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Amersham Biosciences Hoefer DALT User Manual

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Hoefer
DALT System
DALT Electrophoresis Tank
DALT Gradient Maker
DALT Multiple Gel Caster
DALT Blotting Kit
User Manual
80-6431-50
Rev A/12-98

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Summary of Contents for Amersham Biosciences Hoefer DALT

  • Page 1 Hoefer DALT System DALT Electrophoresis Tank DALT Gradient Maker DALT Multiple Gel Caster DALT Blotting Kit User Manual 80-6431-50 Rev A/12-98...
  • Page 3 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.
  • Page 4 Ankündigung zu ändern. Garantie et responsabilité Gewährleistung and Haftung Amersham Biosciences garantit à l’utilisateur que le produit livré a subi Amersham Biosciences garantiert, daß das gelieferte Produkt sorgfältig avec succès tous les essais prévus pour s’assurer qu’il est conforme aux spécifica- auf die Einhaltung der veröffentlichten Spezifikationen getestet wurde.

  • Page 5: Table Of Contents

    Hoefer DALT System ONTENTS Hoefer DALT System Function and Description DALT System Components Unpacking Important Information/Informations Importantes 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...
  • Page 6 Hoefer DALT System 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...

  • Page 7: Hoefer Dalt System Function And Description

    (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.
  • Page 8 Hoefer DALT System Function and Description Hoefer DALT System some leakage current bypassing 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 (+).
  • Page 9 Hoefer DALT System Hoefer DALT System Function and Description Gel Cassettes Figure 3. DALT gel cassettes The DALT 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.
  • Page 10 Hoefer DALT System Function and Description Hoefer DALT System 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.

  • Page 11: 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.

  • Page 12: Important Information/Informations Importantes

    Only accessories and parts approved or supplied by • Si l'instrument n'est pas utilisé en conformité avec les Amersham Biosciences may be used for operating, recommandations du fabriquant, les protections de maintaining, and servicing this product. sécurité qui équipent cet appareil peuvent être rendues inéfficaces.

  • Page 13: Specifications

    Hoefer DALT System Hoefer DALT System Function and Description Specifications DALT Electrophoresis Tank with Buffer Circulation Pump Gel capacity, 1.0 or 1.5 mm thick 10 gels Electrophoresis buffer volume 20 liters Blotting buffer volume 22 liters Dimensions (h × w × d) lid closed: 38 ×...
  • Page 14 *This declaration of conformity is only valid for the instrument when it is: • used in laboratory locations, • used as delivered from Amersham Biosciences, except for alterations described in the User Manual, and • connected to other CE-labeled instruments of products recommended or approved by Amersham Biosciences.

  • Page 15: Required Or Convenient, But Not Supplied

    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.

  • Page 16: Preparing The Gel Caster

    Preparing the Gel Caster 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 when you open it to remove the gels.
  • Page 17 Hoefer DALT System Preparing the Gel Caster 5. 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.

  • Page 18: Casting Homogeneous (Non-Gradient) Gels

    Casting Homogeneous (Non-Gradient) Gels Hoefer DALT System 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.
  • Page 19 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.

  • Page 20: Preparation For Gradient Gel Casting

    Preparation for Gradient Gel Casting Hoefer DALT System 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.

  • Page 21: Calibrating The Peristaltic Pump

    Hoefer DALT System Preparation for Gradient Gel Casting 3. 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. 4. To make a funnel for introducing heavy solution into the left side, bend the remaining top of the divider to the right.

  • Page 22: Casting Gradient Gels

    Casting Gradient Gels 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.

  • Page 23: Pouring Gel Solutions For Gradient Gels

    Hoefer DALT System Casting Gradient Gels 2. Configure the gradient divider for the number of gels you are casting. If necessary, calibrate the gradient pump flow rate. See “Calibrating the Peristaltic Pump” on page 17. 3. Be sure that the faceplate screws on the gradient maker are tightened hand tight and the gradient-maker lines are all clamped off.
  • Page 24 Casting Gradient Gels Hoefer DALT System Figure 11. Priming with light solution Light solution 5. Close both clamps again. All three clamps should now be closed. 6. 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.
  • Page 25 Hoefer DALT System 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. 10. 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).

  • Page 26: Applying Overlay To Dalt Slab Gels

    Casting Gradient Gels 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.

  • Page 27: Unloading The Gel Caster

    Hoefer DALT System 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. 2. Carefully unload the cassettes from the unit.

  • Page 28: Preparing The Dalt Tank For Electrophoresis

    Preparing the DALT Tank for Electrophoresis Hoefer DALT System Preparing the DALT Tank for Electrophoresis 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.
  • Page 29 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.
  • Page 30 Preparing the DALT Tank for Electrophoresis Hoefer DALT System Figure 14. Raising the barrier combs to allow buffer circulation 5. Make a note on 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.

  • Page 31: 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 can be run simultaneously.
  • Page 32 Loading and Running Second Dimension Gels Hoefer DALT System Iodoacetamide alkylates sulfhydryl groups on proteins, preventing their re-oxidation during electrophoresis. Protein re-oxidation during electrophoresis can result in streaking and other artifacts. Iodoacetamide also alkylates residual DTT to prevent point streaking and other silver staining artifacts. Iodoacetamide is introduced in an optional second equilibration step.

  • Page 33: Loading The Ipg Strips Onto The Dalt Slab Gels

    Hoefer DALT System 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.
  • Page 34 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.

  • Page 35: Loading Cassettes Into The Dalt Tank

    Hoefer DALT System 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).

  • Page 36: Electrophoresis Conditions In The Dalt Tank

    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.

  • Page 37: Unloading The Dalt Cassettes

    Hoefer DALT System 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.

  • Page 38: The Dalt Blotting Kit

    The DALT Blotting Kit 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.

  • Page 39: Assemble The Transfer Cassette

    Hoefer DALT System The DALT Blotting Kit Figure 21. DALT Electrophoresis Tank, with barrier combs removed Fill the tank with water before turning the pump on. The pump is not MPORTANT self-priming and can be damaged if run dry. 1. Fill the tank with 15 liters of water or used electrophoresis buffer.
  • Page 40 The DALT Blotting Kit Hoefer DALT System Place one 6-mm-thick foam sponge on the opened submersed cassette and press Note Carefully position the gel. Proteins may begin gently until all air is expelled. Place one sheet of blotting paper on the sponge, and to transfer immediately.

  • Page 41: Loading The Cassettes

    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.
  • Page 42 The DALT Blotting Kit 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.
  • Page 43 Hoefer DALT System 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.
  • Page 44 The DALT Blotting Kit 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.
  • Page 45 Hoefer DALT System 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) 900 g Bis (N, N’ methylenebis-acrylamide, purest grade, FW 154.17) 0.8%...
  • Page 46 Recipes Hoefer DALT System 10% TEMED Final Conc. Amount TEMED (v/v, MW 116.2) 0.5 ml 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 Tris-Cl (1.5M, pH 8.8)
  • Page 47 Hoefer DALT System 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)
  • Page 48 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 317.1 g...
  • Page 49 Hoefer DALT System 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.

  • Page 50: Final

    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.

  • Page 51: 10% Sds

    Hoefer DALT System Homogeneous Gel Solutions 25 gels, 1.0 mm 1500 ml. Amounts shown in ml. Final %T Acryl. Stock 1.5M Tris-Cl, pH 8.8 Water 10% SDS 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% APS 3.68...
  • Page 52 Homogeneous Gel Solutions Hoefer DALT System 23 gels, 1.5 mm 1800 ml. Amounts shown in ml. Final %T Acrylamide 1020 1080 1140 Stock 1.5M Tris- pH 8.8 Water 10% SDS 10% 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 4.41...
  • Page 53 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.

  • Page 54: Glycerol

    Gradient Gel Solutions Hoefer DALT System For 25 gels, 1.0 mm 750 ml each Heavy and Light, by volume in ml. Light Solution Final %T Acryl. Stock 1.5M Tris-Cl, pH 8.8 Water 7.50 7.50 7.50 7.50 7.50 7.50 7.50 7.50 7.50...
  • Page 55 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 Acryl. Stock 1.5M Tris-Cl, pH 8.8 Water 10% SDS Glycerol 10% APS 0.98 0.86 0.76 0.68...
  • Page 56 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 Acryl. Stock 1.5M Tris-Cl, pH 8.8 Water 10% SDS Glycerol 10% APS 2.21 1.93 1.71 1.54...
  • Page 57 Hoefer DALT System Gradient Gel Solutions For 12 gels, 1.5 mm 550 0 0 0 m m m m l l l l each Heavy and Light, by volume in ml. Light Solution Final %T Acryl. Stock 1.5M Tris-Cl, pH 8.8...
  • Page 58 Gel Identification Numbers 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.
  • Page 59 Hoefer DALT System Care and Maintenance Care and Maintenance Cleaning • Do not autoclave or heat any part above 45 °C. • Do not expose the unit or its parts to organic solvents. • If using radioactive reagents, decontaminate the unit with a cleaning agent such as CONTRAD 70 or Decon 90 from Decon Laboratories, Inc.
  • Page 60 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. • Check the foam gasket for cracks or nicks and replace if necessary.
  • Page 61 Hoefer DALT System Troubleshooting Power Supply detects current leak • Cracked or broken heat exchangers. Call your Amersham Biosciences Service representative. Spots are skewed or distorted • Gels run too fast-uneven migration. • Overlay the running gel with water-saturated n-butanol before polymerization begins to avoid forming an uneven gel surface.
  • Page 62 Troubleshooting Hoefer DALT System Poor Spot Resolution • Allow gel to polymerize fully. • Begin electrophoresis as soon as the IPG strips are loaded to prevent low molecular weight species from diffusing. • Conduct the separation at a lower current or voltage setting.
  • Page 63 Hoefer DALT System 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 (≥ 10%), equilibrate the gel before in transfer buffer for 30 minutes to allow it to shrink before assembling the stack.
  • Page 64 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., Görg, A., Westermeier, R., Postel, W. 1982. Isoelectric focusing in immobilized pH gradients: principle, methodology, and some applications.
  • Page 65 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.
  • Page 66 Customer Service Information Hoefer DALT System Qty. Code No. 80-6069-17 DALT Blotting Kit with rack, 5 transfer cassettes and sponges, blotting paper (50 pc) DALT Transfer Cassette, with 2 sponges 80-6069-55 Sponges 80-6069-74 DALT Blotting Paper, 24 × 20 cm (50/pkg)
  • Page 67 14, 19, 42 application 22 creatine kinase charge standards 45 gel storage 23, 42 crosslinker concentration 56 gels culture tubes, screw-cap 11 gradient 18 to 21 non-gradient 14 GelSeal 11, 56 glycerol, in equilibration 27 Amersham Biosciences 63...
  • Page 68 Index Hoefer DALT System gradient gels proteins casting 18 to 21 contamination 26, 35 solutions 49 migration 59 troubleshooting 56 spots on gels 22 transfer from gel 34 gradient maker 16 transfer parameters 38 divider configuration 16 description 6 proteolytic degradation 58...
  • Page 69 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.
  • Page 70 Printed in the USA...

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