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Hoefer SE 600 Chroma User Manual

Standard dual cooled gel electrophoresis unit
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SE 600 Chroma
Hoefer SE 600 Chroma
standard dual cooled gel electrophoresis unit
um
SE600X-IM/Rev. A0/05-04

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Summary of Contents for Hoefer SE 600 Chroma

  • Page 1 SE 600 Chroma Hoefer SE 600 Chroma standard dual cooled gel electrophoresis unit SE600X-IM/Rev. A0/05-04...

  • Page 3: Table Of Contents

    Page finder Gel electrophoresis unit function and description Specifications ..............2 Important information ............3 Unpacking and inventory............5 Operating instructions Prepare the gel sandwich..........9 Construct the gel sandwich & insert into caster.....9 Acrylamide gels..............12 Gradient gels................14 Sample preparation and loading...........16 Final assembly..............18 Separating the sample ...........22 After electrophoresis............24 Care and maintenance .............25...

  • Page 5: Gel Electrophoresis Unit Function And Description

    Gel electrophoresis unit function and description ™ ™ The Hoefer SE 600 Chroma vertical slab gel electrophoresis unit is intended for protein and nucleic acid electrophoresis under commonly used denaturing and non-denaturing conditions. Up to 28 samples can be compared on a single slab gel.

  • Page 6: Specifications

    This declaration of conformity is valid only when the instrument is: • used in laboratory locations, • used as delivered from Hoefer, Inc. except for alterations described in the user manual, and • connected to other CE-labeled instruments or products recom- mended or approved by Hoefer, Inc.

  • Page 7: Important Information

    45 °C continuous duty. Circulate coolant through the heat exchanger during electrophoresis to minimize heating. Overheating will cause irreparable damage to the unit! • Only accessories and parts approved or supplied by Hoefer, Inc. may be used for operating, maintaining, and servicing this product.
  • Page 8 Fig 1. Main components of the leads (2) Hoefer SE 600 Chroma (see Fig 4 for caster compo- nents). safety lid Included but not shown: • Gel Seal compound, 1/4 oz. • Spacer-Mate alignment interlock pins template • Well-locating decal •...

  • Page 9: Unpacking And Inventory

    Unpacking and inventory 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 10 Always install the safety lid before use! Glass plates The SE 600 Chroma accommodates 18-cm-wide plates 16 or 8 cm long. Notched divider plates, ordered separately, divide gel sandwiches to form “club sandwiches” of two gels each, so up to four gels can be run at one time.
  • Page 11 Casting stand The casting stand holds assembled gel sand- wiches upright for casting gels. Adjustable feet level the caster. A laminated gasket in the bottom of each casting cradle seals the bottom of the sandwich when it is clamped into the stand.
  • Page 12 Combs Teflon combs are available in sizes that form 10, 15, 20, or 28 wells. Most combs are avail- able in all three thicknesses: 0.75, 1, and 1.5 mm. Blank combs form a single large well, and preparative combs include one or two reference wells in addition to the preparative well.

  • Page 13: Operating Instructions

    One to four gels (18 × 16 cm) can be assembled and run in the SE 600 Chroma. Both precast gels and self-cast gels can be used. To self-cast multiple gels, kits can be ordered...
  • Page 14  Construct gel sandwiches Fig 2. Sandwich assembly. Inspect glass plates for nicks. Use For each sandwich choose two perfectly clean, unchipped glass only unchipped plates to prevent plates and two spacers. Lay one plate on a flat surface, lay the The glass plates and spacers must be flush with leaking.
  • Page 15  Place the laminated gasket into the casting cradle (See Fig 4) with the foam side down. Place the clamp assembly in the casting cradle, screw side facing out.  Insert a cam into the hole on each side of the casting tray with the ridge (short end) pointing up.

  • Page 16: Acrylamide Gels

    Acrylamide gels  Prepare the monomer solution and pour the gel Prepare the required amount of monomer solution. Deaerate and add the initiator and catalyst just prior to pouring the gel. Pipette the solution into one corner of the sandwich, taking care not to intro- duce any air bubbles.
  • Page 17 Stacking gel preparation Pour the stacking gel while the sandwich is still in the gel caster. Stacking-gel resolution is optimal when poured just before elec- trophoresis.  Remove the overlay by rinsing the top of the gel several times with distilled water.

  • Page 18: Gradient Gels

    Gradient gels Both linear and exponential gradient gels can be poured in the dual-gel caster. We recommend using a Hoefer SG Series Gradient Maker. 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 Hoefer Multiple Gel Caster (see instructions accompanying the caster).
  • Page 19 solution into the mixing chamber and place a stirring bar into this chamber. 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.  Mix the gradient and pump the solution into the sandwich While the solution is stirring, begin pumping from the mixing chamber and open the stopcock to the reservoir chamber.

  • Page 20: Sample Preparation And Loading

    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. When loading Note: With Coomassie Blue ™ it is samples while using divider plates, the samples must be loaded possible to detect 1 µg of protein in a without the upper buffer chamber in place.
  • Page 21 Table 1. Sample volume for standard comb sizes volume of sample (µl) per 1 mm depth no. of comb thickness (mm) wells 0.75 12.4 1/1 (ref/prep) 4/90 6/121 9/183 1/2 (ref/prep) 4/85 6/112 9/171 •...

  • Page 22: Final Assembly

    Final assembly Upper buffer chamber  Rinse both buffer chambers with water and distilled water thoroughly before each use. Note: Before using the first time, disassemble the unit and wash with a dilute solution of a labora- tory detergent and rinse thoroughly first with water and then with distilled water.
  • Page 23 Lower buffer chamber  Fig 6. Attaching gel sandwiches to the upper buffer chamber. Place a magnetic spin bar into the lower buffer chamber (LBC) and If the assembly leaks, take it to a sink place the unit on a magnetic stirrer. Fill the lower chamber with up and partially release the cams to allow to 4 liters of buffer.
  • Page 24 Important assembly notes: • IEF runs: The buffer level in the lower buffer chamber must never reach the upper buffer chamber; maintain at least 2 cm of clearance. • Do not fill the upper or lower chamber above the recommended levels illustrated on the next page.
  • Page 25 Fig 8. Upper and lower buffer cham- ber fill levels. Upper chamber buffer max fill line Lower chamber buffer max fill line Buffer level label  Place the safety lid on the unit by engaging the safety interlock pins before lowering the electrode connections on to the banana plugs.

  • Page 26: Separating The Sample

    ‡ At 25 mA per gel. Voltage The starting voltage for a 1.5 mm slab gel con- nected to a power supply set to 25 mA is usu- ally 80–90 V (using the SE 600 Chroma unit •...
  • Page 27 Time A run is usually complete when the tracking dye reaches the bottom of the gel. In a 16 cm gel (SE 600 Chroma), a 1.5-mm-thick Laemmli SDS gel, run at 25 mA/gel without cooling, usually requires 5 h. Record each run...

  • Page 28: After Electrophoresis

     Unscrew the clamps from the sandwiches and remove. Gently loosen and then slide away both spacers. Use the Hoefer Wonder Wedge Gel Plate Separation tool to separate the plates.  Carefully lift the glass plate with the gel attached. Handle the gel with care to avoid damaging it.

  • Page 29: Care And Maintenance

    Care and maintenance Immediately after each use, rinse the upper and Cleaning lower buffer chambers with water and then rinse thoroughly with distilled water. Handle • Do not autoclave or heat any the upper buffer chamber with care to prevent part above 45 °C.

  • Page 30: Troubleshooting

    Troubleshooting problem remedy possible cause Gel sandwich Dirty or damaged Plates, spacers, and the gasket must be completely leaks while components clean. Wash if necessary. casting Replace chipped plates (especially if chipped near the spacers). Check the caster gasket for cuts or cracks and replace if necessary.
  • Page 31 problem possible cause remedy Solutions with extreme pH values (especially acidic) may not polymerize. Oxygen 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. Temperature Adjust the gel solution temperature to a minimum of 20 °C, especially for low %T gels Upper Mis-aligned parts...
  • Page 32 problem possible cause remedy Protein Particulates in sample Centrifuge or filter sample before loading to remove par- streaks ticulates. vertically Overloading Load less sample. Degradation Add protease inhibitor such as PMSF. Unusually Current leakage around Check for leaks; all plates and spacers must be aligned slow and free of grease and cracks.
  • Page 33 problem possible cause remedy Stained sample collects: Near the Gel concentration Molecules are not sufficiently restricted by the resolving buffer front gel pore size: increase the %T. Degradation Proteins may be degraded by endogenous proteases: use protease inhibitors during the isolation step. Near the top Gel concentration The gel pore size is too small: decrease the %T of the...
  • Page 34 Poor band Sample Store sample on ice before it is denatured. resolution preparation Dialyze or desalt the sample. cont. Heat samples in SDS sample buffer for no more than 1–2 min at 100 °C to improve dissociation of subunits. Store on ice after heating Adjust the sample volume or concentration.

  • Page 35: Bibliography

    Current Protocols in Molecular Biology. (Ausubel, F. A., et al., eds.), OSC 10.6.1–10.6.8 (1991). SDS Polyacrylamide Gel Electrophoresis and Isoelectric Focus- ing Handbook (80-6013-88), Hoefer, Inc. (2001). Non-denaturing gel systems Reisfeld, R. A., et al., Acidic buffer system for resolution of cationic proteins.
  • Page 36 Shapiro, A. L., and Maizel J. V. Jr., Molecular weight estima- tion of polypeptides by SDS-polyacrylamide gel electro- phoresis: further data concerning resolving power and general considerations. Anal. Biochem. Jun; 29(3):505– 514 (1969). Schaegger, H. and Von Jagow, G., Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
  • Page 37 Bjellqvist, B., et al., Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. Methods 6, 317–339 (1982). Görg, A, et al., The current state of two-dimensional electro- phoresis with immobilized pH gradients. Electrophoresis 9, 531–546 (1988). Görg, A.

  • Page 38: Ordering Information

    SE6008B chamber Laminated silicone rubber gaskets SE6054 for casting stand SE6009 Buffer dam SE6032 Upper buffer chamber for SE 600 Chroma 1 SE6054 heat Lower buffer chamber for SE 600 Chroma 1 SE6150X exchanger SE6160 Lid with high-voltage leads for SE 600 Chroma...
  • Page 39 Dual Gel Caster, basic, 2 gels, 18-cm wide SE6015 Includes: 2 blank gaskets for 1 or 2 gels. (One included with each SE 600 Chroma unit.) For up to 4 gels: Gel Caster Kit, 4 gels, 18 × 16 cm...
  • Page 40 choose the appropriate spacer and plate length for your unit gasket universal clamp SE6009 SE6003U basic caster SE6015 spirit level SER11 SE6005L Clamps and cams Clamp and Cam Kit, four 16 cm clamps and 8 black cams SE6003UK Replacement thumbscrews for clamps SE6003U-2 Cams, black, for clamps with cam holes 6403U...
  • Page 41 product quantity product number Glass plate, club sandwich divider, notched SE6102D Teflon combs number thickness width of wells (mm) (mm) 0.75 SE511-10-.75 1.00 SE511-10-1.0 1.50 SE511-10-1.5 0.75 SE511-15-.75 1.00 SE511-15-1.0 1.50 SE511-15-1.5 0.75 SE511-20-.75 1.00 SE511-20-1.0 1.50 SE511-20-1.5 0.75 SE511-28-.75 1.00 SE511-28-1.0 1.50...

  • Page 42: Companion Products

    (mm)(cm) (cm) 0.75 SE6419-2-.75 SE6419-2-1.0 SE6419-2-1.5 0.75 SE6119-2-.75 SE6119-2-1.0 SE6119-2-1.5 SE6118-2-1.0 SE6118-2-1.5 Companion products Hoefer SE 100 Plate Mate washing and storage unit SE100 QuickFit connectors, female 3/8” QF3/8 QuickFit connectors, male 3/8” QFX3/8 •...
  • Page 44 Coomassie is a trademark of ICI plc. 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 •...

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