Materials

• BlotCell
• BA830.2µmporenitrocellulosesheets
• Buffer,PBS-Tween20
• Antigenicproteins,antibodies,andhorserADIshperoxidaselabeledantiglobulins

Procedure21

1. RunanelectrophoreticseparationofknownantigenicproteinsaccordingtotheproceduresinExercise4.1and4.2.

2. Drawaline0.5cmfromthetopedgeofan8x10cmnitrocellulosesheetandsoakitinblotbufferforabout5minutes.

   Nitrocelluloseisbothfragileandflammableandeasilycontaminatedduringhandling.Weargloveswhichareprewashed.

   Whensoakingthenicrocellulosewetfirstonesideandthenturnthesheetoverandwettheother,topreventtrappingairwithinthefilter.

3. Place200mlofblotbufferintoatrayandaddapieceoffilterpaperslightlylargerthantheelectrophoreticgelfromStep1.

4. Removethegelfromtheelectrophoresischamberaftertheproteinshavebeenseparated,andplacethegelintothetraycontainingthefilterpaper.Donotallowthegeltofallontothepaper,butplaceitnexttothepaperinthetray.

5. Gentlyslidethegelontothetopofthefilterpaper.Keepthestackinggeloffofthepaperuntilthelastmoment,sinceittendstostickandmakerepositioningdifficult.

6. Holdingthegelandthefilterpapertogether,carefullyremovethemfromthetrayofblotbufferandtransferthepaperandgeltoapadoftheblotcellwiththegelfacingup.

7. Transferthenitrocellulosesheet(inksidedown)ontothetopofthegelandlineupthelinedrawnonthesheetwiththetopofthestackinggel.

   Oncethegelandnitrocellulosetouchtheycannotbeseparates.

8. Rollaglassrodacrossthesurfaceofthenitrocellulosetoremoveanyairbubblesandinsuregoodcontactbetweenthegelandnitrocellulose.

9. Layanothersheetofwetfilterpaperontopofthenitrocellulosecreatingasandwichofpaper-gel-nitrocellulose-paper,alllyingonthepadoftheblotcell.

10. Addasecondpadtothetopofthesandwichandplacetheentiregroupinsideofthesupportframeoftheblotcell,andassembletheblotcellsothatthenitrocellulosesideofthesandwichistowardthepositiveterminal.

11. Checkthatthebufferlevelsareadequateandthatthecoolingwaterbathisadjustedtoatleast5°C.Subjectthegeltoelectrophoresisfor30minuteswiththeelectrodesinthehighfield-intensityposition.Followthemanufacturerdirectionsduringthisphase.Failuretocloselymonitortheelectrophoresisbufferortemperaturecanresultinafire.Useacirculatingcoldbathappropriatetotheapparatusandholdthevoltagetoaconstant100vdc.

12. Uponcompletionoftheelectrophoresis(timedaccordingtomanufacturer"sdirections),turnoffthepoweranddisassembletheapparatus.Removetheblotpadsfromthesandwichandremovethefilterpaperfromthenitrocelluloseside.

13. Placethesandwich,nitrocellulosesidedown,ontoaglassplateandremovetheotherfilterpaper.

14. Useaballpointpentooutlinetheedgesoftheseparatinggelontothenitrocellulose,includingthelocationofthewells.Carefullyliftthegelawayfromthenitrocelluloseandmarkthelocationsofthepre-stainedmolecularweightstandardsasthegelispeeledaway.Peelthegelfromtheseparatinggelside,notthestackinggel.

15. Washtheblot(thenitrocellulosesheet)atleastfourtimeswith100mlofPBS-Tween20forfiveminuteseachonarockingplatform.

16. Cuttheblotinto0.5cmstrips.

17. Inactivateseracontainingpositiveandnegativeantibodycontrolstotheantigensunderexaminationbytreatingat56°Cfor30minutes.Makedilutionsof1:100and1:1000ofthecontrolswithPBS-Tween20.

18. Place3mlofthedilutedseraorcontrolsontoastripfromStep16andincubatefor1houratroomtemperaturewhilecontinuouslyrockingthesample.

19. Washthestripsfourtimesfor5minuteseachwith10mlquantitiesofPBS-Tween20.Thefirstwashshouldbedoneat50°Cbutthelastthreemaybedoneatroomtemperature.

20. Add3mlofhorseradishperoxidase-labeledantiglobulin,optimallydilutedinPBS-Tweenandincubateatroomtemperaturefor1hourwithcontinuousagitation.

21. Washthestripsfourtimesfor5minuteseachwithPBS-Tween20andonemoretimewithPBSonly.

22. RemovethePBSandadd5mlofsubstratesolution.Positivereactionbandsusuallyappearwithin10minutes.Stopthereactionbywashingwithwater.RefertoFigure4.15foracomparison.

Notes

Oneofthemoredifficulttasksofelectrophoreticseparationsistheidentificationofspecificbandsorspotswithinadevelopedgel.AsobservedwithLDHisozymes,onemethodofdoingthisistoreactthebandswithanenzymesubstratethatcanbedetectedcolorimetrically.

Asarule,however,mostpeptidesaredenaturedduringelectrophoresis,and,ofcourse,nucleicacidshavenoenzymeactivity.Themethodsemployedforidentifyingnon-enzymaticproteinsandnucleicacidshavebeentermedWesternforimmunoblottingofproteins,SouthernfortechniquesusingDNAprobesNorthernwhenusingRNAprobes.Theprobesareradioactivecomplimentarystrandsofnucleicacid.ThefirstofthesetechniqueswastheSouthern,namedforthedeveloperoftheprocedure,EdwardSouthern.NorthernandthenWesternblotswerenamedbyanalogy.

Blottingtechniquesfirstdevelopaprimarygel:proteinonacrylamide;orDNA/RNAonagarose.Thegelpatternsarethentransferredtonitrocellulosemembranefiltersandimmobilizedwithinthenitrocellulosemembrane.Thisprocessoftransfertoanimmobilizingsubstrateiswherethetermblottingoriginated.Theprocessiswidelyusedintoday"slaboratoriesbecausetheimmobilizationallowsforextensivebiochemicalandimmunologicalbindingassaysthatrangefromsimplechemicalcompositiontoaffinitypurificationofmonospecificantibodiesandcell-proteinligandinteractions.

Inpractice,theelectrophoresisgelissandwhichedbetweentwolayersoffilters,twofoampads(forsupport)andtwolayersofastainlesssteelmesh.Thisentireapparatuscanbesubmergedinabufferandtransferallowedtooccurbydiffusion(yieldingtwoblots,oneoneachfilter),orcanbearrangedinanelectro-convectivesystemsothattransferoccursinasecondelectrophoreticfield.

Oncethetransferhasoccurred,theblotscanbeprobedwithanynumberofspecificornon-specificentities.DNAcanbeprobed,forexample,withCDNAorevenaspecificmessengerRNAtoidentifythepresenceofthegeneforthatmessage.