SE 600 Ruby Manual

user manualvertical electrophoresis SE 600 Ruby standard dual cooled gel electrophoresis unit um 80-6472-73/Rev. A0/12-00 Page finder 1. Gel electrophoresis unit function and description Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Important information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. Unpacking and inventory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3. Operating instructions 3.1 Prepare the gel sandwich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.11 Construct the gel sandwich and insert into caster . . . . . . . . . . . . . . . . . . . . 6 3.12 Acrylamide gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.13 Gradient gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.14 Sample preparation and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.2 Final assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.3 Separating the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 3.4 After electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4. Care and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 5. Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Appendices Laemmli system gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Gel recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Customer service information Technical service and repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Companion products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 • pi um 80-6472-73 • pii . and • connected to other CE-labeled instruments or products recommended or approved by Amersham Biosciences. Gel electrophoresis unit function and description The SE 600 Ruby™ series vertical slab gel electrophoresis unit is intended for protein and nucleic acid electrophoresis under commonly used denaturing and non-denaturing conditions. • p1 .• gel electrophoresis unit function and description 1. CSA C22. The focused strips are easily transferred to the second-dimension slab gel for size separation. CE Certified Installation category Pollution degree Dimensions Product certifications This declaration of conformity is valid only when the instrument is: • used in laboratory locations. Up to four gels can be run at one time if sandwiches are paired into “club sandwiches. nucleic acid fractionation.5 in) EN 61010-1.” The heat exchanger allows buffer temperature control in the lower chamber. Applications include protein separations.5 × 11. First-dimension separation of 2-D protein electrophoresis should be performed on Immobiline™ DryStrip Immobilized pH Gradient Gels using the IPGphor™ Isoelectric Focusing System or the Multiphor™ Flatbed Electrophoresis system. UL 3101-1. The gel plates are 18 cm wide by 16 cm long.21010. and the second-dimension separation of 2-D electrophoresis. Specifications Gel plate size Gel size Maximum watt Maximum volt Maximum ampere Maximum temperature Environmental operating conditions 18 × 16 cm 14 or 16 cm × up to 16 cm (w × h) 50 W 1 000 V 500 mA 45 °C Indoor use: 4–40 °C Humidity up to 80% Altitude up to 2 000 m II 2 width × height × depth 32 × 29 × 14 cm (12.5 × 5. • used as delivered from Amersham Biosciences except for alterations described in the user manual. Up to 28 samples can be compared on a single slab gel. • Seulement les accessoires et piéces detachées approuvés ou fournis par Amersham Biosciences sont recommandés pour l’utilisation. l’entretien et réparation de cet appareil. Ne jamais utiliser d’anti-gel ou tout autre solvant organique avec cet instrument. Never introduce antifreeze or any organic solvent into any part of the instrument. • Turn all power supply controls off and disconnect the power leads before removing the safety lid. Les solvants organiques causeraient des dommages irréparables à l’appareil. Circulate coolant through the heat exchanger during electrophoresis to minimize heating. • Ne pas connecter l’échangeur vertical à circulation d’eau à un robinet ou quelque source de refroidissement dont la pression n’est pas régulière. Faire circuler l’eau dans l’échangeur vertical durant l’électrophorèse pour minimiser l’échauffement afin d’éviter des dommages irréparables à l’instrument. maintaining. Informations importantes • Le couvercle de sécurité doit être en place avant de brancher les prises au générateur. Organic solvents will cause irreparable damage to the unit! • Do not connect the heat exchanger to a water tap or any coolant source where the water pressure is unregulated. and servicing this product. • Ne pas utiliser avec un tampon à une température au dessus de 45 °C. • Faire circuler seulement de l’eau ou 50/50 d’eau et d’éthylène glycol dans l’échangeur vertical à cirulation d’eau. Toutes les piéces en plastique sont prévues pour résister à une température constante de 45 °C. • Do not operate with buffer temperature above 45 °C. um 80-6472-73 • p2 . Overheating will cause irreparable damage to the unit! • Only accessories and parts approved or supplied by Amersham Biosciences may be used for operating.SE 600 ruby • important information Important information • The safety lid must be in place before connecting the power leads to a power supply. • Circulate only water or 50/50 water/ethylene glycol through the heat exchanger. All plastic parts are rated for 45 °C continuous duty. • Eteindre le générateur et débrancher les prises avant d’enlever le couvercle de sécurité. which allows visual tracking of electrophoresis process. Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit. Inspect all components for damage that may have occurred while the unit was in transit. contact the carrier immediately. Required but not included: • Magnetic stirrer • Power supply with a minimum rating of 500 V. Lower buffer chamber The lower buffer chamber is transparent. 1/4 oz. 100 mA (constant A or V) Optional: Circulator bath Note: The ordering section lists all accessories and replacement parts.• unpacking and inventory 2. color-coded leads (2) safety lid Fig 1. interlock pins upper buffer chamber with upper electrode heat exchanger with lower electrode lower buffer chamber • p3 . Unpacking and inventory Unwrap all packages carefully and compare contents with the packing list. The chamber is chemically resistant to common electrophoretic buffers but not to organic solvents or strong acids or alkali. Included but not shown: • GelSeal compound. contact your local sales office. making sure all items arrived. Main components of the SE 600 Ruby (see Fig 4 for caster components). • Spacer-Mate assembly template • Well-locating decal • Glass plates (6) • Wonder Wedge plate separation tool • Buffer dam Complete unit also includes spacers (4) and combs (2). Temperatures above 45 °C may cause the chamber to warp. If any part is missing. If any part appears damaged. so up to four gels can be run at one time. (Do not connect to the water tap. The upper chamber requires 0.5–0. to attach the sandwich to the upper buffer chamber. ordered separately.d. but not to organic solvents or strong acids or alkali. Connect only to coolant sources with regulated pressure. the heat exchanger regulates the buffer temperature in the lower chamber.8 l of buffer (fill no higher than the top of the plastic ribs). Cam clamps Cams are used twice: first to secure the assembled sandwich in the casting stand and. The 4 mm shrouded color-coded leads plug into color-coded jacks in the power supply. Casting stand The casting stand holds assembled gel sandwiches upright for casting gels. divide gel sandwiches to form “club sandwiches” of two gels each. um 80-6472-73 • p4 . When connected to a circulator bath. second. distributes pressure evenly. Notched divider plates. The heat exchanger connector ports are 13 mm o. Engage interlock pins before lowering electrode connections on to banana plugs. adjusted with screws. The clamp pressure bar. Coolant passes through the glass tubes. which runs along the bottom of the frame. The upper electrode (cathode) runs along the center ridge and terminates at the banana plug. which are secured with silicone rubber grommets. Clamps Two 16 cm clamps are used to secure the gel sandwich. Always install the safety lid before use! Glass plates The SE 600 Ruby accommodates 18-cm-wide plates 16 or 8 cm long. Adjustable feet level the caster.SE 600 ruby • unpacking and inventory Upper buffer chamber The upper buffer chamber is chemically resistant to common electrophoresis buffers. The heat exchanger is rated to a maximum of 0.8 atmospheres above ambient (12 psig).) Safety lid The banana plug on the heat exchanger connects to the red lead. Heat exchanger The heat exchanger must be installed for every use because it houses the bottom electrode (anode). and the plug on the upper buffer chamber connects into the black lead. A laminated gasket in the bottom of each casting cradle seals the bottom of the sandwich when it is clamped into the stand. Two-dimensional electrophoresis Note: When casting slab gels for 2-D electrophoresis. which are required for higher sample loads. and 20-well combs form wells that are 25 mm deep. and preparative combs include one or two reference wells in addition to the preparative well. Blank combs form a single large well. Table 1 lists sample volumes of wells for all combs (see page 10). and 1. well depth.75. (May be ordered separately. or 28 wells. • p5 . The slotted gaskets fit under the upper buffer chamber and form the seal between the upper and lower chambers. The SE 600 Ruby is especially suited to perform the second dimension on thicker gels. and 1.5 mm) and two widths (1 and 2 cm).5 mm.5 mm spacers to ensure a good fit for film-backed IPG strips. The 28-well comb forms wells that are only 15 mm deep so that wells do not collapse when the comb is removed. 15-. 15. Spacers Spacers determine the thickness of the gel and are available in three thicknesses (0.75.) Spacer-Mate assembly template This template aligns spacers during sandwich assembly.• unpacking and inventory Rubber gaskets There are two sets of two gaskets: The solid laminated gaskets fit into the bottom of the casting stand and form the seal for casting the gel.) All blanks. The ridges on the upper gasket align the gasket slot to maintain an open channel between the top of the gel and the buffer in the upper chamber. Wonder Wedge Gel Plate Separation tool This tool is used to disassemble gel sandwiches and to check spacer and comb thicknesses. Most combs are available in all three thicknesses: 0. 20. and 10-. First-dimension isoelectric focusing of proteins on Immobiline DryStrip IPG strips requires the IPGphor Isoelectric Focusing System or the Multiphor II Electrophoresis System. 1. 12-. Combs Teflon combs are available in sizes that form 10. (May be ordered separately. The sample volume held by each well depends on the gel thickness. preparative combs. Details of each procedure are described in the respective protocol guide (see the ordering information section). use 1 or 1. and the number of wells per comb. 12. 1. Finger-tighten one screw on each clamp. and clamp sets are sized so that the assembled sandwich can be easily aligned to create the seal required first to cast the gel and then to run it. and lay the second glass plate on top. instead of setting the sandwich upright on a flat surface. Inspect glass plates for nicks. Loosen all clamp screws and make space for the sandwich by sliding the pressure plates toward the screws. place a spacer along each edge. Taking great care in alignment will ensure a good seal. set it into the casting cradle. Both precast gels and self-cast gels can be used. Sandwich assembly. two accessory notched divider plates and two additional pairs of spacers are required.11 Construct the gel sandwich and insert into caster 1 Prepare the caster and clamps Place the spirit level into the caster center and adjust the leveling feet. To self-cast multiple gels. Fingertighten all screws. See page 23 for recipes and page 24 for bibliography. lay the Spacer-Mate assembly template onto the plate (wide side at the top of the plate). set the sandwich upright on a flat surface. 3. Lay one plate on a flat surface. Use only unchipped plates to prevent leaking. Tip: Use the casting cradle to hold the sandwich during alignment. spacer clamp ridges glass plates 2 Construct gel sandwiches For each sandwich choose two perfectly clean. Remove the Spacer-Mate. For best results take extra care to align all components when assembling sandwiches. and loosen the screw to align the stack.) To run four gels concurrently. unchipped glass plates and two spacers. Included are instructions for polyacrylamide gels (used with continuous or discontinuous buffer systems) and gradient gels. and the SE 675 Gel Caster Kit holds up to four sandwiches. One to four gels (18 × 16 cm) can be assembled with SE 600 Ruby. (See the accompanying gel caster User Manual for complete instructions.1 Prepare the gel sandwich Glass plates. Remove the laminated gasket from the cradle and. Slide one clamp at a time along the sandwich sides. The glass plates and spacers must be flush with the clamp ridges at both top and bottom for a good seal. kits can be ordered separately: The SE 615 Multiple Gel Caster Kit holds up to 10 single gel sandwiches.SE 600 ruby • operating instructions 3. pressure plate 3 Secure the sandwich with clamps Fig 2. 3. spacers. Operating instructions Gel casting and electrophoresis procedures follow. um 80-6472-73 • p6 . usually 90°–150°. notched center-divider plate (ordered separately) pairs two sandwiches to double the number of gels that can be cast and run. The seal is complete once the glass edge appears darker and nearly transparent against the gasket. screw side facing out. spirit level cam hole levelling feet (4) cam (install ridge end up) cam hole • p7 . 7 Insert a cam into the hole on each side of the casting tray with the ridge (short end) pointing up. cam hole casting cradles (2) Note: When turning the cams.• operating instructions 4 Club sandwich glass plates (at the outer sides of the sandwich) spacers notched center plate A 16-cm-long. Optional: Apply a light film of GelSeal compound only on the bottom corner surfaces created by the spacers and plates if the sandwiches tend to leak. 5 Remove the sandwich and inspect the bottom to make sure that edges are aligned flush to ensure a complete seal. Do not turn past this point. Adjust if necessary. Fig 3. Place the notch so that it will be at the top of the gels.5 mm thick. glass plate spacer cam hole gasket (foam side down) clamp Fig 4. Note: Do not use silicone grease or petroleum jelly to seal the sandwich. These substances are difficult to remove and ultimately cause artifacts. 6 Place the laminated gasket into the casting cradle (See Fig 4) with the foam side down. except before placing the top glass plate. Caster components and setup. The cam action presses the plates down into the gasket to seal the bottom of the sandwich. Seal the gel sandwich against the casting gasket by turning both cams as far as needed. up to 180°. it is easier to keep the caster balanced if you turn both toward the center of the caster. It is essential that the spacers and plates align perfectly in order to seal. lay the divider plate and a second set of spacers on the stack. Club sandwich assembly. Place the clamp assembly in the casting cradle. Side clamps will accommodate two spacers up to 1. Assemble a club sandwich in the same manner as a regular sandwich. Pour the stacking gel onto the resolving gel with a disposable or Pasteur pipette to a level about 2 mm from the top of the plate. This height allows 1 cm of stacking gel below the wells.12 Acrylamide gels 1 Prepare the monomer solution and pour the gel Note: Recipes for the Laemmli gel system are on page 23. prepare the stacking gel as described below. After the gel is set. filling each to the same level below the notched edge.SE 600 ruby • operating instructions 3. Apply the solution near the spacer at one side of the sandwich and allow it to flow across the surface unaided. No stacking gel (Continuous system) Fill solution to just below the top of the upper plate edge. Seal the IPG strip or tube gel in place with agarose dissolved in running buffer. Stacking-gel resolution is optimal when poured just before electrophoresis. 4 Introduce a comb (at a slight angle) into the sandwich.and second-dimension gels. 1 Remove the overlay by rinsing the top of the gel several times with distilled water. Slowly deliver the overlay solution from a glass syringe fitted with a 22-gauge needle. Club sandwich Pipette the solution into both sandwiches. Take care to avoid trapping any air bubbles between the first. um 80-6472-73 • p8 . To ensure a seamless contact between the resolving and stacking gels. 2-D electrophoresis (Discontinuous protein system) Fill monomer solution to about 1 cm below the top of the glass plate to allow 4–5 mm for the IPG strip or tube gel and an agarose seal. and add catalyst and initiator. Allow a minimum of 1 h for the gel to polymerize. water. Pour the gel and apply an overlay (see step 2). Stacking gel preparation Pour the stacking gel while the sandwich is still in the gel caster. remove residual liquid by blotting one corner with a lab wipe. See below for the appropriate solution level according to the application. 2 Calculate the stacking gel monomer solution volume. or diluted gel buffer to prevent gel exposure to oxygen. taking care not to trap air bubbles under the teeth. Stacking gel Fill solution to 3–4 cm below the top of the glass plate. Prepare the required amount of monomer solution. taking care not to trap air under the teeth. 3 Allow the gel to polymerize for a minimum of 1 h. Deaerate and add the initiator and catalyst just prior to pouring the gel. Introduce a comb (at a slight angle) into each sandwich. 3 Prepare the stacking-gel monomer solution. deaerate it. remove with a pipette or syringe. Pipette the solution into one corner of the sandwich. (A stacking gel will require extra space). Invert the caster to drain. 2 Overlay each gel with a thin layer of water-saturated butanol. If bubbles are trapped. taking care not to introduce any air bubbles. and lower-percentage acrylamide solutions.) Place the cannula so that it rests at the bottom of the sandwich. • p9 . Apply the solution near the spacer at one side of the sandwich and allow it to flow across the surface unaided. Note: Gradient gels poured in the SE 615 or SE 675 Multiple Gel Caster are introduced through the bottom. the gel solution may be introduced into the gel sandwich through a pipette tip at a rate that maintains a continuous stream. taking care not to trap air under the teeth. Place the gradient maker onto a magnetic stirrer and begin stirring at a rate that mixes well but does not introduce bubbles into the solution. A stacking gel is then poured over the gradient gel.d. 5 Mix the gradient and pump the solution into the sandwich While the solution is stirring. Pouring a linear gradient gel 1 Assemble sandwich(es) into the dual-gel casters as described in section 3. 2 Set up the monomer solution flow path Run a length of clear vinyl tubing through a peristaltic pump. 4 Pour the “light” solution into the reservoir chamber (the chamber farthest from the outlet). Open the stopcock between the chambers long enough to displace the air and then close. and introduce a comb (at a slight angle) into the sandwich. of the cannula must be less than the spacer thickness. Optional: Adjust the higher-percentage acrylamide solution to 15% (w/v) sucrose or 25% (v/v) glycerol to improve layering. 6 Overlay each gel with a thin layer of water-saturated butanol. position a plunger above the liquid to hold the volume in the mixing chamber constant. 3 Prepare the monomer solution Calculate the volume of monomer solution needed. 7 Allow the gels to polymerize for a minimum of 1 h. keeping the tip at the gel surface. Prepare more gels as required. pour the stacking gel. 8 Prepare the stacking-gel monomer solution. water. begin pumping from the mixing chamber and open the stopcock to the reservoir chamber. Gradient gels are poured from the top of the caster with a cannula if using the provided dual-gel caster or from the bottom if using a Multiple Gel Caster (see instructions accompanying the caster). Pouring a gradient gel. After polymerization. (The o. Divide the total volume in half and prepare this volume of both the higher. Slowly deliver the overlay solution from a glass syringe fitted with a 22-gauge needle.• operating instructions 3. Attach one end of the tubing to the gradient maker outlet port and the other end to a 20 cm cannula. or diluted gel buffer to prevent gel exposure to oxygen. Pour the “heavy” solution into the mixing chamber and place a stirring bar into this chamber. Fig 5.11. We recommend using a SG Series Gradient Maker. Note: When pouring an exponential gradient gel. Allow a minimum of 1 h for the gel to polymerize. pour off the overlay and rinse the gel surface several times with distilled water. Alternatively.13 Gradient gels Both linear and exponential gradient gels can be poured in the dual-gel caster. Raise the cannula as liquid enters the sandwich. midway between the spacers. 3 3.2 4.5 12. 4 Underlay the sample into the wells using a fine-tipped microsyringe or gel-loading pipette tip.1 4/90 4/85 8.1 9/183 9/171 Multi-channel pipette compatible comb 14 – 6. because no stacking gel is used. the samples must be loaded without the upper buffer chamber in place. Note: With Coomassie Blue™ it is possible to detect 1 µg of protein in a single band. so the sample need not be as concentrated.1 2. 3 Note: Once the sample is in the wells. take care to not jar the sandwiches so that the samples are not disturbed. the zone into which each molecular species migrates is sharpened by the stacking gel.7 6/121 6/112 1. With the more sensitive silver stains.SE 600 ruby • operating instructions 3. Store unused sample at 4 °C. Heat the tube in boiling water for 90 s. Heat membrane proteins to 60 °C for 20 min.7 4. Carefully rinse each well with distilled water to remove unpolymerized acrylamide and then drain by inverting the gel sandwich (or caster). To dry protein samples.1 2. it is possible to detect as little as 10 ng of protein. In a continuous buffer system. Fill each well with electrophoresis buffer.0 9.0 10 12 15 20 28 1/1 (ref/prep) 1/2 (ref/prep) 6. add an equal volume of 2X buffer. and the amount of sample expected in each band.7 5.75 1. When loading samples while using divider plates. add equal volumes of 2X sample buffer and high-purity water to achieve the desired concentration. or pyronin Y. Sample volume for standard comb sizes volume of sample (µl) per 1 mm depth no. the sensitivity of the detection method used. Table 1. In a discontinuous buffer system. 1 Prepare the wells Remove the comb by gently rocking it side to side and then lifting it straight up to avoid damaging the well walls. Add a tracking dye such as phenol red. The amount of sample loaded depends on the thickness of the gel.6 6.0 um 80-6472-73 • p10 . the protein sample should be relatively concentrated. use 2X treatment buffer to denature both liquid and dry samples in a test tube.14 Sample preparation and loading The sample can be loaded either while the sandwich is in the caster or after the upper buffer chamber is attached.8 4. bromophenol blue.4 11. then allow to cool to room temperature. To liquid protein solutions. Treated samples can be stored at -40 to -80 °C for future runs. 2 Prepare the sample Increase liquid sample density with 10% glycerol or sucrose. of comb thickness (mm) wells 0. For SDS protein gels.3 7.2 5.5 8. Both slotted gaskets must be used even if running only one gel sandwich. Attaching gel sandwiches to the upper buffer chamber. If you encounter unusual resistance. 3 Note: To help hold the gasket against the upper buffer chamber. Place the upper chamber onto the sandwiches and then insert the cams into the upper cam holes. Install the cams. • p11 . Grooves along each slot help keep the gasket in place. check alignment of all sandwich components. screws facing out. Clamp the sandwich in place by simultaneously turning one cam clockwise and the other counterclockwise a full 180°.2 Final assembly Upper buffer chamber 1 Rinse both buffer chambers with water and distilled water thoroughly before each use. 4 Use a pipette to carefully fill each slot above the sample wells with buffer to minimize disturbing the samples. Clean away any gel adhering to the exterior of the gel sandwiches. Attach the gel sandwich to the upper buffer chamber Turn the upper buffer chamber upside down and place a slotted gasket into both sandwich holder recesses. ridge (short end) pointing down. A.• operating instructions 3. disassemble and inspect clamp and glass alignment along the top of the sandwich. directing the buffer stream toward the side wall. Install the “dummy” gel. Check that no buffer leaks around the gasket. taking care to align the clamp ends and dam edges. Release the sandwiches from the caster by removing all bottom cams (if present). If the assembly leaks. B. dab a small amount of GelSeal at each end of the gasket only and then install. disassemble the unit and wash with a dilute solution of a laboratory detergent and rinse thoroughly first with water and then with distilled water. into the buffer chamber cam holes. and adjust if necessary. Note: Before using the first time. Lower the upper buffer chamber onto the gel sandwiches in the casting stand. Align and reinstall. Fit clamps onto the dam. Remove cams from the lower cam holes. Disassemble. Fig 6. Then pour 100 ml of buffer into the chamber. ridge pointing down. take it to a sink and partially release the cams to allow buffer to drain out of the upper chamber. Important! A smooth fit between the sandwich and gasket is essential to a good seal. 2 If running only one gel: Block the second upper buffer chamber slot by installing the acrylic buffer dam included with the unit. The final cam position (not shown) must be vertical so that the assembly fits into the lower buffer chamber. Note: Do not force the cams. in the second cradle in the dual gel caster. Both the slot in the gasket and the slot in the recess must align. • Use only water or 50/50 water/ethylene glycol as coolant. it is convenient to attach QuickFit connectors to the tubing. 0 -1 Set the circulator bath temperature setting lower than the desired run temperature by the amount indicated on the graph. • Pour buffer slowly and away from the slots in the upper buffer chamber to avoid disturbing the samples. Optional: Prechill the buffer. (The heat exchanger must be in place for all runs because the lower electrode is integrated into the heat exchanger. Never use a commercial antifreeze or any alcohol-based mixture. 2 Lower the heat exchanger into the lower chamber. Fill the lower chamber with up to 4 litres of buffer. bath setting correction. W Example: Run parameters: 200 V. Interpolate the number of degrees to subtract from the desired run temperature.05 A (50 mA) 1.d. changes in power output. or irreparable damage to the heat exchanger will result. Secure the connections with the hose clamps Note: If the cooling option is used frequently. measure the temperature and adjust as necessary. Attach one end of each length of tubing to a heat exchanger port.05 A 2. Use the chart below to estimate a starting point for the circulator bath temperature setting. Attach the free ends of each length of tubing to the circulator bath ports. If the desired temperature is 4 °C. fitting the ports into the notches in the rim. • Do not fill the upper or lower chamber above the recommended levels illustrated on the next page. This should be checked at three points. Connect the heat exchanger to a thermostatic circulator such as the MultiTemp™ III.1 = 22 °C. set the bath to 23 . Slide hose clamps (four total) onto each end of two lengths of 10–12 mm i. set the bath to 4 . • Do not connect the heat exchanger to a water tap or any other source where the water pressure is unregulated. Remove buffer in contact with the electrode posts. (3/8–1/2") vinyl or silicone tubing. The valves in these fittings prevent coolant spillage. If the desired temperature is 23 °C. 10 W intersects the graph at about -1 °C. maintain at least 2 cm of clearance. um 80-6472-73 • p12 . and circulator bath efficiency. 0. Adjust as necessary for variables such as ambient temperature. °C Fig 7. one to the inlet and the other to the outlet.) If no cooling is required. If accurate temperature control is critical. Approximate circulator bath temperature setting -2 -3 -4 -5 -6 -7 0 10 20 30 40 50 60 power supply setting. skip to step 3. Calculate W if your power supply does not display power directly: W=V×A 10 W = 200 V × 0.1 = 3 °C. 1 Place a magnetic spin bar into the lower buffer chamber (LBC) and place the unit on a magnetic stirrer.SE 600 ruby • operating instructions Lower buffer chamber Important assembly notes: • IEF runs: The buffer level in the lower buffer chamber must never reach the upper buffer chamber. In most systems the red lead. Lower buffer chamber (LBC). connected to the top electrode. Fig 8. which is connected to the bottom electrode. and the black lead. This level requires 450–600 ml of buffer— just enough to cover the upper chamber ribs. but not high enough to contact the banana plug. Upper buffer chamber (UBC). Do not fill above UBC MAX fill line. 6 Plug the color-coded leads into the jacks of an approved power supply. is the cathode (–).• operating instructions 3 Fit the upper buffer chamber assembly into the lower buffer chamber. Upper chamber buffer max fill line Lower chamber buffer max fill line Buffer level label 5 Place the safety lid on the unit by engaging the safety interlock pins before lowering the electrode connections on to the banana plugs. Upper and lower buffer chamber fill levels. such as the EPS 601. Plug the red lead into the red output jack and the black lead into the black output jack. The electrode along the upper chamber ridge must be submerged about 1 cm. • p13 . 4 Inspect the installation and check the buffer levels. Fill to LBC MAX fill line. is the anode (+). Use a steady hand to avoid disturbing the samples: Grasp the assembly in the casting stand by the upper buffer chamber and carefully lower it into the lower chamber. Record each run Keep a record of the current or voltage setting. number and thickness of gels. (Two 1. usually requires 5 h. The current must be multiplied by the number of gels. ‡ † Time A run is usually complete when the tracking dye reaches the bottom of the gel.5 mm 25 mA constant current 80–90 V 220–250 V Voltage The starting voltage for a 1. the four gels require four times as much current. keeping an eye on the migration rate of the tracking dye.) *Thicker or thinner gels require proportionally more or less current. and the resulting Joule heating and band distortion. (A small volume of buffer may leak past a nicked plate or gasket. Table 2 lists starting-point guidelines and adjustments for gel thickness. if two club sandwiches are installed. a 1.5 mm slab gel connected to a power supply set to 25 mA is usually 80–90 V (using the SE 600 Ruby unit with a Laemmli discontinuous buffer system for SDS gels). Current Current acts on the total cross-section area of all the gels because the gels are connected in parallel in the electrical circuit. run at 25 mA/gel without cooling. which is half as thick as a 1. and migration rate. The run is complete when the tracking dye reaches the bottom of the gel.SE 600 ruby • operating instructions 3. buffer system. Under these conditions voltage increases as the run proceeds. incorrect buffer concentrations. we suggest a starting current setting of 25 mA. The final voltage is typically 250–400 V.) Refer to page 21 for buffer recipes and electrophoretic conditions. In a 16 cm gel (SE 600 Ruby). and again after 1 h.75 mm gel. if necessary. The gel thickness determines the cross section (and current requirement) for constant current runs.3 Separating the sample Electrophoresis parameters for discontinuous polyacrylamide gels Note: SE 600 Ruby unit uses 18-cm-wide plates. For instance. a 0. Table 2: Laemmli buffer system starting point guidelines Gel thickness* Current per gel† Starting voltage‡ Final voltage 1.5 mm thick. Inconsistent results for the same system and settings indicate potential problems such as leaking current. high salt concentrations. At 25 mA per gel. A lower current setting is recommended for higher resolution. The current can be increased for faster runs if active cooling is used and it can be decreased for slower overnight runs. Check band progress after 5 min. Gels may be run at either constant current or constant voltage settings. do not leave the unit unattended for more than 1 h before checking the progress of the bands and the buffer level. Caution! After initial monitoring. Watch the buffer level and. um 80-6472-73 • p14 . Thus the current setting for one gel must be multiplied by the number of gels of the same crosssection run simultaneously. the main factors that must be balanced include the gel concentration and migration speed. A constant current mode is traditionally used with a discontinuous buffer system so that the rate of electrophoretic migration remains unchanged throughout the run.5 mm gel. The optimal current level must be determined empirically. depending on the length of the gel. For a gel 1.) Note: Cooling may be required to control Joule heating. (See Table 2. replenish it as required to keep the top electrode submerged. and the starting and final current or voltage readings for each run so that results can be compared. or buffer may pass through the gel.5 mm gels = 50 mA.5 mA. or 12. The length of the plate determines the running time. or inconsistent chemical quality.5-mm-thick Laemmli SDS gel. number of gels. requires half as much current. For example. Use the Wonder Wedge Gel Plate Separation tool to separate the plates. 5 Note: Use only flexible plastic prying tools to avoid chipping the glass plates. Pour the buffer into a sink. • p15 . stop the flow and disconnect the fittings or tubing. using finger leverage between the lid and the top of the heat exchanger. if the gel is thick enough to handle. lift it and place it into the tray. To avoid splashing.) 2 If coolant is circulating. turn off the power supply. 4 Unscrew the clamps from the sandwiches and remove.• operating instructions 3. and remove the safety lid. Invert the plate and position the gel low over the staining tray. (Lift straight up to avoid bending the banana plugs. Carefully lift the glass plate with the gel attached. disconnect the leads. add staining or fixative solution to the tray after the gel is transferred. Gently loosen and then slide away both spacers. Install the assembly in the dual gel caster and then release the sandwiches by turning and removing the cams. or. 3 Pull out the upper buffer chamber assembly.4 After electrophoresis 1 Once the tracking dye reaches the bottom of the gel. Handle the gel with care to avoid damaging it. Pry one corner of the gel away from the glass and allow it to drop into the tray. 6 Clean the unit as described in the next section. a piece of cloth. rinse the upper and lower buffer chambers with water and then rinse thoroughly with distilled water. strong cleaning solutions. protect your hand with thick gloves. Then slip the other end into the top grommet. um 80-6472-73 • p16 . Allow to air-dry. then lift the tube out of the lower grommet. until the top end is free of the upper grommet. Replacing a heat exchanger glass tube Note: If the old tube is cracked or broken. Care and maintenance Cleaning • Do not autoclave or heat any part above 45 °C. Carefully guide the tube so that it will clear the assembly. • Do not use organic solvents. 1 Remove the tube by simultaneously twisting and sliding it down as far as possible. abrasives. Immediately after each use.® then rinse thoroughly with tap and distilled water. • Do not soak the laminated gasket. Twist and slide one end of the tube into the lower grommet. Glass plates can also be treated with (but not stored in) acid cleaning solutions. 3 Check that the grommet is not pinched. Handle the upper buffer chamber with care to prevent damaging the banana plug.SE 600 ruby • care and maintenance 4. Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS-35. or paper towels before removing the tube. or strong acids or bases to clean the chambers. gently pushing it with a slight twist until it stops. 2 Lightly grease the outside of both ends of the new tube with silicone grease. Clean gaskets with mild detergent and rinse with distilled water. Sample wells damaged or irregular Air bubbles Remove air bubbles before inserting combs. pH Oxygen Solutions with extreme pH values (especially acidic) may not polymerize.• troubleshooting 5. especially for low %T gels. Rinse out unpolymerized gel with sample buffer. Agarose gels: Lower the comb no more than 1 cm into the gel. Do not use silicone grease. Wash if necessary. add more monomer solution before reinserting the comb. and the gasket must be completely clean. Replace if necessary. Mis-aligned parts Over-clamping Check plate and spacer alignment and realign if necessary. or both. Check that the upper buffer chamber is not warped from prior exposure to excessive heat. Slide comb into solution at an angle. replace with fresh stock. Incomplete or delayed polymerization Debris in wells Comb removal Temperature Upper buffer chamber leaks Mis-aligned parts • p17 . If the dry ammonium persulphate does not crackle when added to water. Increase TEMED or APS concentration. Check the caster gasket for cuts or cracks and replace if necessary. Remove oxygen from the gel environment: Degas the monomer solution 5–10 min before pouring and then overlay the gel surface with water-saturated n-butanol. Troubleshooting problem Gel sandwich leaks while casting possible cause Dirty or damaged components remedy Plates. Dirty or damaged components Check that the gasket is not damaged or pinched. Incomplete gel polymerization Chemicals Use only recent stocks of the highest-quality reagents. On each spacer apply a light film of GelSeal compound to the bottom outside corner only. but up to 180°). spacers. and clamps are aligned and fit snugly into the upper chamber gasket. Check that both gaskets are centered and that the positioning ridges fit inside the grooves. Turn cam only as far as necessary to create a seal (usually 90–150°. Allow acrylamide gels to set for a minimum of 1 h. spacers. Check that the glass plates. Remove the comb at a slight angle and very slowly to prevent damaging the gel. If comb must be removed. Adjust the gel solution temperature to a minimum of 20 °C. Replace chipped plates (especially if chipped near the spacers). Load less sample. Prechill the buffer. (For instance. do not use Tris-HCl instead of Tris for Laemmli tank buffer. Fill the lower buffer chamber to the level appropriate for the run. Check for leaks. Voltage or current settings Bands are skewed or distorted Incomplete gel preparation and polymerization Irregular interface between stacking and running gels Sample preparation Stained sample collects: To increase or decrease the migration rate. do not back-titrate. Proteins may be degraded by endogenous proteases: use protease inhibitors during the isolation step. Near the top of the gel when the buffer front has reached the bottom Gel concentration Precipitation Gel concentration At both top and bottom of the gel um 80-6472-73 • p18 . (See Fig 8. and buffer dilution. Dialyze or desalt the sample. the buffer dam must be secure. The protein has precipitated. Heat the sample at a lower temperature (70 °C or less) for 1–2 min. Check recipes. Run the gel in the cold room. adjust the voltage or current by 25–50%. Overlay the running gel with water-saturated butanol before polymerization begins. Use only freshly deionized urea.SE 600 ruby • troubleshooting problem Power supply detects current leak possible cause Electrical path to outside ground/earth remedy Add more silicone grease to seal heat exchanger grommets. The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the full range of protein sizes. Reagent quality Dispose of older acrylamide solutions and use only stock of the highest quality. Decrease the current or voltage setting. gel concentrations. If used. all plates and spacers must be aligned and free of grease and cracks. Use magnetic stirrer and stir bar to keep buffer well mixed. Discard and prepare fresh buffer. Protein streaks vertically Particulates in sample Overloading Degradation Centrifuge or filter sample before loading to remove particulates. Add protease inhibitor such as PMSF.) Decrease the salt concentration of samples. Degas the stacking-gel solution and avoid trapping air bubbles under the comb teeth. Unusually slow (or fast) run Current leakage around gel Sample or reagent preparation If the required pH of a solution is overshot. Check for leaks or cracks in the heat exchanger. to avoid forming an uneven gel surface. Replace worn grommets. Near the buffer front Gel concentration Degradation Molecules are not sufficiently restricted by the resolving gel pore size: increase the %T. The gel pore size is too small: decrease the %T of the resolving (or stacking) gel. Dye front curves up (smiles) at edges Uneven heat distribution Excessive heat Circulate external coolant. page 13). avoid using solutions with high salt concentrations. Increase glycerol or sucrose to increase sample density. Dialyze or desalt the sample. Add protease inhibitors such as PMSF if necessary to prevent proteolytic degradation of sample. • p19 .5 times the height of the sample in the well. Adjust the sample volume or concentration. Use only gels that were recently prepared. Add a stacking gel or increase height of the stacking gel. Conduct the separation at a lower current or voltage setting to reduce Joule heating. Reagent quality Poor stacking Use only the highest-quality reagents. Store samples to be frozen in aliquots to avoid repeated freezethawing. Do not back-titrate buffers. Store at -40 to -80 °C. Add more mercaptoethanol or dithiothreitol.and stacking-gel solutions. Tracking dye doesn’t sharpen into a concentrated zone in the stacking gel Poor stacking Reagent quality Sample preparation Pour a taller stacking gel. Store sample on ice before it is denatured. Prepare the resolving-gel surface by first rinsing it with stackinggel monomer before pouring the stacking gel to ensure continuity between the gels. allow a stacking-gel height of 2. Heat samples in SDS sample buffer for no more than 1–2 min at 100 °C to improve dissociation of subunits. (For best results. When preparing samples. check sample treatment. Incomplete gel polymerization Sample preparation Allow gel to polymerize fully. Check pH values of the resolving.• troubleshooting problem Poor band resolution possible cause Running conditions remedy Begin electrophoresis as soon as the sample is loaded to prevent low molecular weight species from diffusing.) Dispose of outdated acrylamide solutions and use only the highest grade of acrylamide. Store on ice after heating. The %T in the stacking gel is lower because a larger pore size is required. Polyacrylamide gel composition is indicated by two percentages: %T (w/v) = total acrylamide = g (acryl + bis) × 100 100 ml %C (w/w) = crosslinker = g (bis) × 100 g (acryl + bis) The total percentage of acrylamide [%T (w/v)] in the resolving gel. the amount of crosslinker used [%C (w/w)] is 2.025 M Tris base 0.SE 600 ruby • appendix Appendix Laemmli system gels The Laemmli system is the most common electrophoresis protocol for SDS-denatured proteins. which can range from 5 to 20%.1% electrophoresis buffer *To achieve any other desired final concentration. 2. and the electrophoresis buffer contains Tris-glycine. More examples are given on page 23.N'-Tetramethylethylenediamine.05–0. TrisCl Tris-glycine pH SDS Ammonium persulphate (APS) TEMED† 8.192 M glycine ~8.1% (w/v) 0.N'. All buffers contain 0.6%.6%C 0. 2.* 2.3 0.1% 0. and the trailing ion is glycine.N.1% (v/v) 10%T. um 80-6472-73 • p20 . The leading ion in this discontinuous buffer system is chloride.1% SDS. the resolving gel and the stacking gel contain TrisCl buffers (of different concentration and pH).6%C.8 0. The stacking-gel composition is 4%T. Final concentrations separating gel Acrylamide Conc.8 0.125 M 0. Accordingly. adjust the acrylamide stock and water volumes. which produces a medium pore size. 23. 2. † N. determines the pore size. In the following example.05% (w/v) 0.6%C.05–0.375 M stacking gel 4%T.1% 0. the resolving-gel composition is 10%T. Commonly.05% (v/v) 6.6%C 0. Volumes for different concentrations are listed on p. 8 0.2) Deionized H2O Store at 4 °C away from light.6%C bis.6 g to pH 8.8 1. Remove the aqueous (lower) phase.3 g to pH 6.5 M TrisCl. 500 ml) Tris (FW 121. *N.45 µm filter.1) 4 N HCl Deionized H2O 3 0. 1 l) Tris (FW 121.375 M 3.5 mM 25 ml 1 ml to 100 ml (Resolving-gel overlay. 2 1. 0.44 mM 0.8 to 500 ml (4X stacking-gel buffer.8 (Solution #2) 10% SDS (Solution #4) Deionized H2O — OR — Water-saturated n-butanol Shake butanol and deionized H2O in a separatory funnel. — OR — If an overlay interferes with the preferred protocol. If yours does not.8 0. pH 8.5 M TrisCl. isolate the gel from atmospheric oxygen by placing a preparative comb or resolving-gel former on the gel.N’.375 M TrisCl.Methylene-bis-acrylamide.8%T 2. pH 8. 1 Acrylamide stock solution 30% (w/v) 0. replace it with fresh stock. Bis* (FW 154.35 M 10 g to 100 ml (100 ml) Sodium dodecylsulphate (SDS) (FW 288.8% (w/v) 60 g 1. • p21 .• appendix Solutions Note: Filter solutions 1–4 through a 0. 6 0. 1 ml) Ammonium persulphate (APS) (FW 228.5 M 30.1) 4 N HCl Deionized H2O 4 10% SDS solution 0.1% SDS.4) Deionized H2O 5 10% APS 0.6 g to 200 ml (30. Use the upper phase.5 M TrisCl.08) Caution! Acrylamide is a neurotoxin. Always wear gloves while handling in any form and wear a mask while weighing the powder. Repeat this procedure several times. Never mouth-pipette the solution. 100 ml) 1.1 g to 1 ml (Initiator.5 M 181. Important! Refer to the material safety data sheet (MSDS) accompanying each chemical for detailed handling and safety information. pH 6.2) Deionized H2O Fresh APS crackles when water is added. 200 ml) Acrylamide (FW 71. Prepare just prior to use.8 to 1 000 ml (4X resolving gel buffer. pH 8. 7% acetic acid.125 M 0. especially the sensitive but time.2) Bromophenol Blue (FW 691.192 M 3. used together with PlusOne™ staining kits. 0. 10 ml) 0.9) Divide into 1 ml aliquots and store at -70 °C.4) Glycerol (FW 92. 9 Coomassie Stain Protocol Coomassie stain solution (0.07) SDS (FW 288.3 0.175 mM 7 ml 1g 3 ml 0.8 (Solution #3) SDS (FW 288.and labor-consuming silver staining process.SE 600 ruby • appendix 7 2X Sample treatment buffer (0. pH 6.5 mM 15. pH 6.35 M 0.14 M 20% (v/v) 2% (v/v) 0.4) Deionized H2O The pH of this buffer is approximately 8.5 M TrisCl.3 mM 40% (v/v) 7% (v/v) 0. 9. 40% methanol.2 ml 0.192 M glycine. pH 8. 2 l) Coomassie Blue R-250 (FW 826) Methanol (stir until dissolved) Glacial acetic acid (99%) Deionized H2O 0.35 M TrisCl.3% DTT.2 mg to 10 ml — OR — 6X Sample treatment buffer (0.2) Bromophenol Blue (FW 691.35 M (Solution #4) Glycerol (FW 92. 10% SDS. ~10 ml) 0.5 ml 4 ml 2 ml 0.8. 0.1% SDS. 20% glycerol. pH 6. pH 6.09) 2-mercaptoethanol (FW 78.2 mg 8 0.93 g 1.6 M 0.09) DTT (FW 154.1) Glycine (FW 75. 7% acetic acid.9) Deionized H2O Divide into 1 ml aliquots and store at -40 °C to -80 °C.03 mM 2. Up to 20 l can be prepared and stored for up to 2 mo.8.35 M 30% (v/v) 0.13) [–OR– Dithiothreitol (DTT) (FW 154. 0.025% Coomassie Blue R-250 or R-350.2 mM 0. 1 l) Methanol Note: The Processor Plus with gel staining tray. Do not adjust pH.1 g 72 g 5g to 5 l (Electrophoresis buffer. 5 l) Tris (FW 121. simplifies and automates staining protocols. 30% glycerol.5 g 800 ml 140 ml to 2 l Destaining solution I (40% methanol.125 M TrisCl. 2% 2-mercaptoethanol.8 (Solution #3) 10% SDS.31 g] 0. 4% SDS. 5% methanol) Methanol Glacial acetic acid (99%) Deionized H2O 5% (v/v) 7% (v/v) 50 ml 70 ml to 1 l um 80-6472-73 • p22 .025 M Tris.3.025 M 0. 0.5 M TrisCl. Glacial acetic acid (99%) Deionized H2O 40% (v/v) 7% (v/v) 400 ml 70 ml to 1 l Destaining solution II (7% acetic acid. 0. 3 ml 14.036 ml • p23 .5% Acrylamide stock (Solution #1) 1. adjust as necessary.5–15%T range).1 ml 150 µl 10 µl 30 ml 0.5 M TrisCl.67 ml 1.5 M TrisCl.25 ml 0. The linear gradient recipe is for 100 ml of solution.5–10%T range) as well as smaller-pore gels (12.70 ml 15 g 25 ml 1 ml to 100 ml 0. Tabulated are ingredients and volumes for relatively large-pore gels (7. 18 × 16 cm gel). pH 6. A 4% stacking gel is common.6 ml 150 µl 10 µl 30 ml 7. Linear gradient gel (per 100 ml of solution) 10% T Acrylamide stock (Solution #1) Sucrose 1.• appendix Gel recipes The Laemmli gel recipes are for 30 ml of a single-concentration solution (enough for one 1.300 ml 0.060 ml 0. All gels are crosslinked with 2.5 ml 7.5 ml 7.036 ml 20% T 66.6 ml 150 µl 10 µl 30 ml 0.5 ml 12.5 M TrisCl.8 (Solution #2) 0.8 (Solution #2) 10% SDS (Solution #4) Deionized H2O 10% APS (Solution #5) TEMED 33.5 µl 5 ml For linear gradient gels.5 ml Stacking gel 4% 0. pH 8.1 ml 150 µl 10 µl 30 ml 0. and less still is added to the higher %T solution to allow polymerization to occur from the top down. use equal volumes of low-percentage and highpercentage acrylamide solutions.30 ml — 25 ml 1 ml to 100 ml 0.8 (Solution #3) 10% SDS (Solution #4) Deionized H2O 10% APS (Solution #5) TEMED Final volume 0.3 ml 12.6%C. 10 gel sandwiches can be poured in a multiple-gel caster at a flow rate of 5–10 ml/min. In our experience with the concentrations in the 10–20% gradient example below.5 ml 15% 15 ml 7.5 mm. Laemmli gel (per 30 ml of resolving-gel solution. 5 ml stacking-gel solution) Separating gel 10% 12.5% 10 ml 7. Less APS is added to extend polymerization time.05 ml 3 ml 25 µl 2.5 ml 7. pH 8. The total volume needed depends on the number of gels cast and the gel thickness.3 ml 9.3 ml 7. A.. L. eds. 680–685 (1970). Isoelectric focusing in immobilized pH gradients: principle. Bjellqvist. and Von Jagow. Biochem. Electrophoresis buffers for polyacrylamide gels at various pH values. M. N. J.. Cold Spring Harbor Laboratory. Biol.. Jul. Schreier. et al. T. 29(3):505–514 (1969).. (1998). and Burgess.. OSC 10. The reliability of molecular weight determinators by dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular Cloning: A Laboratory Manual. Staining proteins in gels. V. K. 74:5421–5425 (1977). K. R. May 25. Blomberg. G.6. Chem.. and Rickwood. H. 317–339 (1982). Denaturing gel systems Laemmli.. Jr. Biophys.. Norman G. C. and Gallagher.. H. Biophys. Soc. Arch. L. Electrophoresis 14.. Biochem. et al. K. Matsudaira. Willecke. OSC 10. City IRL Press (1990). Proteins of human urine. Natl.. Large Scale Biology Press (1991). P. High resolution two-dimensional electrophoresis of proteins. Electrophoresis 9. 166. et al. Sci. Initiation of mammalian protein synthesis.. 126. L. Acad. USA.. Nov. More than one-third of the discernible mouse polypeptides are not expressed in a Chinese hamster-mouse embryo fibroblast hybrid that retains all mouse chromosomes. 116(4):727–753 (1977). um 80-6472-73 • p24 . and Bogorad. and Tollaksen. Hedrick. 126.. Standard Formaldehyde Protocol. S. Anderson. 386–396 (1978).. Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins. Methods 6. methodology and some applications. Two-Dimensional Gel Electrophoresis Using the O’Farrell System. Biol. Proc. Biol. A. Anderson. A. Clin. H. Chem. 250(10):4007–4021 (1975). Non-denaturing gel systems Reisfeld. 368–379 (1987). Biochem. High resolution two-dimensional electrophoresis of human plasma proteins.). Mets. Amersham Biosciences (1999). T. 281 (1962). Sci. The current state of two-dimensional electrophoresis with immobilized pH gradients. P. T.. E. Proc. N. Weber. O’Farrell. T. A. L. Shapiro. Nature.SE 600 ruby • appendix Bibliography General Gallagher.. Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis...4. and Tanaka. J. R. Görg. USA. J. A. Fritsch. 94 (1982). and Smith. D.. I. Amersham Biosciences. 2-D Electrophoresis Using Immobilized pH Gradients. 1935–1945 (1995)... J.4. Anal. Nature. J. Jan.. Purification and characterization of seven initiation factors. (Ausubel. Bravo. eds. G. Anal. R.1–10. Anderson.. L.. L. Gel Electrophoresis of Proteins: A Practical Approach: Second edition. Bjellqvist. (Ausubel. 1375–1378 (1993).. B. et al. F. Two-dimensional electrophoresis with immobilized pH gradients: current state. R.. Sasse.. Biochem. Electrophoresis 16. 531–546 (1988). and Osborn. 81:802–806 (1986). W.6. Hames. J.. SDS microslab linear gradient polyacrylamide gel electrophoresis.1–10.).21 (1991). eds. Biochem. Two-dimensional electrophoresis Adams. F. Cleavage of structural proteins during the assembly of the head of bacteriophage T.. S. I.. and Staehelin. R.. Natl. J.. D... et al. OSC pp. NY (1990).). J.2. S. Schafer. Jun.. K. 79(7):2281–2285 (1982). Leigh and Anderson.. J. A. A. and Smith.13 (1992).. and Celis J. Anal. L. R. Acad. B. and Gallagher. et al. Current Protocols in Molecular Biology. Görg. et al. (Ausubel. Anal Biochem. Cold Spring Harbor. D. and Maizel J. Concentration and analysis by two-dimensional electrophoresis.. 155 (1968). McLellan. E. J.2. J. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Current Protocols in Molecular Biology. Anal. 10. Sambrook. Mol. Second Edition. B.8 (1991). D. 25(7): 1199–2210 (1979). S. Electrophoretic separation of proteins. Chem. F. Erni. A. Anderson. 130–132 (1993). Fey S. Trans. R. Biochem. Hurkman. Operation of the ISO-DALT® System. 87... H. Acidic buffer system for resolution of cationic proteins.. Protein Electrophoresis Technical Manual (80-6013-88). 21. Plant Physiology. MacDonald-Bravo. 227. L. Schaegger. A. F. Biochem. In Current Protocols in Molecular Biology. J.. 57(1):200–210 (1974). 224. 195. 4406–4412 (1969). B. Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins. Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two-dimensional gel electrophoresis. and Maniatis.. Two-Dimensional Electrophoresis. M. Size and charge isomer separation and estimation of molecular weights of proteins by discontinuous gel electrophoresis. U. A. Apr.1–10.. Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis: further data concerning resolving power and general considerations. No items can be accepted for servicing or return unless this form is properly completed.• customer service information Customer service information Technical service and repair Amersham Biosciences offers complete technical support for all our products. 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. • p25 . If you have any questions about how to use this product or would like to arrange to repair it. 1/4 oz. tube Spacer-Mate 1 2 2 1 1 1 1 1 4 1 1 1 1 1 1 80-6127-88 80-6174-43 80-6174-62 80-6175-19 80-6176-33 80-6476-15 80-6476-34 80-6177-09 80-6177-28 80-6177-47 80-6183-74 80-6183-93 80-6194-19 80-6421-43 80-6181-65 safety lid with cables 80-6476-34 upper buffer chamber 80-6176-33 heat exchanger 80-6183-74 lower buffer chamber 80-6476-15 um 80-6472-73 • p26 .5 mm thick.SE 600 ruby • customer service information Ordering information product quantity product number SE 600 Ruby Series Electrophoresis Unit for 18 × 16 cm gels SE 600 Ruby basic unit Includes: 3 sets of glass plates. 1 80-6479-57 Replacement Parts Wonder Wedge Gel Plate Separation tool Slotted silicone rubber gaskets for upper buffer chamber Laminated silicone rubber gaskets for casting stand Buffer dam Upper buffer chamber for SE 600 Ruby Lower buffer chamber for SE 600 Ruby Lid with high-voltage leads for SE 600 Ruby High-voltage safety lead set Grommets for heat exchanger/lower electrode assembly Banana plug. dual-gel casting stand with leveling base and level. Spacer-Mate alignment template and Wonder Wedge Gel Plate Separation tool.) 1 80-6479-38 SE 600 Ruby complete unit Includes: basic unit plus two 15-well combs and 2 sets of spacers 1. 6 cams. (Order 2 combs and 2 sets of 16 cm spacers separately. buffer dam. with 2 washers SE 600 Ruby Heat exchanger/lower electrode assembly Glass tube with 2 grommets for heat exchanger/lower electrode assembly Spirit level GelSeal compound. gold. Spacer-Mate alignment template.) For up to 10 gels: Multiple Gel Caster Kit. 16 cm 1 12 4 2 2 80-6173-67 80-6173-48 80-6174-24 80-6187-35 80-6173-29 choose the appropriate spacer and plate length for your unit gasket 80-6174-62 universal clamp 80-6173-29 basic caster 80-6175-00 spirit level 80-6194-19 cam 80-6174-24 • p27 . 18 × 16 cm (Order combs. 2 gels. 100 sheets of wax paper. 5 filler sheets. black. spacers. (One included with each SE 600 Ruby unit. and glass plates separately. for clamps with cam holes Clamp assemblies. and filler plugs.) For up to 4 gels: Gel Caster Kit. 18 × 16 cm Includes: 20 glass plates.) Gel Caster only. 100 sheets of wax paper. 18-cm wide Includes: 2 blank gaskets for 1 or 2 gels. basic. 3 space-saver plates.) Multiple Gel Caster only.• customer service information product quantity product number Gel casters For 1 or 2 gels: Dual Gel Caster. spacers. (Order combs and spacers separately. 4 gels. 18 × 16 cm Includes: 8 glass plates. and Spacer-Mate alignment template. space-saver plate. (Order combs and spacers separately. 18 × 16 cm (Order combs. 10 gels.) 1 80-6182-79 1 80-6191-34 1 80-6175-00 1 80-6191-53 1 80-6183-36 Clamps and cams Clamp and Cam Kit. 5 filler sheets. and glass plates separately. 10 gels. 4 gels. four 16 cm clamps and 8 black cams Replacement thumbscrews for clamps Cams. 8 cm Clamp assemblies. 1 2. Microtiter combs no. Comb depth 15 mm.7 5.50 0.00 1.75 1.75 1.50 0.7 4.7 2. club sandwich divider.3 8.00 1.1 4.50 0.7 2.50 0.7 5.3 8.6 7.50 0. all others 25 mm.6 5.5 width (mm) 6 6 1 1 80-6460-00 80-6460-19 um 80-6472-73 • p28 .00 1.75 1.1 4.75 1.00 1.50 width (mm) 8. notched 2 1 80-6178-99 80-6179-18 2 1 80-6186-59 80-6186-78 Teflon combs number of wells 10 10 10 10a 10a 12 12 12 15 15 15 20 20 20 28 b thickness (mm) 0.6 7.75 1.3 8.3 8.3 7.00 1.SE 600 ruby • customer service information product quantity product number Glass plates 18 × 8 cm Glass plates Glass plate. notched 18 × 16 cm Glass plates Glass plate. of wells 14 14 thickness (mm) 1 1.75 1.7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 80-6159-99 80-6160-18 80-6160-37 80-6092-54 80-6092-73 80-6160-75 80-6459-24 80-6160-94 80-6161-13 80-6161-32 80-6161-51 80-6161-70 80-6161-89 80-6162-08 80-6162-27 80-6162-46 80-6162-65 28b 28b a b Also for use with the PR 200 Deca-Probe Incubation Manifold. club sandwich divider. no.5 0.50 width (mm) prep/ref 121/6 121/6 121/6 113/6 113/6 113/6 1 1 1 1 1 1 1 80-6164-17 80-6164-36 80-6164-55 80-6163-41 80-6163-60 80-6163-79 80-6163-22 Adjustable comb back Required to convert any 25-mm deep comb to 10 or 15 mm depth.75 1. Spacers thickness (mm) 0.5 0.50 0.0 1.0 1.75 1.• customer service information product quantity product number Preparative combs These combs are 25 mm deep.5 length (cm) 8 8 8 8 8 16 16 16 16 16 16 width (cm) 2 2 2 1 1 2 2 2 1 1 1 2 2 2 2 2 2 2 2 2 2 2 80-6187-73 80-6187-92 80-6188-11 80-6443-09 80-6443-28 80-6180-51 80-6180-70 80-6180-89 80-6179-75 80-6179-94 80-6180-13 • p29 .75 1 1.75 1.0 1. adjustable to 10 or 15 mm. of wells prep/ref 1/1 1/1 1/1 1/2 1/2 1/2 thickness (mm) 0.5 1.75 1 1.0 1. base unit 19 × 29 cm staining tray 29 × 35 cm staining tray quantity 1 1 1 2 2 1 1 1 product number 80-6116-29 18-1102-77 18-1102-78 80-6115-15 80-6115-53 80-6444-04 80-6444-99 80-6445-37 2-D Electrophoresis Tube Gel Adaptor Kit (for use with carrier ampholytes) Includes: 24 tubes. 400 mA.d.SE 600 ruby • customer service information Companion products product SE 100 Plate Mate washing and storage unit MultiTemp III Thermostatic circulator 115 VAC 230 VAC QuickFit connectors. 400 mA. × 15 cm. female 3/8" QuickFit connectors. 100 W) 1 1 1 80-6406-99 18-1130-01 18-1130-02 PlusOne reagents Acrylamide PAGE Acrylamide PAGE 40% Solution Acrylamide IEF Acrylamide IEF Acrylamide IEF 40% Solution N. 1. Protein Bromophenol Blue 250 g 1l 250 g 1 kg 1l 25 g 25 g 25 ml 500 g 500 g 100 g 40 1 10 g 17-1302-01 17-1303-01 17-1300-01 17-1300-02 17-1301-01 17-1304-01 17-1311-01 17-1312-01 17-1321-01 17-1323-01 17-1313-01 17-0518-01 17-1150-01 17-1329-01 um 80-6472-73 • p30 .5 mm i. 2 000 mA.Methylene-bisacrylamide Ammonium persulphate TEMED Tris Glycine Sodium dodecylsulphate Coomassie Blue R tablets Silver Staining Kit.N'. 80 W) EPS 601 (600 V. male 3/8" Processor Plus. 200 W) EPS 301 (300 V. and 24 stoppers IPGphor Isoelectric Focusing System 2-D Electrophoresis Technical Manual Protein Electrophoresis Technical Manual Multiphor II Electrophoresis System 1 1 1 1 1 80-6175-38 80-6414-02 80-6429-60 80-6013-88 18-1018-06 Power supplies EPS 2A200 (200 V. . PlusOne. Multiphor.Immobiline. RBS-35 is a registered trademark of Chemical Products. Printed in the USA. Amersham and Amersham Biosciences is a trademark of Amersham plc. IPGphor. All rights reserved. © 2000 Amersham Biosciences Inc.amershambiosciences. MultiTemp. and Ruby are trademarks of Amersham Biosciences Limited or its subsidiaries. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. Coomassie is a trademark of ICI plc. A copy of these terms and conditions is available on request. Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE-751 84 Uppsala Sweden Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D-79111 Freiburg Germany www.com .