Owner manual

EBU-222 Instruction Manual, version 7/28/2011
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SECTION 3
INSTRUCTIONS FOR USE
3.1 BLOTTING UNIT PREPARATION
1. Placetheblottingchamberonalevelworksurfaceinanauthorizedworkarea.
2. Consultapplicationstable4.1fortypeoftransfer,buffersystem,membranetypeandpowersettings.
Coolant Circulation:
Forcoolantcirculation,connectaheavywalltubingwhichwillnotkink(hoseclampsareoptional),tothetubing
adapterssuppliedwiththeunit(excessivepressurewilldamagethesealbetweentheglassandthebaseand
possiblycreateleaks).Attacharegulatedpumporrecirculatingwaterbath(followmanufacturer’sinstructions),
recommendedowis500mlperminuteor15psi.DoNOTusetaporhousewaterasitcanbesubjecttolarge
uctuationsinpressure.
1. Place,doNOTdrop,amagneticspinbar(notsuppliedbyC.B.S.)inthebottomofthetank.Thismaintains
pH, buffer and heat circulation during the electroblot.
2. Placetankontopofthemagneticstirrer.
3. Connecttoacoldwatersupply(seecoolantcirculationabove).
4. Buffer should be pre-cooled to 10°C.
3.2 PREPARING THE GEL FOR TRANSFER
TheEBU-222canholdupto2gelcassettessimultaneouslywithspaceprovidedforamagneticstirbartoallow
buffercirculationandheatexchangeforuniformtransfers.Whentheresolvinggelelectrophoresisiscomplete,
proceedwithstainingandphoto-documentationifapplicable.Cutonecorneroffthegelsothatcorrectorientation
ismaintainedthroughouttheprocedure.
PREPARATION OF ELECTROBLOTTING COMPONENTS
1. Cuttransfermembranetosizeofgelorsufcientdimensionstocovertherelevantbands.Also,pre-cut
Whatman-type3MMlterpapertosamesizeandsoakintransferbufferuntilcompletelysaturated(15to
30minutes).
2. Pre-wetmembranechosenaccordingtomanufacturer’sinstructions.NylonorNylon-supportednitrocellu-
loseshouldbesoakedinddH2O.PVDFinMeOH.Equilibratealltypesintransferbufferfor3minutes.
3. Pre-equilibrategelintransferbuffertobeusedpriortoelectroblottingfor10to30minutesdependingon
gel thickness.
4. Openthetransfercassetteandsubmergethered(+)panelinashallowplasticorglass(Pyrex)dish.Fill
withenoughbuffertocoverentirecassette.SubmergeasingleScotchBrite®padandgentlyteaseto
releasetrappedairbubblesuntilpadiscompletelysaturated.
5. Layeryourtransfermembraneontopofyourspongepad.(Someprefertolayer1to2sheetsofsaturated
3mmlterpaper).Carefullysearchforandremovetrappedairbubblesateachlayerbyrollingapipetover
the surface.
6. Applygelcorrectlyorientedtosubmergedtransfermembrane.Checkforairpocketsandremove.Note:
Someproteinswillbegintransferimmediatelyuponcontractingthetransfermembrane.Disturbingormov-
ingthegel/membraneinterfacecanresultinasmearedblot.
7. Quicklyfollowwith1-2sheetsofsaturated3mmlterpaper(optional).
8. Placesecondsaturatedspongepadoversandwichassemblyandlatchthecassetteintotheclosed
position.