Hoefer SE 400 User Manual
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Hoefer SE 400 User Manual

The sturdier vertical slab gel electrophoresis units
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SE 400/SE 410
Hoefer SE 400/SE 410
the Sturdier vertical slab gel electrophoresis units
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SE400-IM/Rev. A0/06-04

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Summary of Contents for Hoefer SE 400

  • Page 1 SE 400/SE 410 Hoefer SE 400/SE 410 the Sturdier vertical slab gel electrophoresis units SE400-IM/Rev. A0/06-04...

  • Page 3: Table Of Contents

    Page finder 1. Gel electrophoresis unit function and description Unpacking and disassembly ..........2 Annotated inventory ............4 Specifications ..............6 2. Operating instructions 2.1. Gel casting preparation 2.1.1. options—precast and self cast gels .... 7 2.1.2. preliminary casting steps......8 2.2.
  • Page 4 • Only accessories and parts approved or supplied by Hoefer, Inc. may be used for operating, maintaining, and servicing this product. • Utiliser seulement un générateur marqué CE ou Français...
  • Page 5 The basic unit includes one set of glass plates (18 × 16 cm for the SE 400, and 18 × 24 cm for the SE 410), two clamp assemblies (SE 400: two 16 cm clamps; SE 410: two 16 cm clamps and two 8 cm clamps), and two cams.

  • Page 6: Unpacking And Disassembly

    Unpacking and disassembly Unwrap all packages carefully and compare contents with the packing list, making sure all items arrived. If any part is missing, contact your local sales office. Inspect all components for damage that may have occurred while the unit was in transit.
  • Page 7 Fig 1. SE 400 series main components. Included but not shown: safety lid Cams GelSeal grease, 1/4 oz. Spacer-Mate Wonder Wedge Level Required but not included: Approved power supply color-coded connectors (2) upper buffer chamber glass plates (2) clamps casting cradle...

  • Page 8: Annotated Inventory

    Glass plates. Two 18-cm wide glass plates are included. Plates for the SE 400 are 16 cm long, and plates for the SE 410 are 24 cm long. (A notched divider plate, ordered separately, can be used to run two gels at the same time.)
  • Page 9 ing stand and provides the seal for the bottom of the gel sandwich. The slotted gasket fits under the upper chamber and provides the seal between the sandwich and the upper chamber. Two ridges help position this gasket. Spacer-Mate assembly template. Aligns spacers for sandwich assembly.

  • Page 10: Specifications

    Installation category: II Pollution degree: II Dimensions (w × h × d) SE 400: 24 × 28 × 15 cm (9.5 × 11 × 6 in.) SE 410: 24 × 36 × 15 cm (9.5 × 14.2 × 6 in.) Product certifications EN61010–1, UL3101–1,...

  • Page 11: Operating Instructions

    Appendix B gives a bibliography. 2.1. Gel casting preparation 2.1.1. options—precast gels and self-cast gels The SE 400 unit accepts standard precast gels purchased from commercial suppliers as well as self-cast gels, which can be prepared using the built-in casting stand. (To cast multiple 14 ×...

  • Page 12: Preliminary Casting Steps

    A 24-cm sandwich requires two clamp assemblies on each side. Align each end separately. That is, align 16 cm 24 cm (SE 400) (SE 410) one end, finger-tighten the screws, turn the sandwich 180° and align the other end. In each case allow the clamp to slide down and align perfectly with the top Fig 2.
  • Page 13 2-gel sandwich A 16- or 24-cm long notched divider plate (ordered separately) doubles the number of gels that can be cast and run (see Fig 4). Assemble in the same manner as a single gel sand- wich, except before placing the top glass plate, lay the divider plate atop the first set of spacers and a second set of spacers atop the divider plate.
  • Page 14 glass plates (2) spacers clamps (the number required depends on the plate length.) cams (2) insert the cam(s) in the cam holes and turn toward the center to lock the glass plate assembly. gasket (foam side down) casting cradle leveling feet (4) Note: It is easier to keep the caster balanced if you turn both cams toward the center of the caster.

  • Page 15: Acrylamide Gel Preparation

    2.2. Acrylamide gel preparation Table 1. Approximate monomer solution volume required for a single gel Gel thickness (mm) Model 0.75 1.00 SE 400 15 ml 23 ml 30 ml SE 410 23 ml 34 ml 45 ml 2.2.1. resolving gel Prepare the monomer solution and pour the gel.

  • Page 16: Stacking Gel

    If combs are in place, skip to step 4. If no combs are in place, overlay the resolving gel with a thin layer of water-saturated n-butanol, water, or diluted gel buffer to prevent exposure of the top surface of the gel solution to atmospheric oxygen. Slowly deliver the overlay solution from a glass syringe fitted with a 22-gauge needle.

  • Page 17: Gradient Gel

    Linear gradient gels can be poured in the gel caster. For easy gradient mixing, we recommend using one of the Hoefer SG series gradient makers. Gradient gels are poured from the top of the caster with a cannula if using the provided gel caster or from the bottom if using a Hoefer multiple gel caster (see instructions accompanying the caster).
  • Page 18 Mix the gradient. While the solution is stirring, begin pumping (5–10 ml/min) from the mixing chamber and immediately open the stopcock to the reservoir cham- ber. Raise the cannula as liquid enters the sandwich, keeping the tip at the gel surface. Overlay each gel with a thin layer of water-saturated n-butanol, water, or diluted gel buffer to prevent gel exposure to oxygen.

  • Page 19: Sample Preparation

    2.3. Sample preparation The amount of sample loaded depends on the thickness of the gel, the sensitivity of the detec- tion method used, and the amount of sample expected in each band. In a continuous buffer system, the protein sample should be relatively concentrated because no stacking gel is used.

  • Page 20: Final Assembly

    2.4. Final assembly Note: Before the first use, Rinse both buffer chambers with water and distilled disassemble the unit and water thoroughly before each use. wash with a dilute solution of a laboratory detergent and rinse thoroughly, first with water and then distilled water. Install the gel sandwich in the lower buffer chamber Release the sandwich from the caster by removing both cams.
  • Page 21 Fig 7. Upper buffer chamber assembly: First place the upper chamber onto the sandwich assembly, then insert the cams into the cam holes, ridge (short end) pointing down. To secure the assembly, turn the cams a full 180° so that the ridge points up (not shown).
  • Page 22 Pour ~100 ml of electrophoresis buffer into the upper Note: Do not force the cams. chamber, directing the buffer stream against the wall If encountering unusual resis- to avoid disturbing the samples. Inspect the installation tance, disassemble the unit for leaks. Fill both chambers (the final volume for each and inspect clamp and glass chamber is ~350 ml).
  • Page 23 shield guides Fig 8. Safety lid installation. If you are unfamiliar with the installation, please note: The upper electrode is protected by a The safety lid seats effortlessly recessed shield, which rests in the upper buffer if all three features are properly chamber once the lid is installed.

  • Page 24: Resolving The Sample

    Gel length (cm) model final voltage (V) the length of the plate. SE 400 200–250 SE 410 275–325 *Thicker or thinner gels require proportionally more or less current. For example, a 0.75 mm gel , which is half as thick as a 1.5 mm gel, requires half as much current, or 12.5 mA.
  • Page 25 The starting voltage for a 1.5 mm slab gel connected to a power supply set to 25 mA is usually 80–90 V (for the SE 400 model and a Laemmli discontinuous buffer system). The final voltage is typically 200–325 V, depending on the length of the gel.
  • Page 26 Record each run Keep a record of the current or voltage setting, number and thickness of gels, buffer system, and the starting and final current or voltage readings for each run so that results can be compared. Inconsistent results for the same system and settings can indicate potential problems such as current leaks, incorrect buffer concentrations, high salt concentrations, or inconsistent...

  • Page 27: After Electrophoresis

    2.6. After electrophoresis Tip: To avoid splashing, add Once the tracking dye reaches the bottom of the gel, staining or fixative solution turn off the power supply and disconnect the leads. to the tray after the gel is Remove the safety lid, using finger leverage—rest transferred.

  • Page 28: Care And Maintenance

    3. Care and maintenance Cleaning • Rinse with water immediately after use. • Do not autoclave or heat any part of the instrument above 45 °C. • Do not use organic solvents, abrasives, strong cleaning solutions, or strong acids or bases to clean any plastic part.

  • Page 29: Troubleshooting

    4. Troubleshooting problem/possible cause remedy Gel sandwich leaks while casting Dirty or damaged components Plates, spacers, and the gasket must be completely clean. Wash if necessary. Replace chipped plates (especially if chipped near the spacers). Check the caster gasket for cuts or cracks and replace if necessary.
  • Page 30 problem/possible cause remedy Upper buffer chamber leaks Mis-aligned parts Check that the glass plates, 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. Dirty or Check that the gasket is not damaged or pinched.
  • Page 31 problem/possible cause remedy Stained sample collects: Near the buffer front Gel concentration Molecules are not sufficiently restricted by the resolving gel pore size: increase the %T. Degradation 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...
  • Page 32 problem/possible cause remedy Tracking dye doesn’t sharpen into a concentrated zone in the stacking gel Poor stacking Pour a taller stacking gel. (For best results, allow a stacking- gel height of 2.5 times the height of the sample in the well.) Reagent quality Dispose of outdated acrylamide solutions and use only the highest grade of acrylamide.

  • Page 33: Laemmli System Gels

    Appendix A. Laemmli System Gels Table 4. Laemmli gels — final concentrations Electrophoresis Resolving gel Stacking gel buffer Acrylamide conc. 10% T*, 2.6% C 4% T, 2.6% C Tris-Cl 0.375 M 0.125 M Tris-Glycine 0.025 M Tris base 0.192 M glycine ~8.3 0.1% 0.1%...
  • Page 34 Solutions 1. Acrylamide stock solution Note: Filter solutions 1–4 through a 0.45 µm filter. (30.8% T 2.6% C Bis, 200 ml) Acrylamide (FW 71.08) 30% w/v 60.0 g Bis* (FW 154.2) 0.8% w/v 1.6 g Important! Refer to the material safety data sheet Deionized H to 200.0 ml (MSDS) accompanying each...
  • Page 35 6. Resolving gel overlay (0.375 M TrisCl, 0.1% SDS, pH 8.8, 100 ml) 1.5 M Tris-Cl, pH 8.8 (Solution #2) 0.375 M 25.0 ml 10% SDS (Solution #4) 3.5 mm 1.0 ml Deionized H to 100.0 ml Store up to 3 months at 4 °C in the dark. —OR—...
  • Page 36 8. Electrophoresis buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3, 5.0 liters) Tris (FW 121.1) 0.025 M 15.1 g Glycine (FW 75.07) 0.192 M 72.1 g SDS (FW 288.4) 3.5 mm 5.0 g Deionized H to 5.0 liters The pH of this buffer is approximately 8.3.
  • Page 37 14. Silver Nitrate solution (0.1% w/v silver nitrate) 1 g silver nitrate Distilled water 1 to L. 15. 3% Sodium Carbonate solution (3% w/v) 60 g sodium carbonate Bring to 2 L with distilled water, store in glass container. 16. Developing solution (3% sodium carbonate, 0.019% formaldehyde) 200 ml of 3% sodium carbonate 100 µl of 37% formaldehyde...
  • Page 38 Coomassie Stain Protocol A. Stain gel in coomassie stain solution at room temperature overnight. Gels can also be stained rapidly by placing them at 55 °C in a shaking water bath for 30–45 min. B. Place gel in Destain solution I at room tempera- ture.
  • Page 39 Soak the gel in 500 ml of deionized water overnight. The next day, rinse the gel with several changes of deionized water over 30–60 minutes. D. Place the gel in 100–200 ml of 5 µg/ml DTT in deionized water for 30 minutes. E.
  • Page 40 Gel recipes The Laemmli gel recipes are for 30 ml of a single concentration solution (enough for one 1.5-mm 18 × 16 cm gel). Tabulated are ingredients and volumes for relatively large pore gels (7.5 to 10%T range) as well as smaller pore gels (12.5 to 15%T range). A 4% stacking gel is common.

  • Page 41: Bibliography

    Appendix B. Bibliography General Gallagher, S.R., and J.A. Smith., Electrophoretic separation of proteins. In Current Protocols in Molecular Biology. (F.A. Ausubel, et. al, eds.) 10.2.1–10.2.21 (1991). Hames, B. D. and Rickwood, D., Gel Electrophoresis of Proteins, A Practical Approach. Second edition, IRL Press (1990). Sambrook, J, Fritsch, E.F.
  • Page 42 Native gel systems Reisfeld, R.A., et al., Acidic buffer system for resolution of cationic proteins. Nature. 195, 281 (1962). McLellan, T. Electrophoresis buffers for polyacrylamide gels at various pH values. Anal. Biochem. 126, 94 (1982). Hedrick, J.L. and Smith, A.J., Size and charge isomer separation and estimation of molecular weights of proteins by discontinuous gel electrophoresis.

  • Page 43: Ordering Information

    Ordering information product quantity product number Hoefer SE 400 Sturdier Vertical Slab Electrophoresis Units for 18 × 16 cm gels SE 400 Sturdier Vertical Unit, complete. SE400-15-1.5 Includes: one set of glass plates 18 × 16 cm, 2 clamps assemblies, 2 cams and 15-well comb and 2 spacers, 1.5-mm thick.
  • Page 44 1.00 SE511-28-1.0 1.50 SE511-28-1.5 Also for use with the Hoefer PR 200 Deca-Probe Incubation Manifold. Comb depth 15 mm; all others 25 mm. Preparative combs These combs are 25 mm deep, adjustable to 10 or 15 mm. no. of wells...
  • Page 45 Multiple Gel Caster Kit, 10 gels, 18 × 16 cm SE615 Includes: 20 glass plates, space-saver plate, 5 filler sheets, 100 sheets of wax paper, and Space Mate alignment template. Recommended Hoefer SE 100 Plate Mate washing and storage unit. SE100 •...
  • Page 47 Coomassie is a trademark of ICI plc. RBS-35 is a trademark of Pierce Chemical Co. Teflon is a trademark of E.I. du Pont de Nemours & Co. Printed in the USA Hoefer, Inc. 953 Indiana Street San Francisco, CA 94107 USA www.hoeferinc.com •...

This manual is also suitable for:

Se 410

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