SE640 User Manual – English
Contents
1. 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 thickness width mm prep ref mm prep ref guantity code number 1 1 0 75 121 6 1 SE511 R 75 1 1 1 121 6 1 SE511 R 1 0 1 1 1 50 121 6 1 SE511 R 1 5 1 2 0 75 113 6 1 SE511 DR 75 1 2 1 113 6 1 SE511 DR 1 0 1 2 1 50 113 6 1 SE511 DR 1 5 Adjustable comb back 1 SE511 BKA Required to convert any 25 mm deep comb to 10 or 15 mm depth e p40 Spacers thickness width mm length mm quantity code no 0 75 8 2 2 SE6419 2 75 1 00 8 2 2 SE6419 2 1 0 1 50 8 2 2 SE6419 2 1 5 Companion products Hoefer SE100 Plate Mate washing and storage unit 1 SE100 Hoefer TE62 Tank Transfer Unit 1 TE62 Hoefer TE7OXP Semi Dry Transfer Unit 1 TE7OXP Hoefer reagents for gel casting and buffers Acrylamide MB grade 1 kg GR141 1 bis Acrylamide MB grade 100 g GR142 100 TEMED 25g GR151 25 Ammonium persulfate ACS reagent grade 10g GR152 10 Tris Glycine SDS Buffer 10X Solution MB grade TL GR149 1 Tris reagent grade 1 kg GR132 1 Glycine 1 kg GR125 1 Sodium Dodecyl Sulfate SDS 500 g GR126 500 Sodium Dodecyl Sulfate SDS 10 Solution 1L GR155 1 Hoefer reagents for sample loading and gel staining Dithiothreitol DTT MB grade 5g GR122 5 EDTA 0 5 M Solution MB grade 100 ml GR123 100 Bromophenol Blue sodium salt ACS reagent grade 10g GR1202. Gorg A et al The current state of two dimensional electrophoresis with immobilized pH gradients Electropho resis 9 531 546 1988 G rg A Two dimensional electrophoresis with immobilized pH gradients current state Biochem Soc Trans 21 130 132 1993 Bjellgvist B et al Micropreparative two dimensional electro phoresis allowing the separation of samples containing milli gram amounts of proteins Electrophoresis 14 1375 1378 1993 Blomberg A et al Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two dimensional gel electrophoresis Electrophoresis 16 1935 1945 1995 e p37 Ordering information product quantity code no SE640 Dual Vertical Unit basic 1 SE640 Includes 3 sets of glass plates four 8 cm clamp assemblies 6 cams dual gel casting stand with leveling base and level buffer dam Spacer Mate alignment template and Wonder Wedge plate separation tool Order 2 combs and 2 sets of 8 cm spacers separately SE640 Dual Vertical Unit complete 1 SE640 15 1 5 Includes basic unit plus two 15 well combs and 2 sets of 8 cm spacers 1 5 mm thick Replacement parts Wonder Wedge gel plate separation tool 1 SE1514 Slotted silicone rubber gaskets for upper buffer chamber 2 SE6008B Laminated silicone rubber gaskets for casting stand 2 SE6009 Buffer dam 1 SE6432 Upper buffer chamber with electrode fin 1 SE6454 Lid with
3. Allow the gel to polymerize for a minimum of one hour Stacking gel preparation Pour the stacking gel while the sandwich is still in the gel caster Stacking gel resolution is opti mal when poured just before electrophoresis 0 Remove the overlay by rinsing the top of the gel several times with distilled water Invert the caster to drain To ensure a seamless contact between the resolving and stacking gels remove residual liquid by blotting one corner with a lint free tissue a Prepare the reguired amount of stacking gel monomer solution deaerate it and add catalyst APS and initiator TEMED 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 Introduce a comb at a slight angle into the sand wich taking care not to trap air under the teeth Allow a minimum of one hour for the gel to polymerize Fig 5 Pouring a gradient gel The gel solution may be intro duced into the gel sandwich through a pipette tip at a rate that maintains a continuous stream Optional Adjust the higher percentage acrylamide solution to 15 w v sucrose or 25 v v glycerol to improve layering 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 Gra dient gels are poured with a cannula from the top of the dual gel caster see Fig 5 A s
4. Operating instructions Gel casting and electrophoresis procedures follow Included are instructions for polyacryl amide gels used with continuous or discontinu ous buffer systems and gradient gels The gels required for the SE640 must be self cast The Dual Gel Caster included holds two gel sandwiches Prepare the gel sandwich Glass plates spacers 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 For best results take extra care to align all components when assem bling sandwiches both top and bottom sandwich edges must be flush with the clamp guide ridges pressure bar X Fig 2 Sandwich assembly Inspect glass plates for nicks Use only unchipped plates to prevent leaking Tip Use the casting cradle to hold the sandwich during alignment Remove the lami nated gasket from the cradle and instead of setting the sandwich upright on a flat surface set it into the cast ing cradle Construct the gel sandwich and insert into caster 0 Prepare the caster and clamps Place the spirit level into the caster center and adjust the leveling feet Loosen all clamp screws and make space for the sandwich by sliding the pressure plates toward the screws a Construct each gel sandwich For each sandwich choose two perfectly clean unchipped glass plates and two spacers Lay one plate on a flat
5. for the appropriate solution level according to the application No stacking gel Continuous system Fill solution to just below the top of the upper plate edge If bubbles are trapped remove with a pipette or syringe Introduce a comb at a slight angle into each sandwich taking care not to trap air bubbles under the teeth Club sandwich Pipette the solution into both sandwiches filling each to the same level below the notched edge Stacking gel Fill solution to 3 4 cm below the top of the glass plate This height allows 1 cm of stacking gel below the wells Pour the gel and apply an overlay see step 2 After the gel is set prepare the stacking gel as described below 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 A stacking gel will require extra space Seal the IPG strip or tube gel in place with agarose dissolved in running buffer Take care to avoid trapping any air bubbles between the first and second dimension gels e pll Da i pl2 Overlay each gel with a thin layer of water saturated n butanol water or diluted gel buffer to prevent gel exposure to oxygen Slowly deliver the overlay solution from a glass syringe fitted with a 22 gauge needle Apply the solution near the spacer at the side of the sandwich and allow it to flow across the surface unaided
6. manual Hoefer SE640 Wide mini Dual Gel Electrophoresis Unit um SE640 IM Rev B0 07 12 Afro e fe r o Contents Important INFORMATION paia ii Waste Electrical and Electronic Equipment WEEE ee vii Gel electrophoresis unit function and description eee 1 REM 2 Unpacking and INVENTON toiristea ve Bed sir 4 Operating INSHUCTIONS iis ri 7 Caresand maintenance EEN 23 Customer service Information 24 le ei Ee ell 25 Appendix A Laemmli system gels 29 SOUL Le 31 ERT 34 Appendix B Bibliography rn 36 Ordering INFOrmationi i snai ee 38 Important Information English If this equipment is used in a manner not speci fied by Hoefer Inc the protection provided by the equipment may be impaired This instrument is designed for indoor laboratory use only Only accessories and parts approved or supplied by Hoefer Inc may be used for operating main taining and servicing this product Only use a power supply that is CE marked or safety certified by a nationally recognized testing laboratory The safety lid must be in place before connecting the power supply leads to a power supply Turn all power supply controls off and disconnect the power leads before removing the safety lid Circulate only water or 50 50 water ethylene glycol through the heat exchanger if so equipped Do not connect the heat exchanger to a water tap or any coolant source wh
7. C crosslinker g bis x 100 g acryl bis e p29 The total percent of acrylamide T in the resolving gel which can range from 5 to 20 determines the pore size Commonly the amount of crosslinker used C is 2 6 In the fol lowing example system the resolving gel com position is 10 T 2 6 C which results in a medium pore size The stacking gel composition is 4 T 2 6 C The T in the stacking gel is lower because a larger pore size is required Final concentrations Separating gel Stacking gel Electrophoresis 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 pH 8 8 6 8 8 3 SDS 0 1 0 1 0 1 Ammonium persulfate APS 0 05 wiv 0 05 0 1 w v TEMED 0 05 viv 0 05 0 1 v v To achieve any other desired final concentration adjust the acrylamide stock and water volumes Volumes for different concentrations are listed on page 34 fTetramethylethylenediamine p30 Note Filter solutions 1 4 through a 0 45 um filter IMPORTANT Refer to the mate rial safety data sheet MSDS accompanying each chemical for detailed handling and safety information Caution Acrylamide is a neuro toxin Always wear gloves while handling in any form and wear a mask while weighing the powder Never mouth pipette the solution Solutions 1 Acrylamide stock solution 30 8 T 2 6 C Bis 200 ml Acrylamide FW 71 08 30 wiv Bis
8. to attach the sandwich to the upper buffer chamber Rubber gaskets There are two sets of two gaskets The solid laminated gaskets fit into the bottom of the cast ing stand and form the seal for casting the gel The slotted gaskets fit under the upper buffer chamber and form the seal between the upper and lower chambers 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 Spacers May be ordered separately Spacers determine the thickness of the gel and are available in three thicknesses 0 75 1 0 and 1 5 mm Spacer Mate spacer positioning guide Aligns spacers for sandwich assembly Combs May be ordered separately Combs are avail able in sizes that form 10 12 15 20 or 28 wells and are available in three thicknesses 0 75 1 0 and 1 5 mm Preparative combs include 1 or 2 reference wells in addition to the single large preparative well All preparative combs and 10 12 15 and 20 well combs form wells that are 25 mm deep The 28 well comb forms wells that are only 15 mm deep so that wells do not collapse when the comb is removed The sample volume held by each well depends on the gel thickness well depth and the number of wells per comb Table 1 on page 16 lists sample volumes of each well for all combs Wonder Wedge plate separator tool Used to disassemble gel sandwiches and to gauge spacer and comb thickness
9. 2 5 ml 10 SDS 0 35 M Soln 4 0 14 M 4 0 ml Glycerol FW 92 09 20 viv 2 0 ml 2 mercaptoethanol FW 78 13 2 VIN 0 2 ml OR Dithiothreitol DTT 0 2 mM 0 31 gl FW 154 2 Bromophenol Blue FW 691 9 0 03 mM 0 2 mg Deionized H20 to 10 0 ml Divide into 1 0 ml aliquots and store at 40 C to 80 C 6X Sample treatment buffer 0 35 M TrisCl 10 SDS 30 glycerol 9 3 DTT pH 6 8 10 ml 0 5 M Tris Cl pH 6 8 Soln 3 0 35 M 7 0 ml SDS FW 288 4 0 35 M 1 0g Glycerol FW 92 09 30 v v 3 0 ml DTT FW 154 2 0 6 M 0 93 g Bromophenol Blue FW 691 9 0 175 mM 1 2 mg Divide into 1 0 ml aliquots and store at 70 C 8 0 025 M Tris 0 192 M glycine 0 1 SDS pH 8 3 Electrophoresis buffer 5 0 liters Tris FW 121 1 0 025 M 15 1g Glycine FW 75 07 0 192 M 72 0 g SDS FW 288 4 3 5 mM 5 0 g Deionized H20 to 5 0 liters The pH of this buffer is approximately 8 3 Do not adjust pH Up to 20 liters can be prepared and stored for up to 2 months 9 Coomassie stain solutions Coomassie stain solution 0 025 Coomassie Blue R 250 40 Methanol 7 Acetic acid 2 liters Coomassie Blue R 250 FW 826 0 3 mM 0 5g Methanol Stir until dissolved 40 viv 800 0 ml Glacial acetic acid 99 7 VN 140 0 ml Deionized H20 to 2 0 liters Destaining solution 40 methanol 7 acetic acid 1 liter Methanol 40 viv 400 0 ml Glacial acetic acid 99 7 VIN 70 0 ml Deionized H20 to 1 0 liter Destaining solution I
10. Add protease inhibitor such as PMSF Check for leaks all plates and spacers must be aligned and free of grease and cracks If used the buffer dam must be secure Sample or reagent preparation If the required pH of a solution is overshot do not back titrate Discard and prepare fresh buffer Check recipes gel concentrations and buffer dilution For instance do not use Tris HCI instead of Tris for Laemmli tank buffer Decrease the salt concentration of samples Reagent quality Dispose of older acrylamide solutions and use only stock of the highest quality Use only freshly deionized urea Voltage or current settings To increase or decrease the migration rate adjust the voltage or current by 25 50 problem Bands are skewed or distorted possible cause Incomplete gel preparation and polymerization remedy Degas the stacking gel solution and avoid trapping air bub bles under the comb teeth Irregular interface between stacking and running gels Overlay the running gel with water saturated butanol before polymerization begins to avoid forming an uneven gel sur face Sample preparation Stained sample collects Near the buffer front Gel concentration Degradation Dialyze or desalt the sample Molecules are not sufficiently restricted by the resolving gel pore size increase the T Proteins may be degraded by endogenous proteases use pro tease inhibitors during t
11. FW 154 2 0 8 wiv Deionized H20 Store at 4 C away from light N N Methylenebisacrylamide 2 1 5 MTrisCI pH 8 8 4X Resolving gel buffer 1 liter Tris FW 121 1 1 5M 4 N HCI Deionized H20 3 0 5 M TrisCI pH 6 8 4X Stacking gel buffer 500 ml Tris FW 121 1 0 5 M 4 N HCl Deionized H20 4 10 SDS solution 100 ml Sodium dodecylsulfate SDS FW 288 4 0 35 M Deionized H20 5 10 APS Initiator 1 ml Ammonium persulfate 0 44 mM APS FW 228 2 Deionized H20 60 g 1 6 g to 200 0 ml 181 6 g to pH 8 8 to 1000 ml 30 3 g to pH 6 8 to 500 ml 10 0 g to 100 ml 0 1 g to 1 0 ml Fresh APS crackles when water is added If yours does not replace it with fresh stock Prepare just prior to use e p31 p32 6 0 375 M TrisCI 0 1 SDS pH 8 8 Resolving gel overlay 100 ml 1 5 M Tris Cl pH 8 8 Soln 2 0 375M 25 0 ml 10 SDS Soln 4 3 5 mM 1 0 ml Deionized H20 to 100 0 ml OR Water saturated n butanol Shake n butanol and deionized H20 in a separatory funnel Remove the aqueous lower phase Repeat this procedure several times Use the upper phase If an overlay interferes with the preferred protocol isolate the gel from atmospheric oxygen by placing a blank comb or resolving gel former on the gel 7 2X Sample treatment buffer 0 125 M TrisCl 4 SDS 20 glycerol 2 2 mercaptoethanol pH 6 8 10 ml 0 5 M Tris Cl pH 6 8 Soln 3 0 125M
12. down In our experience with the concentrations in the 10 20 gradient example below gels can be poured at a flow rate of 5 10 ml min Linear gradient gel per 100 ml of solution 10 T 20 T Acrylamide stock Solution 1 33 30 ml 66 70 ml Sucrose 15 00 g 1 5 M TrisCI pH 8 8 Soln 2 25 00 ml 25 00 ml 10 SDS Solution 4 1 00 ml 1 00 ml Deionized H20 a 100 00 ml a 100 00 ml 10 APS Solution 5 0 300 ml 0 060 ml TEMED 0 036 ml 0 036 ml e p35 e p36 Appendix B Bibliography General Gallagher S R and Smith J A Electrophoretic separation of proteins In Current Protocols in Molecular Biology Ausubel E A eds OSC 10 2 1 10 2 21 1991 Hames B D and Rickwood D Gel Electrophoresis of Proteins A Practical Approach Second edition City IRL Press 1990 Sambrook J Fritsch E F and Maniatis T Standard Form aldehyde Protocol Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY 1990 Sasse J and Gallagher S R Staining proteins in gels Current Protocols in Molecular Biology Ausubel F A et al eds OSC 10 6 1 10 6 8 1991 Non denaturing 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 isome
13. high voltage leads 1 SE6056 High voltage safety lead set 1 SE6056 HV Lower buffer chamber 1 SE6450 Replacement electrode fin for SE6454 1 SE6870 Banana plug gold with 2 washers 1 SE6067 Spirit level 1 SER11 Gel Seal Ya oz tube 1 SE6070 e p38 product quantity code no Gel Caster for 1 or 2 gels Dual Gel Caster 1 2 gels 18 cm wide 1 SE6015 Includes 2 blank gaskets One included with each SE640 unit Clamps and Cams Replacement thumbscrews for clamps 12 SE6003U 2 Cams black for clamps with cam holes 4 SE6005L Clamp assemblies 8 cm 2 SE6403U Glass Plates 18 x 8 cm Glass plates 2 SE6402 Glass plates low fluorescence 2 SE6402LF Glass plate club sandwich divider notched 1 SE6402D safety lid with cables SE6056 upper buffer chamber with electrode fin SE6454 lower buffer chamber SE6450 universal clamp gasket SE6009 basic caster SE6015 spirit level SER11 SE6005L e p39 Combs number thickness width of wells mm mm quantity code number 0 0 75 8 3 SE511 10 75 0 1 00 8 3 SE511 10 1 0 0 1 50 8 3 SE511 10 1 5 2 0 75 7 6 SE511 12 75 2 1 00 7 6 SE511 12 1 0 2 1 50 7 6 SE511 12 1 5 5 0 75 5 7 SE511 15 75 5 1 00 5 7 SE511 15 1 0 5 1 50 5 7 SE511 15 1 5 20 0 75 4 1 SE511 20 75 20 1 00 4 1 SE511 20 1 0 20 1 50 4 1 SE511 20 1 5 28 0 75 2 7 SE511 28 75 28 1 00 2 7 SE511 28 1 0 28 1 50 27 SE511 28 1 5
14. m zp sob nenapravi teln po kozen jednotka Nejsou provozov na s pufru teplot ch nad maxim ln stanovenou technick mi specifika cemi P eh t zp sob nenapraviteln po kozen jednotka Vigtig Information Danish Hvis dette udstyr bruges i en made ikke specifice ret ved Hoefer Inc den beskyttelse som er blevet forsynet af udstyret kan m ske sv kkes Dette instrument er designet for indendors labora toriumbrug bare Bare tilbeh r og del godkendede eller forsynede ved Hoefer Inc kan m ske bruges for drive funk tionsfejl og betjening dette produkt bruger Bare en str mforsyning der er CE markerede eller sikkerhed som er blevet attesteret af en som nationalt er blevet anerkendt prove laboratorium Sikkerhedlaget m v re p plads for forbinding stramforsyningsblyet til en str mforsyning Drejer alle stromforsyningskontroller af og afbryder kraftblyet for fjerning sikkerhedl get Cirkulerer bare vand eller 50 50 vand ethylene glykol gennem varmeveksleren i s fald udrustet Forbind ikke varmeveksleren til en vandhane eller nogen kolemiddelkilde hvor vandtrykket er unregulated Introducerer Aldrig antifreeze eller noget organisk opl sningsmiddel ind i nogen del af instrumentet Organiske opl sningsmidler vil for rsage uboelig skade til enheden Driver ikke med st dpudetemperaturer over maksimummet specificerede tekniske specifica tions Overheding vil forarsag
15. the run Under these conditions voltage increases as the run proceeds A lower current setting is recommended for higher resolution The optimal current level must be determined empirically the main factors that must be balanced include the gel concentration and migration speed and the resulting Joule heating and band distortion Table 2 lists starting point guidelines and adjust ments for gel thickness number of gels and migration rate Current Current acts on the total cross section area of all the gels because the gels are connected in parallel in the electrical circuit Thus the current setting for one gel must be multiplied by the number of gels of the same gel thickness that are run simultaneously For a gel 1 5 mm thick we suggest a starting current setting of 25 mA Two 1 5 mm gels 50 mA Voltage The starting voltage for a 1 5 mm slab gel con nected to a power supply set to 25 mA is usually 80 to 90 V using the SE600 with a Laemmli discontinuous buffer system for SDS gels The final voltage can typically range from 220 to 400 V depending on the length of the gel See Table 2 Caution After initial monitoring do not leave the unit unattended for more than 1 hour before checking the progress of the bands and the buffer level Time A run is complete when the tracking dye reaches the bottom of the gel A 1 5 mm thick Laemmli SDS gel run at 25 mA gel without cooling usu ally requires 2 5 hours El
16. 10 Glycerol MB grade 1L GR124 1 Protein determination reagent 500 assays 500 ml GR133 500 Coomassie Brilliant Blue G 250 258 GR134 25 Coomassie Brilliant Blue R 250 258 GR135 25 p41 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone 1 508 893 8999 Fax 1 508 893 0176 E mail support hoeferinc com Web www hoeferinc com Hoefer is a registered trademark of Hoefer Inc Coomassie is a trademark of ICI plc RBS 35 is a trademark of Pierce Chemical Co Tygon is a trademark of Saint Gobain Performance Plastics 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
17. Apply a light film of Gel Seal compound only on the bottom corner surfaces created by the spacers and plates if your sandwiches continue to leak after several attempts of alignment Note When turning the cams it is easier to keep the caster balanced if you turn both toward the center of the caster Fig 4 Caster components and setup e plo 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 Seal the gel sandwich against the casting gasket by turning both cams as far as needed usually 90 to 150 up to 180 The cam action presses the plates down into the gasket to seal the bottom of the sandwich The seal is complete once the glass edge appears darker and nearly transparent against the gasket Do not turn the cam past this point clamp glass plate spacer gasket foam side down cam leveling feet install ridge end up Acrylamide gels 0 Prepare the monomer solution and pour the gel See Appendix A for SDS PAGE recipes Prepare the required amount of monomer solution De aerate and add the initiator ammonium persulfate APS and catalyst TEMED just prior to pouring the gel Pipet the solution into one corner of the sandwich taking care not to introduce any air bubbles See below
18. C pp 10 4 1 10 4 13 1992 Anderson N G Anderson N L and Tollaksen S L Proteins of human urine I Concentration and analysis by two dimensional electrophoresis Clin Chem Jul 25 7 1199 2210 1979 Anderson Leigh and Anderson Norman G High resolution two dimensional electrophoresis of human plasma proteins Proc Natl Acad Sci USA 74 5421 5425 1977 Anderson L Two Dimensional Electrophoresis Operation of the ISO DALT System Second Edition Large Scale Biology Press 1991 Bravo R Schafer R Willecke K MacDonald Bravo H Fey S J and Celis J E More than one third of the discern ible mouse polypeptides are not expressed in a Chinese hamster mouse embryo fibroblast hybrid that retains all mouse chromosomes Proc Natl Acad Sci USA Apr 79 7 2281 2285 1982 Hurkman W J and Tanaka C K Solubilization of Plant Membrane Proteins for Analysis by Two Dimensional Gel Electrophoresis Plant Physiology 81 802 806 1986 Mets L J and Bogorad L Two dimensional polyacrylamide gel electrophoresis an improved method for ribosomal proteins Anal Biochem Jan 57 1 200 210 1974 O Farrell P H High resolution two dimensional electropho resis of proteins J Biol Chem May 25 250 10 4007 4021 1975 Bjellqvist B et al Isoelectric focusing in immobilized pH gradients principle methodology and some applications J Biochem Biophys Methods 6 317 339 1982
19. I 7 acetic acid 5 methanol Methanol 5 vv 50 0 ml Glacial acetic acid 99 7 NN 70 0 ml Deionized H20 to 1 0 liter e p33 Laemmli gel Gel recipes The Laemmli gel recipes are for 30 ml of a single concentration solution enough for two 1 5 mm 18 x 8 cm gels Tabulated are ingredients and volumes for relatively large pore gels 7 5 10 T range as well as smaller pore gels 12 5 15 T range A 4 stacking gel is common The linear gradient recipe is for 100 ml of solution The total volume needed depends on the number of gels cast and the gel thickness adjust as necessary All gels are crosslinked with 2 6 C per 30 ml resolving gel solution 5 ml stacking gel solution Separating gel Stacking gel 7 5 10 12 5 15 4 Acrylamide stock Solution 1 7 5m 10 0 ml 12 5 ml 15 0 m 0 67 ml 1 5 M TrisCl pH 8 8 Soln 2 7 5m 7 5 ml 7 5 ml 7 5m 0 5 M TrisCl pH 6 8 Soln 3 1 25 ml 10 SDS Solution 44 0 3m 0 3 ml 0 3 ml 0 3 m 0 05 ml Deionized H20 14 6 m 12 1 ml 9 6 ml 7 1m 3 00 ml 10 APS Solution 5 150 ul 150 ul 150 ul 150 ul 25 ul TEMED 10 ul 10 pl 10 pl 10 ul 2 5 ul Final Volume 30 0 m 30 0 ml 30 0 ml 30 0 m 5 0 ml p34 For linear gradient gels use equal volumes of low and high acrylamide solutions in the 2 chambers of the gradient maker Less APS is added to extend polymerization time and less still is added to the higher T solution to allow polymerization to occur from the top
20. Instrument wird f r den Innenlaborge brauch nur daf r entworfen Nur Zus tze und Teile genehmigten oder lieferten durch Hoefer Inc kann fur das Funktionieren das Aufrechterhalten und die Wartung dieses Produk tes verwendet werden Verwenden Sie nur eine Energieversorgung die CE gekennzeichnet oder durch ein national anerkanntes Probelaboratorium bescheinigte Sicherheit ist Der Sicherheitsdeckel muss im Platz vor dem AnschlieBen der Energieversorgung sein f hrt zu einer Energieversorgung Alle Energieversorgungssteuerungen abdrehen und die Macht trennen f hrt vor dem Entfernen des Sicherheitsdeckels Nur Wasser oder 50 50 Glykol des Wassers Athylens durch den Warmeaustauscher wenn so ausgestattet in Umlauf setzen Verbinden Sie den W rmeaustauscher mit einem Wasserklaps oder jeder Kuhl mittel Quelle nicht wo der Wasserdruck ungeregelt wird F hren Sie nie Frostschutzmittel oder jedes organische L sungsmittel in jeden Teil des Instru mentes ein Organische L sungsmittel werden nicht wiedergutzumachenden Schaden der Einheit verursachen Mit Puffertemperaturen Uber angegebenen technischen Spezifizierungen des Maximums nicht funktionieren Die Uberhitzung wird nicht wiedergutzumachenden Schaden der Einheit verursachen Informazioni Importanti Italian Se quest apparecchiatura usata in un modo specificato da Hoefer Inc la protezione fornito dall apparecchiatura potrebbe essere ind
21. Lift straight up to avoid bending the banana plugs a Pull out the upper buffer chamber assembly Pour the buffer into a sink Install the assembly in the dual gel caster and then release the sandwiches by turning and removing the cams Unscrew the clamps from the sandwiches and remove Gently loosen and then slide away both spacers Use the Hoefer Wonder Wedge plate separator tool to separate the plates o Carefully lift the glass plate with the gel attached Handle the gel with care to avoid damaging it Invert the plate and position the gel low over the staining tray Pry one corner of the gel away from the glass and allow it to drop into the tray or if the gel is thick enough to handle lift it and place it into the tray To avoid splashing add staining or fixative solution to the tray after the gel is transferred Clean the unit as described in the next section Caution Always unplug unit from electrical supply before cleaning or drying the unit Care and maintenance Cleaning Immediately after each use rinse the upper and lower buffer chambers with water and then rinse thoroughly with distilled water Handle the upper buffer chamber with care to prevent damage to the banana plugs and lower electrode fin Clean gaskets with mild detergent and rinse with distilled water Allow to air dry Clean glass plates and spacers with a dilute solu tion of a laboratory cleanser such as RBS 35
22. assa ei m ritetty Hoefer Inc suojelu ehk isty varusteille saattaa olla avuton T m v line suunnitellaan sis laboratoriok yt lle vain Vain lis varusteet ja osat hyv ksyiv t tai toimitti Hoefer Inc oheen voi k ytt k ytt miselle valvoalle ja servicing t m tuote Vain k ytt k ytt j nnitett joka on CE merkitsi tai turvallisuus joka on todistanut aidoksi ohi joka on kansallisesti tunnustettnut testaaminen labo ratoriota e Turvallisuuskansi t ytyy olla paikallaan ennen yhdist minen k ytt j nnitelyijyj k ytt j nnit teeseen Kiert kaikki k ytt j nnitevalvonnat ja irrottaa valtalyijyt ennen poistaminen turvallisuuskantta Kiert vain vesi tai 50 50 vesi ethylene glycol siin tapauksessa varustetun l mm nvaihtimen l pi l yhdist l mm nvaihdinta vesinapautuk seen eik j hdytysnestel hteeseen miss vesi paine on unregulated Pakkasneste eik orgaaninen liuotin v lineen osassa ei esitele Koskaan Orgaaniset liuottimet aiheuttavat korvaamattoman vahingon yksikk n Ei k yt puskuria yll olevia l mp tiloja enint n m ritetyill teknisill t smennyksill Ylikuumen eminen aiheuttaa korvaamattoman vahingon yksikk n Information Importante French Si cet quipement est utilis dans une mani re pas sp cifi par Hoefer Inc la protection fourni par l quipement pourrait tre diminu e Cet instrument e
23. atively concentrated because no stacking gel is used In a discontinuous buffer system the zone into which each molecular species migrates is sharp ened by the stacking gel so the sample need not be as concentrated 0 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 Carefully rinse each well with distilled water to remove unpolymerized acrylamide and then drain by inverting the gel sandwich or caster Fill each well with electrophoresis buffer a Prepare the sample Increase liguid sample density with 10 glycerol or sucrose Add a tracking dye such as phenol red bromophenol blue or pyronin Y For SDS protein gels use 2X treatment buffer to denature both liguid and dry samples in a test tube To liguid protein solutions add an egual volume of 2X buffer To dry protein samples add egual volumes of 2X sample buffer and high purity water to achieve the desired concentration e p15 Note Once the sample is in the wells take care to not jar the sandwiches so that the samples are not disturbed e pl6 Heat the tube in boiling water for 90 seconds then allow to cool to room temperature Treated samples can be stored at 40 to 80 C for future runs Heat membrane proteins to 60 C for 20 minutes Store unused sample at 4 C o Underlay the sample into the wells using a fine tipped microsyringe or sam
24. aturated n butanol Temperature Adjust the gel solution temperature to a minimum of 20 C especially for low T gels e p25 problem Upper buffer chamber leaks Power supply detects current leak Dye front curves up smiles at edges Protein streaks vertically Unusually slow or fast run e p26 possible cause Mis aligned parts remedy 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 position ing ridges fit inside the grooves Dirty or damaged components Electrical path to outside ground earth Uneven heat distribution Check that the gasket is not damaged or pinched Replace if necessary Check that the upper buffer chamber is not warped from prior exposure to excessive heat Add more silicone grease to seal heat exchanger grommets Check for leaks or cracks in the heat exchanger Replace worn grommets Fill the lower buffer chamber to the level appropriate for at edges the run See Fig 7 page 19 Use magnetic stirrer and stir bar to keep buffer well mixed Excessive heat Particulates in sample Circulate ext coolant Decrease the current or voltage setting Prechill the buffer Run the gel in the cold room Centrifuge or filter sample before loading to remove particulates Overloading Load less sample Degradation Current leakage around gel
25. bare Bare tilbeh r og deler godkjente eller forsynte ved Hoefer Inc kan bli brukt for drive vedlikeholde og betjene dette produktet bruker Bare en kraftforsyning som er CE merket eller sikkerhet som ha blitt sertifisert av et som nasjonalt ha blitt anerkjent prover laboratorium piv Sikkerheten lokket ma v re p plass for forbinding kraftforsyningene blyene til en kraftforsyning Vender all kraftforsyningsstyring av og frakopler kreftene blyene f r fjerning sikkerheten lokket Sirkulerer bare vann eller 50 50 vann ethylene glykol gjennom oppvarmingen veksleren i s fall utstyrer Ikke forbind oppvarmingen veksleren til en vanntapp eller noe kj lemiddelkilde hvor vannet trykket er unregulated Introduserer Aldri antifreeze eller noe organisk l semiddel inn i noe del av instrumentet Organi ske l semiddler vil for rsake irreparabel skade p enheten Driver med buffertemperaturer over maksimum ikke spesifiserte teknisk spesifikasjoner overop pheting vil for rsake irreparabel skade p enheten Wazne Informacje Polish Je eli ten sprz t jest wykorzystywany w spos b nie okre lone przez Hoefer Inc do ochrony przewid zianej przez urz dzenie mo e zosta obni ony Instrument ten jest przeznaczony do u ytku w laboratoriach kryty tylko Tylko akcesori w i cz ci zatwierdzone lub dostarc zone przez Hoefer Inc mog by wykorzystane do eksploatacji utrzymania i obs ugi tego prod
26. cks and replace if necessary Check plate and spacer alignment realign if necessary Troubleshooting problem possible cause Gel sandwich Dirty or damaged leaks while components casting Mis aligned parts Over clamping Sample Air bubbles wells damaged or irregular Incomplete gel polymerization Turn cam only as far as necessary to create a seal usually 90 150 but up to 180 On each spacer apply a light film of Gel Seal compound to the bottom outside corner only Do not use silicone grease Remove air bubbles before inserting combs Slide comb into solution at an angle If comb must be removed add more monomer solution before reinserting the comb Incomplete or delayed polymerization Allow acrylamide gels to set for a minimum of 1 h Debris in wells Rinse out unpolymerized gel with sample buffer Comb removal Chemicals Remove the comb at a slight angle and very slowly to prevent damaging the gel Agarose gels Lower the comb no more than 1 cm into the gel Use only recent stocks of the highest quality reagents If the dry ammonium persulfate does not crackle when added to water replace with fresh stock Increase TEMED or APS concentration or both pH Solutions with extreme pH values especially acidic may not polymerize Oxygen Remove oxygen from the gel environment Degas the mono mer solution 5 10 min before pouring and then overlay the gel surface with water s
27. e buffer 2 Fit the upper buffer chamber assembly into the lower buffer chamber 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 buffer chamber Inspect the installation and adjust the buffer levels Upper chamber The electrode along the upper chamber ridge must be submerged to a depth of about 1 cm This level reguires 450 600 ml buffer just enough to cover the upper chamber ribs but not high enough to contact the banana plug Lower chamber The lower buffer level chamber reguires a minimum of 2 1 liters and a maximum of 2 8 liters of buffer enough to cover the wire on the lower electrode fin but maintaining a 1 5 cm clearance from the underside of the upper buffer chamber o Place the safety lid on the unit a Q Plug the color coded leads into the jacks of an approved power supply Plug the red lead into the red output jack and the black lead into the black output jack In most systems the red lead which is connected to the bottom electrode is the anode and the black lead connected to the top electrode is the cathode Important assembly notes e Do not fill the upper or lower chamber above the recommended levels illustrated in Fig 7 Remove buffer in contact with the electrode posts e Pour buffer slowly and away from the slots in the upper buffer chamber to avoid di
28. e uboelig skade til enheden O pii Belangrijke Informatie Dutch Indien deze uitrusting in een manier wordt gebruikt die niet door Hoefer Inc is gespecificeerd de bescherming die door de uitrusting is verzorgd kan worden geschaad Dit instrument is voor binnenlaboratoriumgebruik enkel ontworpen Enkel onderdelen en delen keurden goed of leverden door Hoefer Inc kan voor het bedienen worden gebruikt handhavend en onderhouden van dit product gebruik Enkel een netvoeding die CE is markeerde of veiligheid die door een is gecertificeerd die nationaal is herkend testene laboratorium Het veiligheidsdeksel moet in plaats voor het verbinden van de netvoeding leidt tot een netvoeding zijn Doe alle netvoedingscontroles Uit en koppel los de machtleiding voor het verwijderen van het veiligheidsdeksel Circuleer enkel water of 50 50 water ethyleen glycol door de hitte exchanger zo ja uitrust Verbind de hitte exchanger naar een waterkraan of koelmiddelbron niet waar de waterdruk niet geregulariseerd is Stel Nooit antivriesmiddel of organische oplosmid delen in deel van het instrument voor Organische oplosmiddelen zullen onherstelbare schade aan de eenheid veroorzaken Bedien niet met buffertemperaturen boven het maximum specificeerde technische specificaties Oververhittend zal onherstelbare schade aan de eenheid veroorzaken T rke Tietoa Finnish Jos t t varusteita k ytet n tav
29. ebolita Questo strumento disegnato per l uso di labora torio interno solo Solo gli accessori e le parti hanno approvato o hanno fornito da Hoefer Inc potrebbe essere usato per operare per mantenere e per revisionare questo prodotto usa Solo un alimentatore che CE ha marcato o la sicurezza certificato da un nazionalmente ricon osciuto testando il laboratorio Il coperchio di sicurezza deve essere nel luogo prima di collegare i piombi di alimentatore a un alimentatore Spegne tutto i controlli di alimentatore e disin serisce i piombi di potere prima di togliere il coper chio di sicurezza Circola solo l acqua o 50 50 glicole di acqua etilene attraverso lo scambiatore di calore se cos equipag giato Non collegare lo scambiatore di calore a un rubinetto di acqua o qualunque fonte di refriger ante dove la pressione di acqua sregolata Non introduce mai l antigelo o qualunque solvente organico in qualunque parte dello strumento solventi organici causeranno il danno irreparabile all unit Non opera con le temperature di tampone al di sopra del massimo ha specificato le descrizioni tecniche Il surriscaldamento causer il danno irreparabile all unit Viktig Informasjon Norwegian Hvis dette utstyret blir brukt i en m te ikke spesi fisert ved Hoefer Inc beskyttelsen som ha blitt git av utstyret kan bli svekket Dette instrumentet er utformet for innendors labo ratoriumbruk
30. ectrophoresis parameters for DNA acrylamide gels DNA gels are usually run at a constant voltage setting and since buffer systems are continuous both current and voltage readings remain con stant throughout the run Running conditions are expressed in units of V cm Published run ning conditions vary widely but voltages in the range of 1 to 3 V cm are common for overnight runs 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 set tings indicate potential problems such as leaking current incorrect buffer concentrations high salt concentrations or inconsistent chemical quality Check band progress after 5 minutes and again after an hour keeping an eye on the migration rate of the tracking dye The run is complete when the tracking dye reaches the bottom of the gel Watch the buffer level and if necessary replenish it as required to keep the top electrode submerged A small volume of buffer may leak past a nicked plate or gasket or buffer may pass through the gel e p21 Note Use only flexible plastic prying tools to avoid chipping the glass plates p22 After electrophoresis 0 Once the tracking dye reaches the bottom of the gel turn off the power supply disconnect the leads and remove the safety lid
31. ere the water pressure is unregulated Never introduce antifreeze or any organic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not operate with buffer temperatures above the maximum specified technical specifications Overheating will cause irreparable damage to the unit Dulezit Informace Czech Pokud by toto za zen je pou ito zp sobem kter nen podle Hoefer Inc ochrana poskytovan na z klad za zen m e b t naru ena Tento n stroj je ur en pro vnit n pou it v laborato i pouze Pouze p slu enstv a sti schv len nebo poskyt nut ch Hoefer Inc mohou b t pou ity pro provoz dr bu a dr b tohoto v robku zdroj nap jen pou vaj jen e je opat en ozna en m CE osv d ena nebo bezpe nost vnitrost tn uznan mi zku ebn mi laborato Bezpe nosti lid mus b t zavedena p ed p ipojen m nap jec zdroj nap jen vede k Turn ve ker nap jen kontroly vypnuto a odpojit p ed odb rem energie vede bezpe nostn v ko Rozeslat pouze voda nebo 50 50 voda ethyleng lykolu prost ednictv m v m n k tepla je li to vybav ena Nemaj p ipojen v m n k tepla s vodn mi set epn nebo jak koli chladic kapaliny zdroje kde tlak vody je neregulo Nikdy zav st prost edek proti zamrznut nebo jak koli organick rozpou t dla do jak koli sti z tohoto n stroje Rozpustidl
32. he isolation step Near the top of the gel when the buffer front has reached the bottom Gel concentration Precipitation The gel pore size is too small decrease the T of the resolv ing or stacking gel The protein has precipitated Heat the sample at a lower temperature 70 C or less for 1 2 min At both top and bottom of the gel Tracking dye doesn t sharpen into a concentrated zone in the stacking gel Gel concentration Poor stacking The molecular weight range of the sample requires an acryl amide concentration gradient to resolve the full range of protein sizes 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 Sample preparation When preparing samples avoid using solutions with high salt concentrations e p27 problem Poor band resolution e p28 possible cause Running conditions remedy Begin electrophoresis as soon as the sample is loaded to pre vent low molecular weight species from diffusing Conduct the separation at a lower current or voltage setting to reduce Joule heating Reagent quality Use only the highest quality reagents Poor stacking Use only gels that were recently prepared Add a stacking gel or increase height of the stacking gel Prepare the reso
33. id fractionation and the second dimension separation of 2 D electrophoresis First dimension separation of 2 D protein electrophoresis should be performed on Immobilized pH Gradient Gels The focused strips are easily transferred to the second dimension slab gel for size separation The SE640 gel plates are 18 cm wide and 8 cm in length Up to four gels can be run at one time if sandwiches are paired into club sandwiches Specifications Gel plate size w x h Gel size Maximum watt Maximum volt Maximum ampere Maximum temperature Environmental operating conditions Dimensions w x h x d Product certifications 18x 8 cm 14 x 8 cm 50 W 1000 V 500 mA 45 C Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Installation category Il Pollution degree 2 32 x 22 5 x 14 cm 12 5 x 8 75 x 5 5 in EN61010 1 UL61010A 1 CSA C22 2 1010 1 CE Certified This declaration of conformity is only valid for the instrument when it is e used in laboratory locations e used as delivered from Hoefer Inc except for alterations described in the user manual and e connected to other CE labeled instruments or products recommended or approved by Hoefer Inc Fig 1 Main components of the SE640 series see Fig 4 for caster components Included but not shown e Gel Seal compound Y4 oz e Spacer Mate spacer positioning guide e Glass plates 6 Wonder Wedge plate separati
34. locket Cirkulerar bara vatten eller 50 50 vatten ethylene glycol genom varmen exchanger i sa utrustad fall Inte kopplar varmen exchanger till en vatten kran eller n got kylmedel k lla dar vattnet trycket r unregulated Inf r aldrig kylv tska eller n got organiska l sningsmedel in i n gon del av instrumentet Organiskt l sningsmedel ska orsaka irreparable skada till enheten Anv nd inte med buffert temperaturer ver det h gsta angivna tekniska specifikationerna verhettning skulle orsaka irreparabla skador p enheten e pvi English M French X ii German Dg Exa Italian X Ei Spanish 134 Swedish X ii Waste Electrical and Electronic Equipment WEEE This symbol indicates that the waste of electrical and elec tronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Ce symbole indique que les d chets relatifs l quipement lectrique et lectronique ne doivent pas tre jet s comme les ordures m nag res non tri es et doivent tre collect s s par ment Contactez un repr sentant agr du fabricant pour obte nir des informations sur la mise au rebut de votre quipement Dieses Symbol kennzeichnet elektrische und elektronische Ger te die nicht mit dem gew hnlichen unsortierten Haus m ll entsorg
35. lving gel surface by first rinsing it with stacking gel monomer before pouring the stacking gel to ensure continuity between the gels Check pH values of the resolving and stacking gel solutions Do not back titrate buffers Incomplete gel polymerization Allow gel to polymerize fully Sample preparation Store sample on ice before it is denatured Dialyze or desalt the sample 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 Add more mercaptoethanol or dithiothreitol check sample treatment Add protease inhibitors such as PMSF if necessary to pre vent proteolytic degradation of sample Increase glycerol or sucrose to increase sample density Store samples to be frozen in aliquots to avoid repeated freeze thawing Store at 40 to 80 C Appendix A Laemmli system gels The Laemmli system is the most common elec trophoresis protocol for SDS denatured proteins The leading ion in this discontinuous buffer system is chloride and the trailing ion is glycine Accordingly the resolving gel and the stacking gel contain Tris Cl buffers of different concentration and pH and the electrophoresis buffer contains Tris glycine All buffers contain 0 1 SDS Polyacrylamide gel composition is indicated by two different percentages T total acrylamide g acryl bis x 100 100 ml
36. nalmente recon hecido testando laborat rio A tampa de seguran a deve estar em lugar antes de ligar o estoque de poder leva a um estoque de poder Desliga todos controlos de estoque de poder e desconecta os chumbos de poder antes de retirar a tampa de seguran a Circulam s gua ou 50 50 glicol de gua ethylene pelo exchanger de calor se for assim equiparam N o ligue o exchanger de calor a uma torneira de gua nem qualquer fonte de refrigerante onde a press o de gua n o regulado Nunca introduz anticongelante nem qualquer org nico solvente em qualquer parte do instru mento Org nico solvente causar agress o irrepar vel unidade N o opera com temperaturas de buffer acima do m ximo especificou especifica es t cnicas Super aquecer causar agress o irrepar vel unidade Informaci n Importante Spanish Si este equipo es utilizado en una manera no espe cificado por Hoefer Inc la protecci n proporcio nado por el equipo puede ser da ada Este instrumento es dise ado para el uso interior del laboratorio s lo S lo accesorios y partes aprobaron o suministraron por Hoefer Inc puede ser utilizado para operar para mantener y para atender a este producto S lo utiliza una alimentaci n que es CE marc o la seguridad certificada por un nacionalmente reconocido probando el laboratorio La tapa de la seguridad debe estar en el lugar antes de conectar la alimentaci n lle
37. on tool e Buffer dam Complete unit also includes spacers 4 and combs 2 Required but not included e Magnetic stirrer e Power supply with a minimum rating of 300 V 100 mA constant A or V Note The ordering section on page 38 lists all accessories and replacement parts color coded leads 2 safety lid upper buffer chamber wit h upper electrode and lower electrode fin lower buffer chamber Note Before using the first time disassemble the unit and wash with a dilute solution of a laboratory detergent and rinse thoroughly first with water and then with distilled water Unpacking and inventory Unwrap all packages carefully and compare con tents 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 If any part appears damaged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit Lower buffer chamber The lower buffer chamber is transparent acrylic which allows visual tracking of electrophoresis progress The chamber is chemically resistant to common electrophoretic buffers but not to organic solvents or strong acids and alkali Temperatures above 45 C may cause the chamber to warp Upper buffer chamber The upper buffer chamber is molded polysul f
38. one which is chemically resistant to common electrophoretic buffers but not to organic sol vents or strong acids and alkali The upper elec trode cathode runs along the center ridge and terminates at a banana plug The lower electrode anode runs along the edge of the electrode fin on the underside and terminates at a second banana plug Safety lid The banana plug on the upper buffer chamber at the terminus of the cathode wire connects to the black lead The banana plug on the lower electrode fin at the terminus of the anode wire connects to the red lead The 4 mm shrouded color coded leads plug into color coded jacks in the power supply Always install the safety lid before use Glass plates The plates are 18 cm wide and 8 cm in length Three sets of glass plates are included with each unit Notched divider plates ordered separately pair two gel sandwiches to form a club sand wich so that up to four gels can be run at one time Clamps Clamps are used to secure the plates and spacers together The clamp pressure bar adjusted with screws distributes pressure evenly Casting stand The casting stand holds assembled gel sandwiches 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 cammed into the stand Cams Cams are used twice first to secure the assem bled sandwich in the casting stand and second
39. ple loading pipet tip Table 1 Sample volume for standard comb sizes volume of sample ul per 1 mm depth no of comb thickness mm wells 0 75 1 0 1 5 10 6 2 8 3 12 4 12 5 8 77 11 5 15 4 3 8 6 20 3 1 4 1 6 2 28 2 1 2 7 4 1 1 prep 1 ref 90 4 121 6 183 9 1 prep 2 ref 85 4 113 6 171 9 Fig 6 Attaching gel sandwiches to the upper buffer chamber If the assembly leaks take it to a sink and partially release the cams to allow buffer to drain out of the upper chamber Disassemble check alignment of all sandwich compo nents and adjust if necessary A Remove cams from the lower cam holes Place the upper chamber onto the sandwiches and then insert the cams into the upper cam holes ridge short end pointing down B 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 If encountering unusual resis tance disassemble and inspect clamp and glass alignment along the top of the sandwich Align and reinstall Final assembly Upper buffer chamber 0 Rinse both buffer chambers with water and distilled water thoroughly before each use Clean away any gel adhering to the exterior of the gel sandwiches a If running only one gel Block the second upper buffer chamber slot by installing the acrylic buffer dam included with the unit Fit clamps onto the dam taking care to align the clamp ends and dam edges Ins
40. r separa tion and estimation of molecular weights of proteins by discontinuous gel electrophoresis Arch Biochem Biophys 126 155 1968 Denaturing gel systems Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T Nature 227 680 685 1970 Matsudaira P T and Burgess D R SDS microslab linear gradient polyacrylamide gel electrophoresis Anal Biochem 87 386 396 1978 Schreier M H Erni B and Staehelin T Initiation of mammalian protein synthesis I Purification and charac terization of seven initiation factors J Mol Biol Nov 116 4 727 753 1977 Shapiro A L and Maizel J V Jr Molecular weight estima tion of polypeptides by SDS polyacrylamide gel electropho resis 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 separa tion of proteins in the range from 1 to 100 kDa Anal Biochem 166 368 379 1987 Weber K and Osborn M The reliability of molecular weight determinators by dodecyl sulfate polyacrylamide gel electrophoresis J Biol Chem 224 4406 4412 1969 Two dimensional electrophoresis Adams L D and Gallagher S R Two Dimensional Gel Elec trophoresis Using the O Farrell System Current Protocols in Molecular Biology Ausubel F A et al eds OS
41. rface Prepare more gels as reguired Overlay each gel with a thin layer of water saturated n butanol water or diluted gel buffer to prevent gel exposure to oxygen Slowly deliver the overlay solution from a glass syringe fitted with a 22 gauge needle Apply the solution near the spacer at the side of the sandwich and allow it to flow across the surface unaided o Allow the gels to polymerize for a minimum of one hour After polymerization pour off the overlay and rinse the gel surface several times with distilled water O Prepare the stacking gel monomer solution pour the stacking gel and introduce a comb at a slight angle into the sandwich taking care not to trap air under the teeth Allow a minimum of one hour for the gel to polymerize Note With Coomassie Blue it is possible to detect 1 ug of protein in a single band With the more sensitive silver stains it is possible to detect as little as 10 ng of protein Sample preparation and loading The sample can be loaded either while the sand wich is in the caster or after the upper buffer chamber is attached When loading samples while using divider plates the samples must be loaded without the upper buffer chamber in place 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 rel
42. st con u pour l usage de labora toire int rieur seulement Seulement les accessoires et les parties ont approuv ou ont fourni par Hoefer Inc pourrait tre utilis pour fonctionner maintenir et entrete nir ce produit utilise Seulement une alimentation qui est CET a marqu ou la s curit certifi par un nationale ment reconnu essayant le laboratoire Le couvercle de s curit doit tre sa place avant connecter l alimentation mene a une alimentation Tourner tous contr les d alimentation de et d brancher les avances de pouvoir avant enlever le couvercle de s curit Circuler seulement de l eau ou 50 50 glycol d eau thyl ne par l exchanger de chaleur si si quip Ne pas connecter l lexchanger de chaleur a un robinet d eau ou a la source d agent de refroidissement o la pression d eau est non r gul e Ne Jamais introduire d antigel ou du dissolvant organique dans n importe quelle partie de instrument Les dissolvants organigues causeront e piii des dommages irr parables a l unit Ne pas fonctionner avec les temp ratures de tampon au dessus du maximum a sp cifi des sp cifications technigues La surchauffe causera des dommages irr parables a l unit Wichtige Informationen German Wenn diese Ausr stung gewisserma en nicht angegeben durch Hoefer Inc verwendet wird kann der durch die Ausr stung zur Verf gung gestellte Schutz verschlechtert werden Dieses
43. sturbing the samples Fig 7 Buffer chamber levels upper chamber lt buffer level lt lower chamber buffer level e pl9 Note All SE600 series models use 18 cm wide plates The gel thickness determines the cross section and current requirement The length of the plate determines the running time Table 2 Laemmli buffer system starting point guidelines Gel thickness 1 5 mm Current per gel 25 mA constant current Starting voltage 80 90 V Final voltage 220 400 V Thicker or thinner gels require propor tionally 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 tThe current must be multiplied by the number of gels For instance if two club sandwiches are installed the four gels require four times as much current The current can be increased for faster runs though overheating will eventually become a problem and i can be decreased for slower overnight runs At 25 mA per gel e p20 Separating the sample Electrophoresis parameters for discontinuous polyacrylamide gels Gels may be run at either constant current or constant voltage settings A constant current set ting is traditionally used with a discontinuous buffer system so that the rate of electrophoretic migration remains unchanged throughout
44. surface lay the Spacer Mate spacer positioning guide onto the plate wide side at the top of the plate place a spacer along each edge and lay the second glass plate on top Secure the sandwich with clamps Slide one clamp at a time along the sandwich sides Finger tighten one screw on each clamp set the sandwich upright on a flat surface and loosen the screw to align the stack Take great care in aligning to ensure a seal Finger tighten all screws Remove the Spacer Mate glass plates at the outer sides of the sandwich spacers notched center plate Fig 3 Club sandwich assembly Side clamps will accommodate two spacers up to 1 5 mm thick Note Do not use silicone grease or petroleum jelly to seal the sandwich These substances are difficult to remove and ulti mately cause artifacts Club sandwich A notched center divider plate ordered separately pairs two sandwiches to double the number of gels that can be cast and run Assemble a club sandwich in the same manner as a regular sandwich except before placing the top glass plate lay the divider plate and a second set of spacers on the stack Place the notch so that it will be at the top of the gels It is essential that the spacers and plates align perfectly in order to create a seal o Remove the sandwich and inspect the bottom to make sure that edges are aligned flush in order to ensure a complete seal Adjust if necessary Optional
45. t werden d rfen sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit einem auto risierten Beauftragten des Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchia ture elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalita di smantellamento delle apparecchiature fuori uso contattare un rappresentante autorizzato del fabbricante Este simbolo indica que el equipo el ctrico y electr nico no debe tirarse con los desechos dom sticos y debe tratarse por separado Contacte con el representante local del fabricante para obtener m s informaci n sobre la forma de desechar el equipo Denna symbol anger att elektriska och elektroniska utrust ningar inte far avyttras som osorterat hushallsavfall och maste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information angaende avyttring av utrustningen O pvii Gel electrophoresis unit function and description The Hoefer SE600 series vertical slab gel electrophoresis units SE600 SE640 and SE660 are intended for protein and nucleic acid electro phoresis under commonly used denaturing and non denaturing conditions Up to 28 samples can be compared on a single slab gel Applications include protein separations nucleic ac
46. tacking gel is then poured over the gradient gel Pouring a linear gradient gel 0 Assemble sandwich es into the dual gel caster as described on page 8 a Set up the monomer solution flow path Run a length of Tygon tubing through a peristaltic pump Attach one end of the tubing to the gradient maker outlet port and the other end to a 9 cm cannula The OD of the cannula must be less than the spacer thickness Place the cannula so that it rests at the bottom of the sandwich midway between the spacers Prepare the monomer solution Calculate the volume of monomer solution needed Divide the total volume in half and prepare this volume of both the higher and lower percentage acrylamide solutions e pl3 a e pl4 Pour the light solution into the reservoir chamber the chamber furthest from the inlet Open the stopcock long enough to displace air between the chambers and then close Pour the heavy 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 Raise the cannula as liguid enters the sandwich keeping the tip at the gel su
47. tall the dummy gel screws facing out in the second cradle in the dual gel caster 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 Both the slot in the gasket and the slot in the recess must align Both slotted gaskets must be used even if running only one gel sandwich Grooves along each slot help keep the gasket in place Additionally a small amount of Gel Seal can be applied at each end of the gasket before install to help hold the gasket against the upper buffer chamber Release the sandwiches from the caster by removing all bottom cams if present Lower the upper buffer chamber onto the gel sandwiches in the casting stand Install the cams ridge pointing down into the buffer chamber cam holes Cam the sandwich in place by simultaneously turning one cam clockwise and the other counterclockwise a full 180 e p17 a e p18 Use a pipet to carefully fill each slot above the sample wells with buffer in order to minimize disturbing the samples Then pour 100 ml of buffer into the chamber directing the buffer stream toward the side wall Check that no buffer is leaking around the gasket Lower buffer chamber 0 Place a magnetic spin bar into the lower buffer chamber and place the unit on a magnetic stirrer Fill the lower chamber with a minimum of 2 1 liters of buffer Optional Prechill th
48. then rinse thoroughly with tap and distilled water Glass plates can also be treated with but not stored in acid cleaning solutions e Do not autoclave or heat any part above 45 C e Do not use organic solvents abrasives strong cleaning solutions or strong acids or bases to clean the chambers e Do not soak the laminated gasket e p23 IMPORTANT Request a copy of the Hoefer Inc Health and Safety Declaration form before returning the item No items can be accepted for servicing or return unless this form is properly completed Note A Return Authorization RA number must be obtained from Hoefer Inc before return ing any item to Hoefer Inc p24 Customer service information Technical service and repair Hoefer Inc offers complete technical support for all of our products If you have any ques tions about how to use this product or would like to arrange to repair it please call or fax your local Hoefer Inc representative Check the Hoefer Inc website at www hoeferinc com for the distributor in your area Or contact us directly at Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone 1 508 893 8999 Fax 1 508 893 0176 support hoeferinc com www hoeferinc com remedy 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 cra
49. uktu korzysta jedynie zasilacza e jest nosz ce ozna kowanie CE lub bezpiecze stwa uwierzytelnione przez uznane na poziomie krajowym laboratorium badawcze Bezpiecze stwo lid musi by w miejsce przed pod czeniem zasilania prowadzi do zasilania Za wszystkie r d a zasilania urz dzenia steruj ce off i od czy moc prowadzi przed odbiorem bezpiecze stwa lid Kr tylko wody lub wody 50 50 ethylene glycol wymiennik ciep a poprzez je li tak wyposa one Nie nale y po czy wymiennik ciep a woda z kranu lub jakimkolwiek ch odziwo r d a je eli ci nienie wody jest nieuregulowanych Nigdy nie wprowadza rozpuszczalnika organ icznego przeciw zamarzaniu lub jakichkolwiek na dowoln cz dokumentu Rozpuszczalniki organiczne spowoduje nieodwracalne szkody dla jednostki Nie dzia aj w buforze temperatury powy ej maksymalnego okre lone specyfikacje techniczne Przegrzania spowoduje nieodwracalne szkody dla jednostki Informa es Importantes Portuguese Se este equipamento usado numa maneira n o especificada por Hoefer Inc que a protec o forne cida pelo equipamento pode ser comprometida Este instrumento projectado para uso de interior de laborat rio s S acess rios e partes aprovaram ou forneceu por Hoefer Inc pode ser usada para operar manter e servicing este produto S usa um estoque de poder que CE marcou ou seguran a registrada por um nacio
50. va a una alimentaci n Apaga todos controles de alimentaci n y desco necta los plomos del poder antes de quitar la tapa de la seguridad Circula s lo agua o 50 50 glicol de agua etileno por el intercambiador de calor si se es el caso equiparon No conecte el intercambiador de calor a un toque de la agua ni cualquier fuente del l quido refrigerante donde la presi n del agua est libre Nunca introduce anticongelante ni alg n solvente org nico en cualquier parte del instrumento Los solventes org nicos causar n da o irreparable a la unidad No opera con temperaturas de b fer encima del m ximo especific especificaciones t cnicas Reca lentar causar da o irreparable a la unidad Viktig Information Swedish om denna utrustning anv nds i ett s tt som inte har specificeras av Hoefer Inc skyddet tillhan dah ll vid utrustningen kan skadas Detta instrument formges f r inomhuslaborato rium anv ndning bara Bara medhj lpare och delar godkande eller lever erade vid Hoefer Inc kan anv ndas f r fungera underh lla och servicing denna produkt anv nder bara en kraft tillg ng som r CE markerade eller s kerhet intygade vid en nationellt erk nd testande laboratorium S kerheten locket m ste vara p platsen fore koppla kraften tillg ngen blyen till en kraft tillg ng Vander sig alla kraft tillg ng kontroller av och kopplar bort kraften blyen fore flytta s kerheten
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