Hoefer SE400 / SE410
Contents
1. Cirkulerar bara vatten eller 50 50 vatten ethylene glycol genom v rmen exchanger i s utrustad fall Inte kopplar v rmen exchanger till en vatten kran eller n got kylmedel k lla d r 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 French German Italian Spanish Swedish La Z l 1 La be Waste Electrical and Electronic Eguipment WEEE This symbol indicates that the waste of electrical and electronic eguipment 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 eguipment Ce symbole indique que les d chets relatifs a guipement 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 obtenir 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 Hausm ll entsorgt werden d rfe2. 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 cosi eguipag giato Non collegare lo scambiatore di calore a un rubinetto di acgua o gualungue fonte di refriger ante dove la pressione di acgua 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 II surriscaldamento causera il danno irreparabile all unita 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 bare
3. 11 Destain solution II 7 acetic acid 5 methanol Methanol 5 viv 50 0 ml Acetic acid 7 VIN 70 0 ml Deionized H20 to 1 0 liter 12 Cross linking solution 10 glutaraldehyde 20 ml of 50 glutaraldehyde stock Distilled water to 100 ml 13 DTT dithiothreitol solution 5 ug ml 5 mg DTT Bring to 1 L with ddH20 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 ul of 37 formaldehyde Prepare just before use 17 Stop solution 2 3 M sodium citrate 67 64 sodium citrate dihydrate FW 294 1 Bring to a final volume of 100 ml with deionized water e p33 Note Because this is a highly sensitive staining method it is important to wear gloves when handling gels and to use clean containers To reduce the background use only high purity reagents and remove all buffer from the gels during the fixing and destaining steps e p34 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 at room tempera ture Change the destain solution when it reaches
4. 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 SDS gels pH 8 8 J Mol Biol 116 727 752 1977 Shapiro A L and Maizel J V Jr Molecular weight estimation for polypeptides Anal Biochem 29 505 514 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 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 e p37 L 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 Arch Biochem Biophys 126 155 1968 Two dimensional electrophoresis Adams L D and Gallagher S R Two Dimensional Gel Electrophoresis Using the O Farrell System Current Protocols
5. 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 liguid by blotting one corner with a lint free tissue Calculate the stacking gel monomer solution volume Prepare the stacking gel monomer solution deaerate it and add catalyst and initiator Pour the stacking gel onto the resolving gel with a disposable or Pasteur pipet to a level about 2 mm from the top of the glass plate 4 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 SCC ME 0008 Fig 6 Pouring a gradient gel Note With Coomassie Blue it is possible to detect 1 pg in a single band with the more sensitive silver stains it is possible to detect as little as 10 ng 2 2 3 Gradient gels Linear gradient gels can be poured in the gel caster For easy gradient mixing we recom mend 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 accompany ing the caster Once the gradient gel polymer izes a stacking gel is poured o Assemble the glass plate assembly into the caster as de
6. Order comb and spacers separately Replacement parts Slotted silicone rubber gasket for upper buffer chamber 1 SE4008B Blank silicone rubber gasket for casting stand 1 SE4009 Lid with electrodes for SE400 16 cm ul SE4156 Lid with electrodes for SE410 24 cm 1 SE416 Lower buffer chamber casting stand 1 SE4151 Upper buffer chamber with gasket 1 SE4154 High voltage safety lead set 1 SE6056 HV Wonder Wedge plastic gel plate separation tool 1 SE1514 GelSeal 1 4 oz tube 1 SE6070 Clamps and Cams Clamp and Cam Kit four 16 cm clamps and 8 black cams 1 SE6003UK Replacement thumbscrews for clamps 12 SE6003U 2 Cams black for new style clamps with cam holes 4 SE6005L Clamp assemblies 8 cm 2 SE6403U Clamp assemblies 16 cm 2 SE6003U Glass Plates 18 x 16 cm Glass plates 2 SE6102 Glass plate club sandwich divider notched 1 SE6102D Glass Plates 18 x 24 cm Glass plates 2 SE6602 Glass plate club sandwich divider notched 1 SE6602D e p39 Combs number thickness width of wells mm mm guantity product number 10 0 75 8 3 1 SE511 10 75 10 00 8 3 1 SE511 10 1 0 10 50 8 3 1 SE511 10 1 5 12 0 75 7 6 1 SE511 12 75 12 00 7 6 1 SE511 12 1 0 12 50 7 6 1 SE511 12 1 5 15 0 75 5 7 1 SE511 15 75 15 00 B 7 1 SE511 15 1 0 15 50 5 7 1 SE511 15 1 5 20 0 75 4 1 1 SE511 20 75 20 00 4 1 I SE511 20 1 0 20 50 4 1 1 SE511 20 1 5 28 0 75 2 7 1 SE511 28 75 28 00 2 7 1 SE511 28 1 0 28 50 2 7 1
7. depth and the number of wells per comb Table 2 on page 15 lists volume per 1 mm depth for wells created by each comb size See the ordering information for additional comb specifications Specifications Glass plate size Approximate gel size Max wattage Max voltage Max amperage Max temperature Environmental operating conditions Dimensions w x h x d Product certifications SE400 18 x 16 cm SE410 18 x 24 cm SE400 14 x 15 cm SE410 14 x 23 cm 20W 500 V at 40 mA 30 mA gel 60 mA total at 325 V 45 C Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Installation category II Pollution degree II SE400 24 x 28 x 15 cm 9 5 x 11 x 6 in SE410 24 x 36 x 15 cm 9 5 x 14 2 x 6 in EN61010 1 UL3101 1 CSA C22 2 1010 1 CE 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 alter ations described in the user manual and e connected to other CE labeled instruments or prod ucts recommended or approved by Hoefer Inc 2 Operating instructions Procedures for casting gels and electrophoretic separation follow Included are instructions for both single percentage homogeneous and gradient polyacrylamide gels Appendix A lists recipes and Appendix B gives a bibliography 2 1 Gel casting preparation 2 1 1 Options precast gels and self cast gels The SE400 unit accepts
8. 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 O 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
9. Bare tilbehor 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 O piv Sikkerheten lokket m 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 produktu kor
10. 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 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 e p25 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 damaged components 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 Dye front curves up smiles at edges Excessive heat Prechill the buffer Decrease the current or voltage setting Run the gel in the cold room Protein streaks vertically Particulates in sample Centrifuge or filter sample before loading to remove particulates Overloading Load less sample Degradation Add protease inhibitor such as PMSF Unusually slow or fast run Current leakage around gel Check for leaks all plates and spacers must be aligned and free of grease and crack
11. in Molecular Biology pp 10 4 1 10 4 13 1992 Anderson N G Anderson N L and Tollaksen S L Clin Chem 25 1199 1210 1979 Anderson N L and Anderson N G Proc Natl Acad Sci USA 74 5421 5425 1977 Bravo R et al Proc Natl Acad Sci USA 79 2281 2285 1982 Hurkman W J and Tanaka L K Plant Physiology 81 802 906 1986 Mets L J and Bogorad Anal Biochem 57 200 210 1974 O Farrell P H J Biol Chem 250 4007 4021 1975 e p38 Ordering information product guantity product number for 18 x 16 cm gels SE400 Sturdier Vertical Unit complete 1 SE400 15 1 5 Includes one set of glass plates 18 x 16 cm 2 clamp assemblies 2 cams and 15 well comb and 2 spacers 1 5 mm thick Other size combs and spacers ordered separately SE400 Sturdier Vertical Unit basic A SE400 Includes one set of glass plates 18 x 16 cm 2 clamp assemblies 2 cams Order comb and spacers separately for 18 x 24 cm gels SE410 Sturdier Vertical Slab Electrophoresis Unit complete 1 SE410 15 1 5 Includes one set of glass plates 18 x 24 cm two 16 cm and two 8 cm clamp assemblies 2 cams 15 well comb and 2 spacers 1 5 mm thick Other size combs and spacers ordered separately SE410 Sturdier Vertical Slab Electrophoresis Unit basic i SE410 Includes one set of glass plates 18 x 24 cm two 16 cm and two 8 cm clamp assemblies and 2 cams
12. 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 indend rs 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 Sikkerhedl get m v re p plads for forbinding stramforsyningsblyet til en stramforsyning Drejer alle stramforsyningskontroller 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 for rsage uboelig s
13. 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 Dule it 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 m zp sob
14. side of the unit The size of the gel is 14 x 15 cm if using the SE400 and 14 x 23 cm if using the SE410 After cast ing the sandwich is transferred into the lower buffer chamber for electrophoresis The basic unit includes one set of glass plates 18 x 16 cm for the SE400 and 18 x 24 cm for the SE410 two clamp assemblies SE400 two 16 cm clamps SE410 two 16 cm clamps and two 8 cm clamps and two cams The complete unit includes one 15 well comb and two spacers 1 5 mm thick in addition to the basic unit 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 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 This unit is partially assembled to protect components during shipping To disassemble o Position the unit so that the electrical connectors face you 2 Note the holes at each side on the upper buffer chamber Rest your thumbs in these holes and use your index fingers to lift the sides of the safety lid gently until the electrode connectors unplug First lift the lid straight up so that the upper electrode shield clears the upper chamber and then lift the lid out
15. 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 x 16 cm gels the Multiple Gel Caster Kit which holds up to 10 sandwiches and the Gel Caster Kit which holds up to four sandwiches can be ordered separately Gels for the SE410 must be self cast Glass plates spacers and clamp sets are sized so that the assembled sandwich can be easily aligned to create the reguired seal When assem bling sandwiches take extra care to align all componenis for best results 6666666 BA o 0 Of0 00000 000000 16 cm 24 cm SE400 SE410 Fig 2 A 24 cm sandwich requires two 16 cm and two 8 cm clamps 2 1 2 Preliminary casting steps o Prepare the caster Place the spirit level into the lower buffer chamber and adjust the leveling feet 2 Prepare the clamps Loosen all clamp screws and make space for the sandwich by sliding the pressure plates toward the screws Construct each gel sandwich For each sandwich choose two perfectly clean unchipped glass plates and two spacers Lay one plate on a flat surface lay the Spacer Mate assembly template onto the plate wide side at the top place a spacer along each edge and lay the second glass plate on top 4 Secure the sandwich with clamps Slide one clamp at a time along the sandwich sides Finger tighten one screw on each
16. toward you to remove it completely Lift out the upper buffer chamber and then the glass plate assembly Remove the clamps by loosening the thumb screws Fig 1 SE400 series main components Included but not shown Cams GelSeal grease W oz Spacer Mate Wonder Wedge Level Reguired but not included Approved power supply safety lid color coded connectors 2 upper buffer chamber glass plates 2 clamps casting cradle lower buffer chamber color coded leads 2 leveling feet 4 Annotated inventory Buffer chambers Both buffer chambers are chemically resistant to common electrophoretic buffers but not to organic solvents or strong acids and alkalis Safety lid The lid contains both electrodes and both electrode connectors The electrode connec tors plug into the lead connectors on the lower buffer chamber The color coded leads plug into color coded jacks on the power supply Glass plates Two 18 cm wide glass plates are included Plates for the SE400 are 16 cm long and plates for the SE410 are 24 cm long A notched divider plate ordered separately can be used to run two gels at the same time Clamps Two 16 cm clamps are reguired to secure the 16 cm long sandwich These and an additional pair of 8 cm clamps are required to secure a 24 cm long sandwich Casting stand The caster can be leveled with the adjustable leveling feet on the bottom of th
17. Brama Hoefer SE400 SE410 The Sturdier vertical slab gel electrophoresis units um SE400 IM Rev CO 06 12 Adu O e fe r Contents Important Information sees ceviche rincones ii Waste Electrical and Electronic Equipment WEE vii 1 Unit function and description 1 Annotated INVENTO rsss iarna rn tadas 4 e EIDA iio Senna 6 2 Operating instructions 3 Care and maintenance 4 Troubleshooting ccccccceseesceeeeeeeeeeseeeeeeeseees Appendix A Laemmli System Gels 29 Gel ole 36 Appendix B Bibliography sss eee eee eee 37 Ordering Informatica 39 Important Information English If this eguipment is used in a manner not speci fied by Hoefer Inc the protection provided by the eguipment 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 eguipped Do not connect the heat exchanger to a water tap or any coolant source where the water
18. I pH 8 8 1 liter Tris base FW 121 1 1 5M 181 5g HCI to pH 8 8 Deionized H20 to 1000 ml Store up to 3 months at 4 C in the dark 3 4X Stacking gel buffer 0 5 M TrisCI pH 6 8 500 ml Tris base FW 121 1 0 5 M 30 3g HCI to pH 6 8 Deionized H20 to 500 m Store up to 3 months at 4 C in the dark 4 10 SDS solution 100 ml SDS FW 288 4 0 35 M 10 0 g Deionized H20 to 100 ml Store up to 6 months at room temperature Sodium dodecylsulfate 5 10 APS Initiator 1 mi APS FW 228 2 0 44 mm 0 1 9 Deionized H20 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 Ammonium persulfate p30 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 5mm 1 0 ml Deionized H20 to 100 0 ml Store up to 3 months at 4 C in the dark 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 preparative comb or resolving gel former on the gel 7 2X Sample treatment buffer 0 125 M TrisCl 4 SDS 20 glycerol 0 2 mM DTT pH 6 8 10 ml 0 5 M Tris Cl pH 6 8 Solution 3 0 125 M 2 5 ml 10 SDS 0 35
19. M Solution 4 0 14M 4 0 ml Glycerol FW 92 09 20 vw 2 0 ml Dithiothreitol DTT FW 154 2 0 2 mM 0 318 Bromphenol Blue FW 691 9 0 3 mM 2 0 mg Deionized H20 to 10 0 ml or 2 mercaptoethanol FW 78 13 2 v v 0 2 ml Divide into 1 0 ml aliquots and store at 40 C to 80 C for up to 6 months 6X Sample treatment buffer 0 35 M TrisCI 10 SDS 30 glycerol 9 3 DTT pH 6 8 10 ml 0 5 M TrisCI pH 6 8 Solution 43 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 Bromphenol Blue FW 691 9 0 175 mm 1 2 mg Divide into 1 0 ml aliquots and store at 70 C e p31 Caution Glutaraldehyde should only be handled in a fume hood e p32 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 721g SDS FW 288 4 3 b 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 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 Acetic acid 7 VN 140 0 ml Deionized H20 to 2 0 liters 10 Destain solution 40 methanol 7 acetic acid 1 liter Methanol 40 viv 400 0 ml Acetic acid 7 VIN 70 0 ml Deionized H20 to 1 0 liter
20. SE511 28 1 5 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 product number 1 1 0 75 121 6 1 SE511 R 75 1 1 1 00 121 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 00 113 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 length width mm cm cm guantity product number 0 75 16 2 2 SE6119 2 75 1 0 16 2 2 SE6119 2 1 0 1 5 16 2 2 SE6119 2 1 5 1 0 16 1 2 SE6118 2 1 0 1 5 16 1 2 SE6118 2 1 5 0 75 24 2 2 SE6619 2 75 1 00 24 2 2 SE6619 2 1 0 1 50 24 2 2 SE6619 2 1 5 Gel Casters Order combs and spacers separately For up to 4 gels Gel Caster Kit 4 gels 18 x 16 cm 1 SE675 Includes 8 glass plates 3 space saver plates 5 filler sheets 100 sheets of wax paper Spacer Mate alignment template and filler plugs For up to 10 gels Multiple Gel Caster Kit 10 gels 18 x 16 cm 1 SE615 Includes 20 glass plates space saver plate 5 filler sheets 100 sheets of wax paper and Spacer Mate alignment template Recommended Hoefer SE100 Plate Mate washing and storage unit 1 SE100 Hoefer PS300B Power Supply 1 PS300B e p41 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800
21. a deep blue color until clear background results C Store the gel in Destain solution II For a more sensitive method silver stain protocol is recommended Silver Stain Protocol adapted from Morrissey 1981 Gentle agitation is recommended throughout this procedure A Stain the gel as usual with Coomassie Blue Destain the gel with several changes of Destain solution II Fix the gel in 100 ml Destain solution for 30 minutes then place the gel in 100 200 ml Destain solution II for 30 minutes Discard the solution refill and wash with Destain solution II a second 30 minutes B Transfer the gel to 100 ml crosslinking solution for 30 minutes C Decant the glutaraldehyde and rinse the gel with several changes of deionized water over a period of two hours Note Some bleaching may occur if using Destain solution II as a stop solution 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 ug ml DTT in deionized water for 30 minutes E Pour off the DTT solution but do not rinse the gel Add 100 ml of silver nitrate solution directly to the gel Shake gently for 30 minutes and then rinse the gel for 1 2 seconds with deionized water F Add 50 ml of developer solution guickly swirl the gel and pour off developer Repeat once more Add 100 ml of developer and agitate until the bands are
22. acers atop the divider plate Place the notch so that it will be at the top of the gels As with a regular sandwich it is essential that the spacers and plates align perfectly in order to create a seal Inspect the bottom of the sandwich to make sure that edges are aligned flush in order to ensure a complete seal Adjust if necessary see Fig 4 Optional Apply a light film of GelSeal only on the bottom outside corners if your sandwiches tend to leak Do not use silicone grease or petroleum jelly to seal the sandwich because these substances are difficult to remove and ultimately may cause artifacts in the gel Place the laminated gasket into the casting cradle with the foam side down Place the glass plate assembly in the casting cradle screw side facing out see Fig 5 24 cm plates Place the sandwich so that the short clamps are at the bottom o Insert a cam into the hole on each side of the casting tray with the ridge short end pointing up Seal the gel sandwich by turning both cams as far as needed usually 90 to 150 up to 180 The cam action presses the plates 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 tighten the cam past this point Fig 5 Caster components and assembly 1 Lower the assembled sandwich into the casting cradle 2 Insert cams into the cam hole
23. amber Release the sandwich from the caster by removing both cams Clean away any gel adhering to the exterior of the gel sandwich Install the sandwich in the lower buffer chamber clamp screws facing toward the leads Carefully fill each sample well with electrophoresis buffer then underlay prepared sample into the wells using a fine tipped microsyringe or gel loading pipet tip Q Attach the upper buffer chamber to the gel sandwich Invert the upper chamber and press the slotted gasket into the grooves for a precise fit Proceed with care so that the samples are not disturbed Lower the upper chamber onto the gel sandwich Install the cams ridge pointing down into the cam holes as shown on page 17 Simultaneously turn one cam clockwise and the other counterclock wise a full 180 to secure the assembly 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 cam starting position final clamping position e p17 Note Do not force the cams If encountering unusual resistance disassemble the unit and inspect clamp and glass alignment along the top of the sandwich Align and reinstall the upper chamber Note If the assembly leaks take the assembly to a sink and partially releas
24. 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 24 cm sandwich SE410 A 24 cm sandwich requires two clamp assemblies on each side Align each end separately That is align 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 or bottom edge of the glass plates For 24 cm long plates position the 8 cm clamp at the bottom see Fig 2 glass plates at the outer sides of the sandwich notched D divider spacers Fig 3 2 gel sandwich assembly 2 gel sandwiches are limited to thinner gels no spacers thicker than 1 5 mm can be used both top and bottom sandwich edges must be flush with the clamp guide ridges pressure bar Fig 4 Sandwich assembly Inspect glass plates for nicks Use only unchipped plates to prevent leaking Tip Remove the laminated gasket from the cradle and use the casting cradle to hold the sandwich for alignment 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 3 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 sp
25. e unit A laminated gasket seals the bottom of the glass plate assembly when it is locked into the stand Cams Cams are used twice first to secure the assembled sandwich in the casting stand and again to lock the sandwich and the upper buffer chamber together Rubber gaskets There are two gaskets The laminated gasket fits into the bottom of the cast 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 Wonder Wedge Plate Separator Tool Use to disassemble gel sandwiches and to gauge spacer and comb thickness Spacers May be ordered separately Spacers determine the thickness of the gel They are 2 cm wide and are available in three thicknesses 0 75 1 0 and 1 5 mm Combs May be ordered separately Combs are available in sizes that form 10 12 15 20 or 28 wells Preparative combs include 1 or 2 reference wells in addition to a preparative well Most combs are available in all three thicknesses 0 75 1 0 and 1 5 mm All preparative combs and combs with fewer than 28 wells 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
26. e the cams to allow buffer to drain Remove the upper chamber check alignment of all sandwich components and adjust if necessary e pl8 Pour 100 ml of electrophoresis buffer into the upper chamber directing the buffer stream against the wall to avoid disturbing the samples Inspect the installation for leaks Fill both chambers the final volume for each chamber is 350 ml Safety lid installation Built in safety features reguire that all three guides are properly placed see Fig 8 o Plug the color coded leads into the jacks of an approved power supply min 50 mA 300 V 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 gt shield guides Fig 8 Safety lid installation If you are unfamiliar with the installation The safety lid seats effortlessly please note S W s features are properly The upper electrode is protected by a recessed ZE shield which rests in the upper buffer chamber 1 The recessed upper electrode once the lid is installed It is easiest to install the shield slides into the upper lid by first approaching the upper buffer chamber buffer chamber from the front and then sliding the safety lid down 2 The lower electrode shield fits straight down onto the conn
27. each well with electrophoresis buffer to remove unpolymerized acrylamide and then drain by inverting the gel sandwich or caster Fill each well with electrophoresis buffer 2 Prepare the sample Increase liquid 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 liquid and dry samples in a test tube To liquid protein samples add an equal volume of 2X treatment buffer To dry protein samples add equal volumes of 2X treatment buffer and deionized water to achieve the desired concentration 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 e p15 Note Before the first use disassemble the unit and wash with a dilute solution of a laboratory detergent and rinse thoroughly first with water and then distilled water Note To help hold the gasket against the upper buffer chamber dab a small amount of GelSeal at each end of the gasket only and then install Important A smooth fit between the sandwich and gasket is essential for a good seal e pl6 2 4 Final assembly o Rinse both buffer chambers with water and distilled water thoroughly before each use 2 Install the gel sandwich in the lower buffer ch
28. ectors If the lid does into the lower buffer chamber not fit properly check the position of the lower and rests in front of the shield electrode shield which must clear the connectors guides and rest in the lower buffer chamber in front of the the shield guides Once all guides are in place 3 The electrode connectors align press gently to connect the plugs and seat e pl9 Note The cross section and current reguirement is determined by gel thickness The running time is determined by the length of the plate e p20 2 5 Resolving the sample Electrophoresis parameters for discontinuous polyacrylamide gels Gels may be run at either constant current or constant voltage settings A constant current setting is traditionally used with a discontinuous buffer system so that the rate of electrophoretic migration remains unchanged throughout the run Under constant current conditions the voltage increases as the run proceeds A lower current setting is recommended for higher reso lution The optimal current level must be deter mined empirically the main factors that must be balanced are the gel concentration and migration speed and the resulting Joule heating and band distortion Table 3 lists starting point guidelines and adjustments for gel thickness number of gels and migration rate Table 3 Laemmli buffer system starting point guidelines Gel thickness 1 5 mm Current per gelt 25 mA constant current Startin
29. er or diluted gel buffer to prevent gel exposure to oxygen S owly deliver the overlay solution from a glass syringe fitted with a 22 gauge needle Apply the solution near the spacer and allow it to flow across the surface unaided o Allow the gel s to polymerize for a minimum of one hour After polymerization pour off the overlay and rinse the gel surface several times with distilled water 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 Table 2 Well volume pl per 1 mm depth for each comb size Comb thickness mm No of wells 0 75 1 0 15 10 62 83 12 4 12 58 7 7 115 15 43 57 8 6 20 31 41 6 2 28 21 27 4 1 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 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 o 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
30. f the resolving or stacking gel Precipitation At both top and bottom of the gel Gel concentration The protein has precipitated Heat the sample at a lower temperature 70 C or less for 1 2 min The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the full range of protein sizes Poor band resolution Running conditions Begin electrophoresis as soon as the sample is loaded to prevent 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 resolving 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 e p27 problem possible cause remedy Poor band resolution continued 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 mercaptoetha
31. g 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 3 After the gel is set prepare the stacking gel as described in the next section 2 D electrophoresis Discontinuous system For the second dimension resolving gel fill solution to 1 0 cm below the top of the glass plate leave extra space for a stacking gel if reguired One centimeter allows enough space for the first dimension IPG strip or tube gel and an agarose seal While transferring take care to avoid trapping air between the tube gel and slab gel seal the tube gel into place with agarose in elec trophoresis buffer e pll L e p12 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 Apply the solution near the spacer and allow it to flow across the surface unaided 4 Allow the gel to polymerize for a minimum of one hour 2 2 2 Stacking gel Pour the stacking gel before removing the sandwich from the gel caster Stacking gel resolution is optimal when prepared just before electrophoresis o
32. g voltage 80 90 V Gel length cm model final voltage V 16 SE400 200 250 24 SE410 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 The current must be multiplied by the number of gels For instance if a 1 mm 2 gel sandwich is installed twice as much current is required than for a single 1 mm gel at the same voltage Note Passive cooling such as running the unit in a cold room may be reguired to reduce the effects of Joule heating Important After initial monitoring do not leave the unit unattended for more than 1 hour without checking the progress of the bands and the buffer level Current Current acts on the total cross sectional area of all the gels and in terms of a circuit the gels are considered to run in parallel Therefore any current setting for one gel must be multiplied by the number of gels run For a gel 1 5 mm thick we suggest a starting point current setting of 25 mA Two 1 5 mm gels 50 mA Voltage 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 SE400 model and a Laemmli discontinuous buffer system The final voltage is typically 200 325 V depending on the length of the gel See Table 3 on page 20 Time A run is complete when the tracking dye reaches the bottom of the gel A 16 c
33. h tap and distilled water Glass plates can also be treated with but not stored in acid cleaning solutions 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 Mis aligned parts Check plate and spacer alignment and realign if necessary Over clamping Turn cam only as far as necessary to create a seal usually 90 1507 but up to 180 On each spacer apply a light film of GelSeal compound to the bottom outside corner only Do not use silicone grease Sample wells damaged or irregular Air bubbles 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 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 Incomplete gel polymerization Chemicals Use only recent stocks of the highest quality reagents If the dry ammonium persulphate does not crackle when
34. kade 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 tata varusteita k ytet n tavassa ei m
35. 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 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 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 e piii instrument Les dissolvants organigues causeront 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 techniques 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 Instrument
36. lamide solutions 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 down In our experience with the concentrations in the 10 20 gradient example below ten gel sandwiches can be poured in a multiple gel caster at a flow rate of 5 10 ml min Table 6 Linear gradient gel recipes per 100 ml 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 Solution 2 25 00 ml 25 00 ml 10 SDS Solution 4 1 00 ml 1 00 ml Deionized H20 to 100 00 ml to 100 00 ml 10 APS Solution 5 0 300 ml 0 060 ml TEMED 0 036 ml 0 036 ml e p36 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 and Maniatis T Standard Formaldehyde 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 F A Ausubel R Brent R E Kingston D D Moore J G Seidman J A Smith and K Struhl eds 10 6 1 10 6 8 1991 Denaturing gel systems
37. m long 1 5 mm thick Laemmli SDS gel run at 25 mA gel with out cooling usually reguires 5 hours A 24 cm gel reguires about 8 hours e p21 p22 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 chemical quality Check band progress after 5 minutes and again after an hour noting 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 Tip To avoid splashing add staining or fixative solution to the tray after the gel is transferred Note Use only flexible plastic prying tools to avoid chipping the glass plates 2 6 After electrophoresis o Once the tracking dye reaches the bottom of the gel turn off the power supply and disconnect the leads Remove the safety lid using finger leverage rest your thumbs on the top of the cams and gently pull the lid up with your index fingers Once loose lift the lid straigh
38. mentaci 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 godk nde 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 f re koppla kraften tillg ngen blyen till en kraft tillg ng V nder sig alla kraft tillg ng kontroller av och kopplar bort kraften blyen f re flytta s kerheten locket
39. n 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 lleva a una ali
40. n sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten des Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalit di smantellamento delle apparecchiature fuori uso contattare un rappresentante autorizzato del fabbricante Este s mbolo 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 utrustningar inte f r avyttras som osorterat hush llsavfall och m ste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information ang ende avyttring av utrustningen O pvii 1 Unit function and description The Hoefer SE400 and SE410 Sturdier Vertical Slab Gel Electrophoresis Units are intended for electrophoretic separation of proteins and nucleic acids under both denaturing and native conditions Up to 28 samples can be compared on a single slab gel One gel or two gels if using the divider plate ordered separately is cast in the casting stand
41. nol or dithiothreitol check sample treatment Add protease inhibitors such as PMSF if necessary to prevent proteolytic degradation of sample Increase glycerol or sucrose to increase sample density Store samples to be frozen in aliguots to avoid repeated freeze thawing Store at 40 to 80 C Tracking dye does not 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 guality 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 p28 Caution Acrylamide is a neurotoxin Always wear gloves while handling in any form and wear a mask while weighing the powder Never mouth pipette the solution 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 pH 8 8 6 8 83 SDS 0 1 0 1 0 1 APS 0 05 w v 0 05 0 1 w v TEMED 0 05 v v 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 in Table 5 Ammonium persulfate Tetrameth
42. ritetty Hoefer Inc suojelu ehk isty varusteille saattaa olla avuton T m valine 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 Turvallisuuskansi t ytyy olla paikallaan ennen yhdist minen k ytt j nnitelyijyj k ytt j nnit teeseen Kiert kaikki k yttoj 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 est con u pour
43. s 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 Bands are skewed or distorted Incomplete gel preparation and polymerization Degas the stacking gel solution and avoid trapping air bubbles 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 surface Sample preparation e p26 Dialyze or desalt the sample 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 Near the top of the gel when the buffer front has reached the bottom Gel concentration Proteins may be degraded by endogenous proteases use protease inhibitors during the isolation step The gel pore size is too small decrease the T o
44. s glass plates 2 ridge end up 3 Turn cam up to 180 until spacers the glass plates seal against the gasket 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 E leveling feet 4 p Note It is easier to keep the caster balanced if you turn both cams toward the center of the caster e plo Note Appendix A on page 29 lists recipes for the Laemmli gel system 2 2 Acrylamide gel preparation Table 1 Approximate monomer solution volume required for a single gel Gel thickness mm Model 0 75 1 00 1 5 SE400 15 ml 23 ml 30 ml SE410 23 ml 34 ml 45 ml 2 2 1 Resolving gel o Prepare the monomer solution and pour the gel Prepare the reguired amount of monomer solution deaerate and add the initiator and catalyst just prior to pouring the gel 2 Pipet the solution into one corner of the sandwich taking care not to introduce any air bubbles See below for the appropriate solution level No stacking gel Continuous system Fill solution to just below the top of the upper plate edge If bubbles are trapped remove with a pipet or syringe Introduce a comb at a slight angle into each sandwich taking care not to trap air bubbles under the teeth 2 gel sandwich Pipet the solution into both sand wiches fillin
45. scribed in section 2 1 2 2 Set up the monomer solution flow path Run a length of clear vinyl tubing through a peristaltic pump Attach one end of the tubing to the gradient maker outlet port and the other end to a 20 cm cannula The outside diameter 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 total volume needed Prepare one half of this volume of higher and the other half of lower acrylamide solu tion Optional Add 15 sucrose or 25 glycerol final concentration to the higher solution to improve layering Q 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 on a magnetic stirrer and begin stirring at a rate that does not introduce bubbles in the solution e p13 L e pl4 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 liguid enters the sandwich keeping the tip at the gel surface Overlay each gel with a thin layer of water saturated n butanol wat
46. t up and then out to clear the ledge on the upper buffer chamber 2 Pour out the buffer by inverting the unit over a sink Release the upper buffer chamber by removing the cams Lift the chamber off and lift the sandwich out of the lower chamber Unscrew the clamps from the sandwiches and remove Gently loosen and then slide away both spacers Use the Wonder Wedge plate separator tool to separate the plates Carefully lift off one glass plate Handle the gel with care to avoid damaging it Over an empty stain tray either invert the plate holding the gel near the bottom of the tray and lift one corner so that the gel drops into the tray or if the gel is thick enough to handle lift it and place into the tray Add enough fixative or stain to completely submerge the gel Clean the unit as described in Care and maintenance on page 24 e p23 p24 3 Care and maintenance Cleaning e Rinse with water immediately after use e Do not autoclave or heat any part of the instrument above 45 C e Do not use organic solvents abrasives strong cleaning solutions or strong acids or bases to clean any plastic part e Do not soak the gaskets Clean with a mild detergent and allow to air dry e Handle the safety lid with care to prevent damage to the electrode connectors Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS 35 then rinse thoroughly wit
47. visible Be sure to stop the development before the background becomes significant by neutralizing the solution with 5 ml of stop solution Alternatively pour off developer and add 100 ml Destain solution II G Wash the gel in 2 3 changes of deionized water Keep the gel in Destain solution II or dry it for permanent storage e p35 Gel recipes The Laemmli gel recipes are for 30 ml of a single concentration solution enough for one 1 5 mm 18 x 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 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 Table 5 Laemmli gel recipes per 30 ml resolving gel solution 5 ml stacking gel solution Resolving gel Stacking gel 7 5 10 12 5 15 4 Acrylamide stock Soln 1 7 5 ml 10 m 12 5 m 15 ml 0 67 ml 1 5 M TrisCl pH 8 8 Soln 2 7 5 ml 7 5m 7 5m 7 5 ml 0 5 M TrisCl pH 6 8 Soln 3 1 25 ml 10 SDS Soln 4 0 3 ml 0 3m 0 3m 0 3 ml 0 05 ml Deionized H20 14 6 ml 12 1 m 9 6m 7 1 ml 3 00 ml 10 APS Soln 5 150 ul 150 U 150 u 150 ul 25 ul TEMED 10 ul 10 U 10 U 10 ul 2 5 ul Final Volume 30 0 30 0 m 30 0 m 30 0 ml 5 0 ml For linear gradient gels use egual volumes of low and high acry
48. wird f r den Innenlaborge brauch nur daf r entworfen Nur Zus tze und Teile genehmigten oder lieferten durch Hoefer Inc kann f r 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 W rmeaustauscher wenn so ausgestattet in Umlauf setzen Verbinden Sie den W rmeaustauscher mit einem Wasserklaps oder jeder K hlmittel Ouelle 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 ber angegebenen technischen Spezifizierungen des Maximums nicht funktionieren Die berhitzung 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 indebolita
49. ylethylenediamine The Laemmli system is the most common electropho resis 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 electropho resis buffer contains Tris glycine All buffers contain 0 1 SDS Polyacrylamide gel composition is indicated by two different percentages g acrylamide bisacrylamide 100 ml T x 100 g bisacrylamide g acrylamide bisacrylamide The total percent of acrylamide T in the resolving gel which can range from 4 to 20 determines the pore size Commonly the amount of crosslinker used C is 2 6 In the following system example the resolving gel composition 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 e p29 AAA Solutions Note Filter solutions 1 4 1 Acrylamide stock solution through a0 45 um filter 30 8 T2 6 C Bis 200 ml Acrylamide FW 71 08 30 w v 60 0 g Important Refer to the Bis FW 154 2 0 8 ww 1 68 material safety data sheet Deionized H20 to 200 ml MSDS accompanying each chemical for detailed handling Store at 4 C away from light and safety information N N Methylenebisacrylamide 2 4X Resolving gel buffer 1 5 M TrisC
50. zysta 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 chtodziwo 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 nacionalmente reco
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