 67 53 40 27 13 0 10 SDS 4 4 4 4 4 4 4 4 4 4 4 4 4 Glycerol 27 27 27 27 27 27 27 27 27 27 27 27 27 1096 APS 2 00 2 00 2 00 2 00 2 00 2 00 2 00 2 00 2 00 2 00 2 00 2 00 2 00 10 TEMED 0 28 0 26 0 23 0 21 0 19 0 18 0 16 0 15 0 14 0 14 0 13 012 011 Amersham Biosciences 51 Gradient Gel Solutions Hoefer DALT System For 23 gels 1 5 mm 900 ml each Heavy and Light by volume in ml Light Solution Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 210 240 270 300 330 360 390 420 450 480 510 540 570 1 5MTris Cl 225 225 225 225 225 225 225 225 225 225 225 225 225 pH 8 8 Water 445 415 385 355 326 296 266 236 206 176 146 116 86 10 SDS 9 9 9 9 9 9 9 9 9 9 9 9 9 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 9 9 9 9 9 9 9 9 9 9 9 9 9 10 2 21 1 93 1 71 1 54 1 40 1 29 1 19 1 10 1 03 0 96 0 91 0 86 0 81 Heavy Solution Final T 8 9 10 11 12 13 14 15 16 17 18 19 20 Acryl Stock 240 270 300 330 360 390 420 450 480 510 540 570 600 1 5MTris Cl 225 225 225 225 225
25. 225 225 225 225 225 225 225 225 pH 8 8 Water 360 330 300 270 240 210 180 150 120 90 60 30 0 10 SDS 9 9 9 9 9 9 9 9 9 9 9 9 9 Glycerol 6l 61 6l ei 6l 61 6l 61 6l 61 6l 61 61 10 APS 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 4 50 10 TEMED 0 64 0 58 0 51 0 47 0 42 0 40 0 37 0 34 0 32 0 31 00 29 0 27 0 25 52 Amersham Biosciences Hoefer DALT System Gradient Gel Solutions For 12 gels 1 5 mm 550 ml each Heavy and Light by volume in ml Light Solution Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 128 147 165 183 202 220 238 257 275 293 312 330 348 1 5MTris Cl 138 138 138 138 138 138 138 138 138 138 138 138 138 pH 8 8 Water 272 254 235 217 199 181 162 144 126 108 89 71 53 10 SDS 55 ss 55 5 5 55 5 5 55 5 5 55 55 55 ss 55 0 0 0 0 0 0 0 0 0 0 0 0 0 1095 APS 55 55 55 55 55 5 5 55 ss 55 55 55 55 55 10 TEMED 1 35 1 35 1 35 1 35 135 135 1 35 135 1 35 135 1 35 1 35 1 35 Heavy Solution Final T 8 9 10 11 12 13 14 15 16 17 18 19 20 Acryl Stock 147 165 183 202 220 238 257 275 293 312 330 348 367 1 5MTris Cl 138 138 138 138 138 138 138 138 138 138 138 138 138 pH 8 8 Water 220 201 183 165 147 128 110 92 73 55 37 18 0 10 SDS 55 55 55 55 55 55 55 55 55 55 55 55 55 Glycerol o 37 NEM 37 DO c Me vc ERA v BO c E 1095 APS 275 275 275 275 275 275 275 275 275 275 275 275 275 10 TEMED 0393 0 35 0 31 0 29 0 26 0 24 0 28 0 21 019 0 19 0 18 017 0 15 A
26. HCI in place of Tris for the SDS electrophoresis buffer e Dispose of older acrylamide solutions and use only stock of the highest quality e Only use freshly deionized urea e To increase or decrease the migration rate adjust the voltage or current by 25 50 Protein spots are diffuse or broader than usual Use only high quality acrylamide and bis Ensure that polymerization is complete Fully equilibrate IPG strips before second dimension Incomplete IPG focusing Make sure the IPG strip rests on the gel surface without gouging or separating the gel Stained Sample Collects Near the buffer front Molecules are not sufficiently restricted by the resolving gel pore size increase the T e Proteins may be degraded by endogenous proteases use protease inhibitors during sample separation e Adjust the pH of 1 5 M Tris Cl to pH 8 8 Samples migrate faster when pH 8 8 Near the top of the gel when the buffer front has reached the bottom The gel pore size is too small Decrease the T of the resolving or stacking gel e Adjust the pH of 1 5 M Tris Cl to pH 8 8 Samples migrate slower when pH gt 8 8 At each end of the gel The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the full range of proteins Amersham Biosciences 57 Troubleshooting Hoefer DALT System Poor Spot Resolution Allow gel to polymerize fully Begin electrophoresis as soon as the IPG strips are loaded to p
27. Ordering information Index Hoefer DALT System 38 39 39 40 41 46 49 50 51 52 53 54 55 55 55 56 60 61 61 61 63 Amersham Biosciences Hoefer DALT System Figure 1 The DALT Electrophoresis Tank Amersham Biosciences Hoefer DALT System Function and Description Hoefer DALT System Function and Description In 2 D protein electrophoresis proteins are separated first according to isoelectric point by isoelectric focusing most reliably on immobilized pH gradient IPG strips The second dimension electrophoresis separates the proteins on the basis of their molecular mass on a slab gel containing the denaturing detergent sodium dodecyl sulfate SDS The Hoefer DALT System is designed to simplify the handling of multiple second dimension gels and improve the reproducibility of the second dimension separation DALT System Components The Hoefer DALT System comprises amultiple vertical slab electrophoresis tank amultiple gel caster agradient maker with peristaltic pump gel cassettes e blotting kit Electrophoresis Tank Hinged lid B Platinum core electrode panels two Barrier combs two with silicone rubber flap seals Buffer circulation flutes Alumina heat exchanger Siphon pump included but not shown The DALT Electrophoresis Tank accommodates up to ten 23 x 19 cm slab gels for separation under identical conditions The sample following focusing in
28. acked heat exchanger can leak coolant into the tank or buffer out of the tank either of which might be hazardous Connecting a Refrigerated Circulating Bath To maintain a constant buffer temperature connect a refrigerated circulating bath such as the MultiTemp III to the heat exchanger fittings at the back of the DALT Tank The pump in the bath circulates chilled liquid through the alumina heat exchanger The pump on the DALT Tank circulates the buffer in the central chamber over the heat exchanger and the gel cassettes ensuring even gel temperature during the run 1 Prepare two lengths of vinyl or silicone tubing Slide hose clamps 4 total onto each end of two lengths of tubing Attach one end of each length of tubing to the Quick fit connector attached to the heat exchanger port Attach the free ends of each length of tubing to the circulator bath ports one to the inlet and the other to the outlet Secure the connections to the Quick fit connectors and the circulating water bath with the hose clamps 2 Setthe circulating bath temperature to 10 C IMPORTANT Thecirculator pump must not generate a pressure greater than 0 7 bar 10 psi above atmospheric pressure If you use a temperature below 10 C use a 50 50 water ethylene glycol solution in the circulating bath Do not use any organic solvent other than 50 50 ethylene glycol water as a coolant Most solvents including methanol and isopropanol will craze the plastic parts
29. amide the pH of the equilibration solution is 8 8 to compensate for this acid production Tracking dye bromophenol blue allows monitoring of electrophoresis Equilibration Steps Use the gel number labels that were polymerized into the separating gel to help orient the IPG strips Conventionally the acidic or pointed end of the IPG strip is on the left or label side NOTE To avoid protein contamination wear gloves when handling the IPG strip and hold only the protruding end of the gel backing film when moving the strip 1 Prepare SDS equilibration buffer see page 43 Just prior to use add DTT to the buffer at a concentration of 100 mg DTT per 10 ml SDS equilibration buffer 2 Place the IPG strips in individual tubes with the support film toward the wall Screw cap culture tubes 25 x 200 mm work well 3 Add 10 ml of the DTT containing solution to each tube Cap the tube or seal it with flexible paraffin film and place is on its side on a rocker 4 Equilibrate for 10 15 minutes Do not over equilibrate as proteins can diffuse out of the strip during this step An optional second 10 min equilibration with iodoacetamide will alkylate sulfhydryl groups and prevent disulfide reformation The second equilibration also removes excess DTT which can lead to vertical point streaking when silver staining 5 Second Equilibrium optional Prepare a solution of 25 mg iodoacetamide per 10 ml SDS equilibration buffer Ad
30. an IPG strip is attached to the cathodic surface of the gel by embedding in agarose then the gel is turned 90 and run in a sideways orientation with the IPG strip standing vertically at one edge The tank is divided into three chambers by silicone rubber flap seals on the barrier combs The seals are not designed to be liquid tight but they do provide a good barrier to electrical current causing most current to flow through the gels Although there is Hoefer DALT System Function and Description Hoefer DALT System Figure 2 Rear view of electrophoresis tank some leakage current by passing the gels this has little effect on system operation The barriers can be raised or removed by loosening the nylon screws which secure them in place The left and right chambers contain platinum wire electrodes cathode and anode The center chamber provides cooling via circulating buffer and a tube type heat exchanger Since the gel cassettes are almost entirely exposed to the buffer in the center chamber with only their ends protruding into the side chambers cooling of the center chamber buffer provides excellent temperature control of the gel slabs during the run Electrode panels can be removed by loosening the nylon screws securing them in place Removal is not typically necessary unless the platinum wire becomes broken and requires replacement See Removing the Electrode Panels from the DALT Tank on page 55 Pump shield with f
31. antes Specifications Required or Convenient But not Supplied Preparing the Gel Caster Casting Homogeneous Non Gradient Gels Preparation for Gradient Gel Casting Configuring the Gradient Divider Calibrating the Peristaltic Pump Casting Gradient Gels Gradient Casting Setup Pouring Gel Solutions for Gradient Gels Applying Overlay to DALT Slab Gels Polymerization Unloading the Gel Caster Preparing the DALT Tank for Electrophoresis Connecting a Refrigerated Circulating Bath Filling the Tank with SDS Electrophoresis Buffer Loading and Running Second Dimension Gels Equilibrating IPG Strips Loading the IPG Strips onto the DALT Slab Gels Loading Cassettes into the DALT Tank Electrophoresis Conditions in the DALT Tank Unloading the DALT Cassettes Cleaning DALT Cassettes The DALT Blotting Kit Preparing the DALT Tank for Transfer Assemble the Transfer Cassette Loading the Cassettes CON U 12 14 16 16 17 18 18 19 22 22 23 24 24 24 27 27 29 31 32 33 33 34 34 35 37 Connecting the Power Supply Transfer Conditions Completing the Transfer Factors Affecting the Transfer Recipes Homogeneous Gel Solutions Gradient Gel Solutions For 25 gels 1 mm For 12 gels 1 mm For 23 gels 1 5 mm For 12 gels 1 5 mm Gel Identification Numbers Care and Maintenance Cleaning Removing the Electrode Panels from the DALT Tank Troubleshooting References Customer Service Information Technical Service and Repair
32. bes 3 Heat six of the tubes for 4 6 8 10 12 and 15 minutes at 95 C in a heating block or in a boiling water bath After heating place tubes in an ice bucket 4 Mix the contents of the 7 tubes together and aliquot 50 uL of the pool into small microcentrifuge tubes for storage at 70 C 5 Thaw out a tube for each experiment and load 2 uL CK mix per IPG strip During electrophoresis the CK charge standards produce a carbamylation chain of spots separated according to pl dar ss RR de ae de ait db 4 45 Homogeneous Gel Solutions Hoefer DALT System Homogeneous Gel Solutions 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 change these volumes for your lab because of differences in temperature and reagent quality Perform a small scale test before first using a new composition to check that your solution polymerizes in about 10 minutes 1000 ml Amounts shown in ml Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 233 267 300 333 367 400 433 467 500 533 567 600 633 1 5MTris Cl 250 250 250 250 250 250 250 250 250 250 250 250 250 pH 8 8 Water 494 461 428 395 362 329 295 262 229 196 162 129 9 10 SDS I 10 Mos 10 BOM 10 EUN 10 10 BV 10 EIC 10 APS 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 00 10 TEMED 245 2 14 1 90 1 71 1 55 143 1 32 122 114 107 1 01 0 95 0 90 46 Am
33. d 10 ml of solution to each tube containing an IPG strip Cap the tube or seal it with flexible paraffin film and place is on its side on a rocker to equilibrate for 10 minutes IMPORTANT After equilibration load IPG strips directly onto the DALT slab gels as soon as possible Amersham Biosciences Hoefer DALT System Figure 16 For reference place the pointed end of the IPG strip above the gel label Amersham Bioscienes Loading and Running Second Dimension Gels Loading the IPG Strips onto the DALT Slab Gels Place the DALT gels in the dish rack in alphabetical numerical order with respect to the identification label with the sample application surface of the slab up and the label readable from the front Be sure to record gel identification numbers 1 Dip the equilibrated IPG strip in SDS electrophoresis buffer to lubricate it then place the IPG gel strip onto the DALT gel cassette See Figure 16 Position the IPG strip between the plates touching the surface of the second dimension gel with the plastic backing against one of the glass plates For a convenient reference place the pointed acidic end of the IPG strip on the same side as the gel label Pointed acidic end on same end as gel label Use a thin plastic spatula or ruler to push against the plastic backing of the IPG strip not the gel itself and move the strip down into contact with the surface of the second dimension gel The edge
34. d to it replace with fresh stock Solutions with extreme pH values especially acidic may not polymerize Remove oxygen from the gel environment Degas the monomer solution 5 minutes before pouring and then overlay the gel surface with water saturated n butanol Adjust the gel solution temperature to a minimum of 20 C especially for low T gels Increase both APS and TEMED by 30 50 Make up fresh APS daily Gel is too soft too brittle or white e Adjust crosslinker concentration Crosslinker should be at 2 6 C for standard SDS gels where C g bis x 100 g monomer g bis Gel contains swirls e f gel polymerized in 10 min too much catalyst Reduce concentration of ammonium persulfate and TEMED by 25 e f gel polymerized in gt 50 min not enough catalyst Increase concentration of ammonium persulfate and TEMED by 50 e Make up fresh acrylamide stock solution Dye front curves up smiles at the edges e Fill the lower buffer chamber to the level appropriate for the run e Circulate coolant e Pre chill the buffer e Decrease the current or voltage setting Vertical protein streaks e IPG Strip not properly placed on gel surface Avoid gouging separating gel while loading strips e Perform iodoacetamide treatment e Make sure IPG strip uniformly contacts the gel surface along the entire length of the strip Gels cast simultaneously are different sizes Wait one minute before overlayi
35. dient in hydrostatic equilibrium with the feed tube removed Displacing solution fills the sloped trough at the bottom of the caster Amersham Biosciences 10 11 12 15 Displacing solution 14 15 16 Casting Gradient Gels Light solution should begin to flow through the feed tube and mixer towards 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 a level 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 within 4 cm from the top of the cassettes or the gradient maker is empty whichever comes first turn off the pump and close the feed tube clamp c Stop the pump before air enters the feed tube Pull the gradient feed tube out of the balance chamber grommet Place its end in a waste container to collect 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 casettes is more than 1 cm below the top of the cassettes you may add up to 50 ml more displacing solution to the balance chamber The gradient is now in hydrostatic equilibrium in the unit ready to polymer
36. ditions were developed using 12 T gels 193 mM glycine 25 mM Tris 20 methanol 0 1 SDS Transfers were made onto nitrocellulose A refrigerated circulating water bath set at 4 C was used for all transfers 1000 mA for 2 3 h For rapid transfer of proteins M lt 70 k 1000 mA for 5 6 h For moderate transfer of proteins M lt 100 k 400 mA for 18 h For most efficient transfer of high and low molecular weight proteins Completing the Transfer 1 When the transfer is complete turn the voltage and current settings to zero and turn off the power supply Disconnect the leads from the power supply jacks 2 Open the lid and lift out the cassettes 3 Open each cassette carefully and remove the gels Lift the membranes with blunt forceps Discard the blotter paper Rinse the sponges with ddH O and reuse them indefinitely 4 With a soft lead pencil label each membrane and indicate the sample side 5 Rinse the unit immediately after use 39 The DALT Blotting Kit 40 Hoefer DALT System Factors Affecting the Transfer Sample characteristics membrane type gel pore size and the transfer buffer used affect the efficiency of macromolecule transfers The most widely used buffer system for transferring proteins is that of Towbin et al Conditions required for efficient elution may not coincide with optimal conditions for binding To find the optimum conditions for transferring your sample balance these effects
37. e If the sample elution rate is slow a longer transfer period may be required In our experience high current transfers for short periods of time are superior to low voltage transfers for longer periods e If sample binding is inadequate try different buffer conditions methanol or SDS concentration For a comprehensive review see Gershoni and Palade 1983 If the transfer buffer system is different from the electrophoresis buffer system the gel should be equilibrated briefly with the transfer buffer before the transfer to assure swelling or shrinking occurs before the gel contacts the transfer membrane If this step is skipped band distortion or loss of resolution could result Amersham Biosciences Hoefer DALT System Amersham Biosciences Recipes Recipes DALT Acrylamide Stock 30 8 T WARNING Acrylamide is a neurotoxin Always use mechanical pipettes and wear gloves when working with acrylamide solutions Final Conc Amount Acrylamide best affordable grade MW 71 08 30 900 g Bis N N methylenebis acrylamide purest grade FW 154 17 0 8 24g Water purest available up to 3000 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 1 5 M 545g 6N HCI to pH 8 8 about 150 ml Water to 3000 ml Adjust to pH 8 8 store at 4 C 1096 SDS Final Conc Amount Sodium dodecylsulfate MW 288 38 10
38. e Only accessories and parts approved or supplied by Amersham Biosciences may be used for operating maintaining and servicing this product Hoefer DALT System Informations Importantes e Raccorder l instrument une prise de terre appropri e e Le couvercle de s curit doit tre en place avant de brancher les prises au g n rateur e Eteindre le g n rateur et d brancher les prises avant d ouvrir le couvercle de s curit e Rinser seulement les lectrodes pas les banana plugs avec de l eau distill e avant et apr s l utilisation e Toujours d connecter le cordon d alimentation avant de r parer l instrument e Ne pas faire fonctionner la pompe de circulation tampon lorsque la cuve d lectrophor se DALT est vide La pompe ne s amorce pas automatiquement et peut tre endommag e si elle tourne sec e Faire circuler seulement de l eau ou 50 50 d eau et d thyl ne glycol dans l changeur vertical cirulation d eau Ne jamais utiliser d anti gel ou tout autre solvant organique avec cet instrument Les solvants organiques causeraient des dommages irr parables l appareil e Ne pas connecter l changeur vertical circulation d eau un robinet ou quelque source de refroidissement dont la pression n est pas r guli re e Ne pas utiliser avec un tampon une temp rature au dessus de 45 C Toutes les pi ces en plastique sont pr vues pour r sister une temp rature constante de 45 C Faire
39. ease binding to the membrane The pH of this buffer may vary from 8 2 8 4 Do not adjust the pH Adjusting the pH alters the conductivity of the buffer Depending on the membrane type adding 10 20 methanol can improve protein binding and reduce gel swelling during transfer Buffers containing methanol may deteriorate if stored for long periods Towbin Buffer for Transfer Cassette Assembly 25 mM Tris 192 mM glycine approximate pH 8 3 4 liters optional 20 v v methanol Final Conc Amount Tris FW 121 14 25 mM 12 11g Glycine FW 75 07 192 mM 57 65 Dissolve in 3 liters distilled water and mix in a carboy with a magnetic stirbar Add methanol as required Bring to 4 liters with water The pH of this buffer may vary from 8 2 8 4 Do not adjust the pH Adjusting the pH alters the conductivity of the buffer If recommended by the membrane manufacturer you may add 10 20 methanol Amersham Biosciences Hoefer DALT System Figure 23 An example of electrophoresis of CK charge standards for testing pl separation Amersham Biosciences Recipes Preparation of Creatine Kinase CK Charge Standards Use CK charge standards to evaluate first dimension isoelectric focusing and second dimension separation 1 Dissolve 5 mg of rabbit muscle creatine phosphokinase Sigma in 1 ml of a solution of 8 M urea and 1 mercaptoethanol to give a CK concentration of 5 mg ml 2 Aliquot the above mixture into 7 tu
40. en you open it to remove the gels Alternatively place the unit on a plastic or rubber kitchen drain board that empties into a sink The DALT Gel Caster can accommodate up to twenty five 1 mm or twenty three 1 5 mm gel cassettes with separator sheets between each cassette before the first cassette and after the last cassette If you are not preparing the maximum number of gels use filler blocks and separator sheets to take up the excess caster volume Place separator sheets between the filler blocks Cast at least 22 cassettes for two 10 sample runs to allow for the possibility of one or two imperfect gels 1 Check that the caster is level Remove the front plate and rest the caster on its back with its feet facing you Place the triangular sponge in the base of the V shaped feed channel Plates are more easily loaded in this orientation Check that the caster is clean and free of dust 2 Lubricate the foam sealing gasket with a light coating of GelSeal to help assure leak proof sealing Place the gasket in the groove along the front side of the caster Avoid stretching the gasket 3 Start filling the gel caster by placing a separator sheet against the inside wall to make it easier to remove the cassettes and filler blocks after polymerization If you are casting fewer than the maximum number of gels first add a selection of filler blocks with a separator sheet between each block For example to cast twelve 1 0 m
41. ersham Biosciences Hoefer DALT System 25 gels 1 0 mm 1500 ml Amounts shown in ml Homogeneous Gel Solutions Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 350 400 450 500 550 600 650 700 750 800 850 900 950 1 5MTris Cl 375 375 375 375 375 375 375 375 375 375 375 375 375 pH 8 8 Water 741 692 642 592 544 493 443 393 343 293 243 194 144 3 10 SDS 15 15 15 15 15 15 15 15 15 15 15 15 l5 10 APS 15 00 15 00 15 00 15 50 15 00 15 00 15 00 15 00 15 00 15 00 15 00 15 00 15 00 10 TEMED 3 68 3 21 285 2 57 233 215 1 98 1 83 171 161 1 52 143 1 35 12 gels 1 0 mm 800 ml Amounts shown in ml Final T 7 8 9 10 dl 12 13 14 15 16 17 18 19 Acrylamide 187 213 240 267 293 320 347 373 400 427 453 480 507 Stock 1 5M Tris 200 200 200 200 200 200 200 200 200 200 200 200 200 CI pH 8 8 Water 395 369 342 316 289 263 236 210 183 156 130 103 77 10 SDS 8 8 8 8 8 8 8 8 8 8 8 8 8 10 APS 8 00 8 00 8 00 8 00 8 00 8 00 8 00 8 00 8 00 8 00 8 00 8 00 8 00 10 TEMED 1 96 1 71 1552 1 37 1 24 1 14 1 06 0 98 0 91 0 86 0 81 0 76 0 72 Amersham Biosciences 47 Homogeneous Gel Solutions 23 gels 1 5 mm 1800 ml Amounts shown in ml Hoefer DALT System Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acrylamide 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 Stock 1 5M Tris 450 450 450 450 450 450 450 450 450 450 450 450 450 8 8 Water 890 830 771 711 651 591 532 472 412 352 292 232 172 1096 SDS 18 18
42. es Hoefer DALT System Figure 18 Inserting loaded cassette into DALT Tank Figure 19 Lift the lid slightly at its center hinge to make sure the banana leads make contact with the recessed plugs Amersham Biosciences Loading and Running Second Dimension Gels Loading Cassettes into the DALT Tank 1 Carefully load the cassettes after the agarose overlay has fully solidified The cassettes are correctly loaded in running orientation in the DALT Tank slot with the IPG strips vertical along the left or cathode side and the rubber cassette hinge along the bottom Figure 18 Dip the hinge side of the cassette into the tank buffer first to lubricate it before inserting it into the flap seals Use both hands to slide cassettes firmly to the bottom Be careful The plates slip easily once your hands are immersed in tank buffer Do not drop the plates into the tank and onto the circulation flutes Cassette IPG strip A E RAAT Barrier comb Hinge Buffer circulation flutes Adjust the buffer level after all the cassettes are loaded in position IMPORTANT The tank buffer level should be even with the uppermost spacer of the cassette and neither above the top of the cassette nor below the level of the top edge of the gel If the level is too low or too high add SDS electrophoresis buffer or use the siphon pump to correct the buffer level After the last cassette has been introduced into
43. es Limited All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form by any means without permission in written form from the company Amersham Biosciences Renseignements importants d utilization Pour une bonne compr hension et une utilisation en s curit maximale il convient de lire enti rement ce manuel Francais mation dans un triangle quilat ral a pour but d attirer l attention de l utilisateur sur des instructions importantes de fonctionnment ou de maintenance Dans la documentation qui accompagne l instrument un point d excla q pag P Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsahilit Amersham Biosciences garantit l utilisateur que le produit livr a subi avec succ s tous les essais pr vus pour s assurer qu il est conforme aux sp cifica tions et normes en vigueur La garantie incluse dans les conditions de livraison n est valable que si le produit a t install et utilis conform ment aux instruc tions fournies par Amersham Biosciences La soci t Amersham Biosciences ne sera en aucun cas responsable de tout dommage caus directement ou indirecteme
44. esota Street San Francisco CA 94107 USA Amersham Biosciences beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and Haftung Amersham Biosciences garantiert da das gelieferte Produkt sorgf ltig auf die Einhaltung der ver ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gew hrleistungsanspr che gelten nur dann wenn das Produkt gem den von Amersham Biosciences gelieferten Anweisungen installiert und benutzt wurde Amersham Biosciencces bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbufen Einkom mensverluste entgangene Gesch ftsabschl sse Verlust der Gebrauchsf higkeit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verursacht wurden O 1998 Amersham Biosciences Limited Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorhergehender schriftlicher Gene hmigung durch das Unternehmen vervielf ltigt in einem Abrufsystem gespe ichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attentam ente l intero contenuto del presente manuale AN Si prega di inviare eventuali commenti al presente manuale a Italiano Il punto esclamativo all interno di un
45. 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 Figure 11 19 Casting Gradient Gels Figure 11 Priming with light solution Figure 12 Both chambers of the gradient maker filled and the balance chamber of the casting box loaded with overlay solution 20 Hoefer DALT System A A 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 side balance chamber with 150 ml dense displacing solution Figure 12 The grommet seal and gradient feed tube should prevent leakage of displacing solution into the caster ight solution AA Open the clamp after the mixer to open the feed tube 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 Amersham Biosciences Hoefer DALT System Figure 13 The gra
46. fondo para comprobar el cumplimiento de las especificaciones publica das La garant a incluida en las condiciones de entrega s lo es v lida si el producto se ha instalado y utilizado de acuerdo con las instrucciones entregadas por Amersham Biosciences Amersham Biosciences no ser responsable bajo ning n concepto de da os directos o indirectos incluyendo sin limitaci n la p rdida de beneficios la p rdida de ingresos la p rdida de oportunidades de negocio la p rdida de uti lizaci n y otras consecuencias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto O 1998 Amersham Biosciences Limited Reservados todos los derechos No est permitida la reproducci n ni el almacenaje en un sistema de recuper aci n ni la transmisi n de parte alguna de esta publicaci n sin la autorizaci n por escrito de la empresa Hoefer DALT System Wichtige Benutzerinformationen F r ein vollst ndiges Verst ndnis und eine sichere Handhabung dieses Produktes ist es notwendig dafs der Benutzer dieses Handbuch vollst ndig durchliest AN Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Deutsch Ein Ausrufezeichen in einem gleichseitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und Wartungsan weisungen in der dem Ger t beiliegenden Dokumentation hin weisen Amersham Biosciences Inc Marketing Department 654 Minn
47. fter each use Allow the unit to air dry completely Periodically wash with a dilute solution of a mild detergent Clean gaskets with mild detergent and rinse with distilled water Allow to air dry Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS 35 from Pierce Chemical Company Rinse thoroughly with tap and distilled water Glass plates can also be treated with but not stored in acid cleaning solutions Removing the Electrode Panels from the DALT Tank You can remove each electrode panel for more thorough cleaning or to replace damaged electrodes 1 Usea Phillip s head screwdriver to loosen the white plastic retaining screws on the exterior of the DALT Tank You do not need to remove the screws 2 Lift and slide the panel out Electrode panel Retaining screws Handle the electrode panel carefully to avoid stretching or breaking the platinum wire 55 Troubleshooting Hoefer DALT System Troubleshooting Gel Casting Gel caster leaks Apply a light film of GelSeal to the foam gasket each time the unit is used e Check the foam gasket for cracks or nicks and replace if necessary Ifthe stack is too tall the front plate may not seat firmly against the gasket Remove filler plates or cassettes until the gasket seals Incomplete gel polymerization Use only recent stock of the highest quality reagents e dry ammonium persulfate does not crackle when water is adde
48. gradient gel requirements Amersham Biosciences Hoefer DALT System Figure 6 The DALT Blotting Kit Amersham Biosciences Hoefer DALT System Function and Description Blotting Kit The DALT Blotting Kit consists of a five place acrylic cassette holding rack and five transfer cassettes complete with cassette packing material sponges and blotting paper The cassettes hold the gel and the transfer membrane secure with just enough pressure to assure even transfer Unpacking Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local Amersham Biosciences 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 Rinse the DALT Tank and Gel Caster with distilled water and let air dry Before initial use carefully clean glass plates and spacers with a dilute solution of laboratory cleanser to remove fingerprints Rinse thoroughly with tap and distilled water See Care and Maintenance on page 55 for more detailed instructions Important Information e Plug the instruments into a properly grounded outlet e safety lid must be in place before connecting the power leads to a power supply e Turn all power supply contr
49. gth of the electrodes If bubbles do not form along the entire length of the electrode replace the electrode Grid pattern on membrane Add sheets of blotting paper to increase the clearance between the cassette panel and the gel Take care not to overstuff the cassette The gel should be held firmly and evenly between the sponges but not so tightly that it is squeezed Molecules do not migrate out of gel Increase the field strength Increase or double the transfer period Donotuse staining or fixing agents on the gel before transfer Useathinner gel Reduce the gel acrylamide concentration e Check that the buffer pH is close to the intended pH Most buffers should not be titrated Make fresh buffer Use 3 5 mM SDS 0 1 in the transfer buffer Use reagent grade chemicals Increase the net charge on the protein by changing to a transfer buffer with a different pH Lower pH lt 6 increases the positive charge on proteins Higher pH gt 6 increases the negative charge on proteins 58 Amersham Biosciences Hoefer DALT System Amersham Biosciences Troubleshooting Diffuse band patterns Transfer immediately after electrophoretic separation If equilibrating before the transfer shorten or eliminate the equilibration time or move the gel to the cold room during equilibration If transfer buffer contains methanol gt 10 equilibrate the gel before in transfer buffer for 30 minutes to allow it to sh
50. helps maintain humidity near the gels A dry gel breaks and is useless for electrophoresis Remove and clean the thin separator sheets Examine the gels for air spaces uneven top surfaces or other defects and discard any unsatisfactory gels Put the extra good gels in gel storage solution see page 42 at 4 C for later use Extra gels may also be stored by wrapping the cassettes individually in plastic wrap and putting them in a sealed plastic box with several milliliters of diluted Tris buffer Take the foam funnel out of the bottom of the gel caster and rinse with water to remove displacing solution Rinse the gel caster and all tubing with water and mild detergent followed by a deionized water rinse 23 Preparing the DALT Tank for Electrophoresis Hoefer DALT System Note For quick and easy connections install Quick fit connectors with valves in the line 24 Preparing the DALT Tank for Electrophoresis NOTE Position the DALT Tank near a sink for easy rinsing and draining A magnetically coupled centrifugal pump mounted on the back of the DALT Tank provides buffer circulation across the heat exchanger tubes and the gels in the center chamber IMPORTANT Never lift or move the tank by holding the pump or the tubing at the back of the tank The heat exchanger consists of four ceramic tubes below the buffer circulation flutes It is quite robust but dropping a heavy sharp object into the tank could damage it A cr
51. ient pump flow rate See Calibrating the Peristaltic Pump on page 17 Be sure that the faceplate screws on the gradient maker are tightened hand tight and the gradient maker lines are all clamped off There are three clamps one coming from each chamber and one after the bow tie mixer Close all three Prepare a sufficient volume of gel overlay solution water saturated n butanol You need 0 75 ml of overlay for each cassette or about 20 ml for a set of 25 cassettes Make up 200 ml of displacing solution page 42 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 tetramethylethylene diamine TEMED See Gradient Gel Solutions on page 49 Pouring Gel Solutions for Gradient Gels Prepare the gel caster as described on page 12 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 minutes to cast the gradient before 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 wider at the top Light in Right Fill the tubing between the light and heavy chambers with light solution Carefully open the clamp on the light chamber
52. is correctly adjusted and you are comfortable with the gel casting procedure clean all parts of the caster and gradient maker including sponges separating sheets and filler blocks with a solution of mild detergent followed by a fresh water rinse Configuring the Gradient Divider You can adjust the shape of the gradient divider to suit the number of gels to be cast and 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 70 For fewer gels decrease the divider angle Figure 9 Whatever angle you use a straight divider gives a linear gradient Divider angle approx 40 50 60 70 Number thickness 12 gels 1 0 cm 12 gels 1 5 cm 25 gels 1 0 cm 23 gels 1 5 cm 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 configurations in Figure 9 as a guideline to 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 Amersham Biosciences Hoefer DALT System Amersham Biosciences Preparation for Gradient Gel Casting For a linear gradient straighten the divider with its upper end almost touching the left wall at the height determined by the volume of water To
53. ize Figure 13 Apply overlay as soon as possible page 22 Water Y VES Quickly reopen clamp c and restart the pump to empty the gradient maker completely of any excess polymerizing acrylamide from the gradient maker 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 about a liter of water into each chamber of the gradient maker and open all clamps Start the pump to flush the system Flush 2 liters more water through the gradient maker and tubing 21 Casting Gradient Gels 22 Hoefer DALT System Applying Overlay to DALT Slab Gels The flatness of the top gel surface is a major determinant of the quality and resolution of SDS slab gels Imperfect gel tops can lead to irreproducible protein spot 2 D gel patterns A convex or tilted slab top surface can give rise to double spots as protein at the front and back gel surfaces starts moving at different points To avoid this quickly and carefully pipette identical volumes of water saturated n butanol overlay solution onto each gel This results in very flat slab gel tops 1 Immediately after removing the feed tube from the caster slowly deliver 0 75 ml of water saturated n butanol to the surface of each gel The overlay should spread evenly across
54. lectroendosmosis occurs due to the presence of fixed the charges of the IPG matrix in the electric field and can interfere with protein transfer from the IPG strip to the second dimension gel Glycerol 30 together with urea reduces electroendosmosis and improves transfer of protein from the first to the second dimension Dithiothreitol DTT preserves the fully reduces state of denatured unalkylated proteins Sodium dodecyl sulfate SDS denatures proteins and forms negatively charged protein SDS complexes The amount of SDS bound to protein and therefore the additional negative charge is directly proportional to the mass of the protein Thus electrophoresis of proteins through a sieving gel in the presence of SDS separates proteins on the basis of molecular mass 27 Loading and Running Second Dimension Gels Hoefer DALT System Note It takes approximately 10 minutes to prepare Agarose Sealing Solution page 43 Prepare the solution during IPG equilibration 28 lodoacetamide alkylates sulfhydryl groups on proteins preventing their re oxidation during electrophoresis Protein re oxidation during electrophoresis can result in streaking and other artifacts lodoacetamide also alkylates residual DTT to prevent point streaking and other silver staining artifacts lodoacetamide is introduced in an optional second equilibration step NOTE The reaction between iodoacetamide and DTT creates hydroiodic acid When using iodoacet
55. lled by circulating cooled liquid through the heat exchanger in the base Coolant is pumped through the alumina heat exchanger located at the base of the unit Temperature is maintained by buffer circulation through tubes in the base of the tank We strongly recommend connecting a constant temperature circulation bath to the heat exchanger to maintain the correct temperature during transfer A set temperature between 20 and 30 C is recommended although lower temperatures can also be used Never operate the unit for more than one hour under high power conditions gt 250 mA without active cooling See page 44 for the recipe for Towbin Buffer Depending on the membrane type and the manufacturer s recommendations you may add 0 196 SDS and 10 20 methanol Preparing the DALT Tank for Transfer IMPORTANT Gels should be transferred immediately after electrophoresis to avoid sample diffusion Do not soak gels in fixative Equilibrate gels with transfer buffer before they are placed in the cassette if necessary 1 Configure the unit for active cooling See Connecting a Refrigerated Circulating Bath on page 24 Active cooling is optional but strongly recommended Start the circulating bath at the same time as the transfer The circulator pump must not generate a pressure greater than 0 7 bar 10 psi above atmospheric pressure 2 Loosen the white nylon screws on the exterior of the DALT Electrophoresis Tank and lift out the ba
56. m gels you may need four 25 mm blocks and one 3 mm block with separator sheets between each block To cast twelve 1 5 mm gels you may need three 25 mm blocks one 12 mm block one 6 mm block and one 3 mm block with separator sheets between each block Next add the cassettes with all hinge strips vertical and aligned on the side of the unit opposite the feed tube Insert a gel separator sheet between each cassette pair End with a separator sheet between the final cassette and the gel caster face plate If necessary use additional separator sheets to bring the level of the stack even with the edge of the caster 4 Put the removable front plate of the gel caster in place and screw on the black knurled knobs Tighten these hand tight not over tight then carefully place the gel caster in an upright position Be sure the sealing gasket on the front plate forms a tight seal against the face plate Amersham Biosciences Hoefer DALT System Figure 7 Attaching the feed tube to the gel caster Amersham Biosciences Preparing the Gel Caster Prepare a set of gel labels on filter paper as described on page 53 Cut the labels apart leaving little excess around the characters and place them in order in front of the gel caster Then taking care to keep track of which cassette will be numbered next drop the numbers in order into the cassettes on the side opposite the gradient inlet port As the gradient is introduced the number
57. mersham Biosdences 53 Gel Identification Numbers 54 Hoefer DALT System Gel Identification Numbers For positive identification of gels label each slab by incorporating a small label printed on thin filter paper in the bottom corner of the gel Use a carbon typewriter ribbon photocopier or laser printer to make these labels since many liquid based inks are electrophoresed off paper during an SDS electrophoresis run A variety of numbering schemes are possible In our experience the easiest uses three parts as follows e An upper case letter to identify the investigator or an extended gel series e Atwo or three digit serial number to identify the slab gel batch e lower case letter to identify a gel in the batch Since a maximum of 22 gels can be made in a batch use the letters a v The resulting numbers in the format A63a A63b etc provide a useful system for keeping track of and cross indexing experiments and gel production Amersham Biosciences Hoefer DALT System Figure 24 Removing the electrode panels Amersham Biosciences Care and Maintenance Care and Maintenance Cleaning e Do not autoclave or heat any part above 45 C e Do not expose the unit or its parts to organic solvents e If using radioactive reagents decontaminate the unit with a cleaning agent such as CONTRAD 70 or Decon 90 from Decon Laboratories Inc Rinse the DALT Tank cassettes and sponges with distilled water immediately a
58. ml min for 12 gels 1 2 Place the inlet side of the tubing in a beaker that contains one liter of water Place the outlet side of the tubing in a 1 liter graduated cylinder Set the flow speed at approximately 3 3 5 on the dial and start the pump Stop the flow of liquid after exactly 2 minutes 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 times the flow speed used in step 3 Example You want to cast a full tank of gels and determine the flow rate is 500 ml min when you set the flow rate at 3 For a flow rate of 440 ml min 440 500 x 3 2 6 the appropriate flow rate setting to deliver 440 ml min 17 Casting Gradient Gels Figure 10 The pump assisted gradient maker 18 Hoefer DALT System Casting Gradient Gels The gradient maker is a simple unit with two chambers defined by a silicone rubber gasket clamped between two acrylic plates The chambers are separated by a movable divider which you can modify to define the shape of the gel gradient Solutions flow out of the two chambers join at the Y connector and then thoroughly mix in a bow tie in line pipe 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 pe
59. n t catch and no circulation is observed you have an airlock in the pump Quickly turn off the pump Wait a moment for the air to bubble out through the small bypass tube that comes up from the pump outlet and enters the back of the tank about halfway up then restart the pump You may have to do this a few times Once the pump is circulating leave it on 3 Weigh out the correct SDS electrophoresis buffer mixture See page 42 Add the dry powder directly to the water in the center chamber distributing it evenly from front to back The pump circulates the contents enough to dissolve the solids in about 1 2 hours Some solids may lie on the bottom for awhile but they gradually disappear You may want to make a number of these buffer packets in 1 liter plastic bottles and have them on hand 4 Once the buffer is dissolved use a Phillip s head screwdriver to loosen the white plastic retaining screws on the exterior of the DALT Tank that hold the barrier combs in place Raise or remove the two barrier combs to mix the total DALT Tank contents with the dissolved contents of the center chamber See Figure 14 on page 26 You may tighten the retaining screws to secure the barrier combs in the raised position while the buffer circulates throughout the tank Amersham Biosciences 25 Preparing the DALT Tank for Electrophoresis Hoefer DALT System Figure 14 Raising the barrier combs to allow buffer circulation i
60. ne TEMED See Homogeneous Gel Solutions on page 46 Load the gel caster with cassettes separator sheets and filler blocks if necessary Place a gel label in each cassette See page 12 for directions Connect the feed tube to a funnel held in a ring stand at a level about 12 inches above the top of the gel caster Figure 8 Insert the other end of the feed tube in the grommet in the bottom of the balance chamber Funnel E F g Balance chamber A IN _ Grommet Feed tube Load the balance chamber with 150 ml heavy displacing solution Add the appropriate volumes of APS and TEMED only when you are ready to pour the gel not before Once these reagents are added polymerization begins You have about 10 minutes before the gels begin to solidify Vary the amount of TEMED added to control the rate of polymerization Slowly pour the gel solution into the funnel taking care to avoid introducing any bubbles into the feed tube line Amersham Biosciences Hoefer DALT System Casting Homogeneous Non Gradient Gels 10 Once the pouring is complete remove the feed tube from the balance chamber grommet As soon as the feed tube is removed the dense blue displacing solution flows down the connecting tube to the unit fills 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 casettes is more than 1 cm below the top of the casse
61. ng each gel so that the solution settles Use the same amount of overlay on each separation gel Add the overlay as rapidly as possible Make sure the cassettes and foam sponges are not clogged with polyacrylamide Gradient gels uneven layering e Add sucrose 15 final concentration or glycerol 25 final concentration to the high percent monomer solution e Add a small amount of bromophenol blue to the heavy solution to track the gradient formation Excessive amounts of bromophenol blue inhibit polymerization 56 Amersham Biosciences Hoefer DALT System Troubleshooting Power Supply detects current leak e Cracked or broken heat exchangers Call your Amersham Biosciences Service representative Spots are skewed or distorted e Gels run too fast uneven migration e Overlay the running gel with water saturated n butanol before polymerization begins to avoid forming an uneven gel surface e Uneven gel polymerization or gradient formation Heavy background after silver staining Use reagents specified as electrophoresis purity e Use only double distilled water Unusually slow or fast run e Check for leaks all plates and spacers must be clean dry and free of grease e Make sure buffer level is not above the level of the upper spacer e fthe required pH of a solution is exceeded do not back titrate Prepare fresh buffer e Check recipes gel concentrations and buffer dilutions For instance do not use Tris
62. nsfers are performed at high currents considerable heat is generated during a run Do not allow the buffer temperature to exceed approximately 40 C Buffer Towbin Current A up to 1 Voltage V approx 70 Transfer time approx 5 hours Coolant temperature 10 C Empirically determine the parameters for your sample and buffer system If you are planning to do a second transfer or set of transfers in the same buffer be sure to maintain constant temperature or to cool the buffer to room temperature between the two runs Do not allow the buffer to overheat and damage the unit Set the timer Most transfers are complete with 5 hours but larger molecules or thicker gels may require longer transfer times Determine the optimum transfer time for your system empirically NOTE For overnight transfers set the current at 400 mA with active cooling Amersham Biosciences Hoefer DALT System Table 2 Typical Protein Transfer Conditions Note Transfer buffer may be re used two to three times Store buffer in a separate container and cool to 10 C before reuse Amersham Biosciences The DALT Blotting Kit Transfer Conditions The blotting transfer conditions shown in Table 2 are only suggestions Efficiency of transfer depends on the percentage of gel used for the electrophoresis run the physical characteristics of the proteins being transferred and on how many times the transfer buffer has been used The con
63. nt par toute utilisation incor recte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives commerciales tout emp chement d utilisation et tout autre risques ayant un rapport avec Putil isation du produit mais sans aucune limitation quant la nature de ces dommages O 1998 Amersham Biosciences Limited Tous droits r serv s La reproduction le stockage dans un syst me de r cup ration d informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publication en totalit ou en partie sont strictement interdits sans autorisation pr alable crite de la soci t Informaci n importante para el usuario Para comprender el producto y utilizarlo con segu ridad es necesario leer este manual en su totalidad Espa ol El signo de admiraci n en un tri ngulo NM equil tero en el manual advierte al usuario sobre la presencia de instrucciones importantes de operaci n y mantenimiento del aparato Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se reserva el derecho a modificar las especifica ciones sin previo aviso Garant a y responsabilidad Amersham Biosciences garantiza que el producto entregado ha sido pro bado a
64. ols off and disconnect the power leads before opening the safety lid e Rinse only the electrodes not the banana plugs with distilled water before and after use e Always disconnect the power cord before servicing e Do not operate the circulation pump if the DALT Tank is empty The pump is not self priming and can be damaged if run dry e Circulate only water or 50 50 water ethylene glycol through the heat exchanger Never introduce anti freeze or any organic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit e Do not connect the heat exchanger to a water tap or any coolant source where the pressure is unregulated e Do not operate with buffer temperature above 45 C All plastic parts are rated for 45 C continuous duty Circulate coolant through the heat exchanger during electrophoresis to minimize heating Overheating will cause irreparable damage to the unit For longer runs you can control heating somewhat by chilling the buffer before use running the unit in a cold room or both e Do not lift the DALT Tank by the buffer pump Do not autoclave or boil this unit or any of its parts e casting unit when filled with glass plates and gel solutions is very heavy Use caution when trying to move or lift the casting unit e If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired
65. on of conformity is only valid for the instrument when it is used in laboratory locations e used as delivered from Amersham Biosciences except for alterations described in the User Manual and e connected to other CE labeled instruments of products recommended or approved by Amersham Biosciences 10 Amersham Biosciences Hoefer DALT System Amersham Biosciences Hoefer DALT System Function and Description Required or Convenient But not Supplied Refrigerated Cooling Bath DALT Tank cooling is provided by circulating chilled liquid through the heat exchanger on the floor of the central chamber With the aid of Quick fit connectors you can connect a refrigerated circulating bath such as the MultiTemp III to the fittings at the back of the DALT Tank We strongly recommend active cooling for protein transfers Focused IPG Strips Immobiline DrySrips provide a stable gradient pH gradient IPG immobilized onto an acrylamide matrix and supported by a plastic film backing They are readily available easy to handle and not prone to breakage and stretching IPG strip focusing is accomplished on the IPGphor or Multiphor II with the Immobiline DryStrip tray accessory Power Supply For overnight or full day runs a conventional electrophoresis power supply delivering 600 V at 400 mA such as the EPS 601 is suitable For multiple tanks or faster runs 8 9 h a higher current power supply such as the EPS 2A200 is desirable
66. oot for open lid support Buffer circulation pump Pump priming line Quick fit connectors attach to circulating water bath WARNING The liquid in the tank must cover the opening to the pump priming line when the circulation pump is in operation Buffer moves into the pump through the middle of the three flutes running along the length of the tank s center chamber and is expelled back into the tank through the left and right flutes The holes in the output flutes are aligned to generate maximum circulation over both the cassettes and the heat exchanger on the floor of the tank s centre chamber beneath the flutes As described on page 24 run the pump only when the tank is filled with liquid to prevent damage to the pump The Quick fit connectors on the ends of the tank port tubing provide a leak proof seal when disengaged eliminating spillage of coolant solution Amersham Biosciences Hoefer DALT System Hoefer DALT System Function and Description Gel Cassettes Figure 3 DALT gel cassettes The DAIT gel cassettes are pre assembled Two glass plates are held together along one edge by a strip of silicone rubber and the glass spacers 1 5 or 1 0 mm thick are glued in position To complete assembly close the two plates like a book Gels are removed by opening the book after the run and lifting out the slab The cassette is easily cleaned as a unit and can be stood upright in the open to dry The cassettes a
67. orm is properly completed Ordering information Qty Code No Hoefer DALT Multiple Electrophoresis Tank with buffer circulation pump 115 V 1 80 6068 79 230 V 1 80 6068 98 DALT Multiple Gel Caster 1 80 6330 61 with filler blocks and separator sheets Cassettes not included DALT Gel Cassette for 1 0 mm thick gel 25 x 20 cm 1 80 6067 27 for 1 5 mm thick gel 25 x 20 cm 1 80 6067 46 Separator sheets 30 pkg 1 pkg 80 6436 63 Filler block set four 25 mm one 12 mm one 6 mm one 3 mm lset 80 6436 82 Knobs 4 pkg 1 pkg 80 6437 58 Sponge funnel 8 pieces 1 pkg 80 6437 01 Acrylic feed tube 4 6 mm o d 178 mm 1 80 6437 20 Foam sealing gasket 1 80 6023 76 Silicon tubing set two pieces pkg 9 mm o d 178 mm long 1 pkg 80 6437 39 and 12 5 mm o d 16 mm long DALT Gradient Maker with peristaltic pump 115 VAC 1 80 6067 65 230 VAC 1 80 6067 84 Gradient maker gasket divider 1 80 6068 41 Gasket adjuster rod 1 80 6068 60 Knobs 4 pkg 1 pkg 80 6437 58 Plastic feed tubing 6 feet 1 80 6068 03 Bow tie mixer kit 1 80 6068 22 Acrylic feed tube 4 6 mm o d 178 mm 1 80 6437 20 61 Customer Service Information 62 DALT Blotting Kit Hoefer DALT System Qty 1 with rack 5 transfer cassettes and sponges blotting paper 50 pc DALT Transfer Cassette with 2 sponges Sponges DALT Blotting Paper 24 x 20 cm 50 pkg Replacement Parts GelSeal 1 4 oz tube Wonder Wedge Knobs for Gel Caster or Gradient Maker 4 pkg Quick
68. rd the anode in the Towbin buffer system independent of the presence of SDS 1 Place the support rack for the DALT Blotting Kit into the tank with the rack straddling the heat exchanger flutes in the bottom of the tank and the side engraved pump closest to the circulation pump 2 Lift the cassette sandwich from the tray of buffer and quickly insert it into one of the sets of vertical slots in the submerged cassette holding rack As many as five gels can be transferred at once in the DALT Blotting Kit For three or less gels use the cassette positions nearest the center 3 Place the cassettes into the unit so that all gels are oriented the same way with the clip side facing up and all anode sides of the cassettes facing the same side of the transfer unit 4 After inserting a cassette tap it a few times to release any air bubbles that may have been introduced in moving from the buffer tray to the tank A few bubbles left in the sponges should not interfere with the transfer 5 Check the buffer level to make sure buffer covers the cassettes WARNING The banana plugs corrode if exposed to buffer during transfer Carefully blot away any buffer that falls into the banana plug recesses 6 When all transfer cassettes are in the tank place an empty glass gel cassette horizontally across the tops of the cassettes to anchor them in the tank If you are using only one transfer cassette for blotting place another emp
69. re dishwasher safe Cassettes are 20 4 x 25 5 cm and produce a gel about 19 x 23 cm 46 ml for 1 mm thick 68 ml for 1 5 mm thick The gels make the best use of the standard 20 x 25 cm X ray film Multiple Gel Caster Figure 4 The DALT Gel Caster The DALT Gel Caster holds up to 25 gel cassettes 1 mm thick or up to 23 cassettes 1 5 mm thick with separating sheets for casting homogeneous or gradient gels Fewer cassettes can be filled at one time by inserting filler blocks to occupy unneeded volume Amersham Biosciences 5 Hoefer DALT System Function and Description Hoefer DALT System Figure 5 The DALT Gradient Maker A removable front plate and cassette separating sheets simplify loading and unloading cassettes from the unit A hydrostatic balance chamber allows accurate gradient positioning and provides a means to flush the fill tubing before polymerization occurs The chamber also provides a make up solution to accommodate the volume shrinkage during polymerization Pump Assisted Gradient Maker Divider adjusting rod The DALT Gradient Maker casts gradients of arbitrary shape up to 2 0 liters total volume The solution is delivered at a controlled rate by a peristaltic pump A flexible liquid tight divider partitions the gradient maker into two chambers of complementary shapes for casting both linear and non linear gradients With the divider adjusting rod you can configure the gradient divider to adapt to your
70. revent low molecular weight species from diffusing Conduct the separation at a lower current or voltage setting Reduce the temperature setting Reagent quality and gel preparation Useonly the highest quality reagents Only use gels that were recently prepared Check pH values of the stacking gel solutions Do not back titrate buffers Sample preparation Store IPG strips at 40 C or below Add a protease inhibitor such as phenylmethylsulfonyl fluoride PMSF if necessary to prevent proteolytic degradation of sample Insufficient equilibration Extend equilibration to 15 minutes Add DTT and iodoacetamide fresh before use Tracking Dye Doesn t Sharpen into a Concentrated Zone in the Stacking Gel Dispose of outdated acrylamide solutions and use only the highest grade of acrylamide Buffer reused too many times Prepare fresh each time for best results Incomplete Transfer Blank areas on the membrane Remove all trapped air pockets in the transfer stack assembly Assemble the stack while it is submerged in transfer buffer Gently press on each sponge as it is added to the stack Roll a glass pipette or test tube over the membranes and gel to eliminate all bubbles Process only one strip or membrane in each tray or cassette to prevent overlapping Use buffer with a lower ionic strength Check electrode continuity During the transfer a continuous stream of gas is released along the entire len
71. rink before assembling the stack Because methanol causes the gel to shrink slightly large molecules may migrate more slowly Make sure the gel is held firmly against the membrane and that it does not shift once contact is made If excess heating occurs during the transfer lower the temperature setting of the circulating cooler Check that the preferred binding surface of the membrane if any contacts the gel Inefficient binding to membrane Chemical parameters Prepare protein transfer buffer without SDS Verify the optimal amount of methanol required for the membrane type and check the buffer solution Add 10 2096 methanol to the transfer buffer to enhance binding to nitrocellulose Membrane parameters Wear gloves when handling membranes Store membranes at ambient temperature out of direct sunlight to keep the membranes activated Use a membrane with a smaller pore size 0 10 0 20 um if proteins pass through the membrane or use a different membrane type If you suspect one protein is moving in the opposite direction from the majority of the proteins place a membrane both over and under the gel Check both membranes for proteins Check if too much sample is available for the binding surface area by applying two membranes instead of one If blow through occurs reduce the sample load 59 References 60 Hoefer DALT System References Ames G F L and Nikaido K 1976 Two dimensional gel electrophoresis of
72. ristaltic pump Figure 10 Light chamber Heavy chamber 1 4 Gradient divider Clamp b si 3 z 4 F Clamp a LE i 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 assure 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 2 cm less than the height of the light solution Under these conditions the two solutions are in hydrostatic equilibrium See Configuring the Gradient Divider on page 16 Gradient Casting Setup WARNING Acrylamide is a neurotoxin Always use mechanical pipettes and wear protective gloves when working with acrylamide solutions IPG strips or surfaces that come in contact with acrylamide solutions 1 Be sure the entire gel casting system is clean dry and free from any polymerized acrylamide Amersham Biosciences Hoefer DALT System Amersham Biosciences Casting Gradient Gels Configure the gradient divider for the number of gels you are casting If necessary calibrate the grad
73. rrier combs See Figure 21 Preparing the Transfer Buffer Because the DALT Tank has its own circulating pump you can make the tank buffer within the tank itself In the tank it may take up to two hours for the buffer solids to dissolve As an alternative mix the transfer buffer in a carboy with a magnetic stirbar You may reuse the buffer in which you ran the original electrophoresis 25 mM Tris 192 mM glycine 0 196 SDS for the transfer if you first adjust the SDS and methanol concentrations for optimum binding Prepare an additional 4 liters of Towbin Buffer to use for cassette assembly See page 44 for this recipe Amersham Biosciences Hoefer DALT System Figure 21 DALT Electrophoresis Tank with barrier combs removed The DALT Blotting Kit IMPORTANT Fill the tank with water before turning the pump on The pump is not self priming and can be damaged if run dry Fill the tank with 15 liters of water or used electrophoresis buffer Add the appropriate volumes of water and or methanol to bring up to 22 liters Turn on the pump after the tank is filled At first the pump blows some bubbles through the circulating flutes in the tank and then establishes a vigorous circulating action in the tank If the pump doesn t catch and no circulation is observed you have an airlock in the pump See the instructions on page 25 If preparing fresh buffer add the dry buffer mixture NOTE Even if no cooling is required
74. rs attached run a single edge razor blade down between gel and spacers to assure that the gel is not sticking to either spacer Carefully peel the gel from the glass lifting it by the bottom high T end if a gradient and place it in a tray of fixative solution Use a fixative appropriate for your staining technique In case of a tear in the gel remove the torn section last working toward the tear Cleaning DALT Cassettes 1 Soak the used cassettes in distilled water if you can t clean them immediately To avoid clogging the sink drain use a fine removable sink trap to catch shreds of polymerized acrylamide To clean the cassettes thoroughly rinse them in warm water going over all surfaces with a non scratching plastic dish cleaning pad The SDS already on the plates is usually sufficient detergent for cleaning Finally rinse the cassettes with distilled water and air dry them in the open position on the lab bench or in a drying rack Store the cassettes in a closed cabinet to keep them free from dust 33 The DALT Blotting Kit 34 Hoefer DALT System The DALT Blotting Kit The DALT Blotting Kit supports the transfer of proteins from up to five large format polyacrylamide gels onto a membrane Gels and membranes are held by a cassette which is submerged into the transfer tank Molecules migrate under an electric field to the membrane where they are bound The transfer buffer temperature can be contro
75. s fall to the floor of the caster but remain in the respective cassettes and ultimately are polymerized into the gels Insert the end of the plastic feed tube supplied with the gel caster into the grommet in the floor of the side hydrostatic balance chamber of the caster Figure 7 Feed tube Ee EN 4 Balance chamber Grommet y Identification numbe 7 a EJ V well The feed tube must be snugly in place so that there is no leakage from the balance chamber into the caster at this point The feed tube should be connected directly to the gradient maker tubing or to a funnel with flexible vinyl tubing 13 Casting Homogeneous Non Gradient Gels Hoefer DALT System Figure 8 Setting up the caster for a non gradient gel 14 Casting Homogeneous Non Gradient Gels WARNING Acrylamide is a neurotoxin Always use mechanical pipettes and wear protective gloves when working with acrylamide solutions IPG strips or surfaces that come in 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 water saturated n butanol You need 0 75 ml of overlay for each cassette or about 20 ml for a set of 25 cassettes Make up 200 ml of displacing solution page 42 Make up the gel acrylamide stock solution without adding the 10 ammonium persulfate APS or 10 N N N N tetramethylethylenediami
76. t Solution Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 175 200 225 250 275 300 325 350 375 400 425 450 475 1 5MTris Cl 188 188 188 188 188 188 188 188 188 188 188 188 188 pH 8 8 Water 371 346 321 296 271 246 222 197 172 147 122 97 72 10 SDS 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 1095 APS 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 10 TEMED 1 84 1 61 1 43 1 28 1 16 11 07 0 99 0 92 0 86 00 80 0 76 0 71 0 68 Heavy Solution Final T 8 9 10 11 12 13 14 15 16 17 18 19 20 Acryl Stock 200 225 250 275 300 325 350 375 400 425 450 475 500 1 5MTris c1 188 188 188 188 188 188 188 188 188 188 188 188 188 pH 8 8 Water 300 275 250 225 200 175 150 125 100 75 50 25 0 10 SDS 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 Glycerol 51 51 51 51 51 5 51 co 51 51 NEM 51 51 1095 APS 3 75 375 375 375 375 375 375 375 375 375 375 3 75 375 10 TEMED 0 53 0 48 0 43 0 39 0 35 0 33 0 31 0 29 0 26 0 26 0 24 0 23 0 21 50 Amersham Biosciences Hoefer DALT System Gradient Gel Solutions For 12 gels 1 0 mm 400 ml each Heavy and Light by volume in ml Light Solution Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 93 107 120 133 147 160 173 187 200 213 227 24 253 1 5M Tris Cl 100 100 100 100 100 100 100 100 100 100 100 100 100 pH 8 8 Water 198 184 171 158 145 131 118 105 92 78 65 5
77. ting paper Place the gel which contains a sample that has been electrophoretically separated and equilibrated if required with transfer buffer on the membrane Gently roll a glass pipet or test tube over the gel to expel trapped air between the membrane and gel Cover the gel with a sheet of blotting paper and an additional 6 mm sponge see Figure 22 while pressing gently to expel trapped air Check that the gel membrane and sponges fit properly within the blotting cassette 2 Cathode aoe Sponge 595 A Blotting paper Tp Gel Pat Membrane Blotting paper Ar Sponge HI dal RSS 5 Close the cassette and press lightly to lock the tabs The assembled cassette should hold the gel in firm contact with the membrane without squeezing the gel If the stack seems loose add sheets of blotting paper Amersham Biosciences Hoefer DALT System The DALT Blotting Kit Loading the Cassettes Work quickly when moving the assembled cassette s to the tank to avoid draining the sponges Place the tray holding the cassette s near the tank lift out one cassette at a time and slide each one into a vertical slot Do not discard the buffer The direction of molecule migration depends on both the characteristics of the sample and the pH of the transfer buffer If the species of interest is negatively charged in the transfer buffer and the stack is assembled so that the membrane faces the anode Most proteins migrate towa
78. triangolo equilatero indica all operatore la presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione allegata al prodotto Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciecnes si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Garanzia e responsabilit Amersham Biosciences garantisce che prima della consegna il prodotto amp stato collaudato a fondo per soddisfare i requisiti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodotto stato instal lato ed utilizzato nel rispetto delle istruzioni fornite da Amersham Biosciences Amersham Biosciences non potr essere ritenuta responsabile di incidenti o danni consequenziali inclusi ma non limitati a perdite di profitti mancato guadagno perdite di affari difetti di funzionamento e relative esposizioni dovuti ad un utilizzo non corretto del prodotto 1998 Amersham Biosciences Limited Tutti i diritti riservati Nessuna parte della presente pubblicazione pu essere riprodotta conservata in sistemi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autorizzazione scritta del produttore Amersham Biosciences Hoefer DALT System Amersham Biosciences CONTENTS Hoefer DALT System Function and Description DALT System Components Unpacking Important Information Informations Import
79. ttes you may add up to 50 ml more displacing solution to the balance chamber The displacing solution pushes the remaining acrylamide solution out of the V well into the gel cassettes As gels contract during polymerization the displacement solution is drawn in from the bottom IMPORTANT Apply overlay immediately See Applying Overlay to DALT Slab Gels on page 22 11 Allow non gradient gels to polymerize for at least 1 hour Amersham Biosciences 15 Preparation for Gradient Gel Casting Hoefer DALT System Figure 9 Gradient divider configurations for half full and full Gel Caster tanks 16 Preparation for Gradient Gel Casting Successful gel casting requires planning timing and practice A full DALT Gel Caster requires approximately two liters of acrylamide stock Polymerization begins as soon as you add TEMED and APS to the acrylamide stock Once you have added these reagents 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 you set up the unit as described on page 12 and configure the gradient divider as described on this page Follow the gradient pouring procedure on page 18 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
80. ty cassette in a vertical slot to help support the glass cassette anchor Amersham Biosciences 37 The DALT Blotting Kit Table 1 Typical Transfer Parameters for Proteins 38 Hoefer DALT System Connecting the Power Supply If using a power supply that can be set to either constant current or constant voltage mode we recommend that it be set to operate in constant current mode Buffer conductivity increases with temperature During blotting in an uncooled chamber at constant voltage Joule heating and rising conductivity may result in dangerous overheating If a constant voltage power supply must be used monitor and adjust the voltage to maintain a current at 1000 mA or less and voltage should not exceed 100 V 1 Close the plastic lid on the chamber The cassettes are labeled to match the leads in the lid To transfer toward the anode position the lid so that the side of the cassette faces the anode or red lead and the side of the cassette faces the cathode or black lead Connect the leads to your power supply Set the power supply See Table 1 for typical transfer parameters for proteins Constant current mode is recommended If constant voltage mode is selected carefully monitor the current Increased current increases Joule heating If the current exceeds 1 0 A decrease the voltage IMPORTANT The plastics used in the DALT unit may warp at high temperatures Because electrophoretic tra

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