2D protocol.docx

2D protocol.docx

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2Dprotocol

TwoDimensionalElectrophoresis

1.SamplePreparation:

GrowthecelltoOD600=0.6~0.8

Collectthecellpelletbycentrifugationat3000g,10min,4℃

Washthecellgentlyonicewith50mMPBS（pH7.2）

Separatethecellinto1.5mltube（e.g.10OD/1.5mltube）

Centrifugetocollectthepelletat4℃,3000g,10min

Ifthepelletwillnotbeusedimmediately,storethepelletat-80℃

Resuspendthepelletwithlysisbufferonice（e.g.500ulinto10ODpellet）

Sonicationinice-watermixture:

1’’on,9’’pause,energy:

25%,30times

Aftersonication,staythesampleatroomtemperaturefor30min

Centrifugeat40,000gfor1hourat20℃

Collectthesupernatantandallotintoseveral1.5mltubes.

Ifthesamplewillnotbeusedimmediately,storethesampleat-80℃.

2.Concentrationdetermination:

Kit:

2-DQuantKit（80-6486-22,Amersham）

Steps:

PrepareastandardcurveaccordingtoTable1usingthe2mg/mlBovine

serumalbumin（BSA）standardsolutionprovidedwiththekit.Setupsix

tubesandaddstandardsolutionaccordingtoTable1.Tube1isthe

assayblank,whichcontainsnoprotein.ConcentrationofBSAstandard

solution:

2mg/ml.

Table1:

Preparationofstandardcurve

Note:

Theaccuracyoftheassayisunaffectedbythevolumeofthesample

aslongasthesamplevolumeis50µlorless.Itisthereforeunnecessaryto

dilutestandardorsamplesolutionstoaconstantvolume.

Preparetubescontaining1–50µlofthesampletobeassayed.

Duplicatesarerecommended.Theusefulrangeoftheassayis0.5–50µganditisalsorecommendedthatmorethanonesamplevolumeordilutionbeassayedforeachsampletoensurethattheassayfallswithinthisrange.

Add500µlprecipitanttoeachtube（includingthestandardcurvetubes）.Vortexbrieflyandincubatethetubes2–3minatroomtemperature.

Add500µlco-precipitanttoeachtubeandmixbrieflybyvortexingor

inversion.

Centrifugethetubesataminimumof10000×gfor5min.Thissediments

theprotein.

Removethetubesfromthecentrifugeassoonascentrifugationiscomplete.Asmallpelletshouldbevisible.Decantthesupernatants.Proceedrapidlytothenextsteptoavoidresuspensionordispersionofthepellets.

Carefullyrepositionthetubesinthemicrocentrifugeasbefore,withthe

cap-hingeandpelletfacingoutward.Centrifugethetubesagaintobringany

remainingliquidtothebottomofthetube.Abriefpulseissufficient.Usea

micropipettetoremovetheremainingsupernatant.Thereshouldbeno

visibleliquidremaininginthetubes.

Add100µlofcoppersolutionand400µlofdistilledorde-ionizedwaterto

eachtube.Vortexbrieflytodissolvetheprecipitatedprotein.

Add1mlofworkingcolorreagenttoeachtube（See“Preliminary

preparations”forpreparingtheworkingcolorreagent）.Ensureinstantaneousmixingbyintroducingthereagentasrapidlyaspossible.Mixbyinversion.

Incubateatroomtemperaturefor15–20min.

Readtheabsorbanceofeachsampleandstandardat480nmusingwateras

thereference.Theabsorbanceshouldbereadwithin40minoftheaddition

ofworkingcolorreagent（step9）.

Note:

Unlikemostproteinassays,theabsorbanceoftheassaysolutiondecreaseswithincreasingproteinconcentration.Donotsubtracttheblankreadingfromthesamplereadingorusetheassayblankasthereference.

Generateastandardcurvebyplottingtheabsorbanceofthestandardsagainstthequantityofprotein.Usethisstandardcurvetodeterminetheproteinconcentrationofthesamples.

3.Iso-ElectricFocusing（IEF）

AddXulsampleintostripholder:

Forsilverstainingaddproteinlessthen200ug,forcoomassiebluestainingadd400to1000ugprotein.

Addrehydratationbuffer:

For18cmstripholder:

Add350-Xulrehydratationbuffer.For24cmstripholder:

Add450-Xulrehydratationbuffer

Addstrip:

Carefullytakeofftheplasticmembraneonthestrip.Addstripintostripholderwiththegelsidedowntoattachthe“sample-buffer”mixture.Makesuretheliquidallunderthegel.

Addcoverfluid:

Addenoughcoverfluidontotheplasticsideofthestrip（e.g.2mlapprox.）

Addplasticcoverforthestripholder

IEF:

PutthestripholderscontainingstripsontoIPGphorwithcorrectelectrodedirection.PlacethestripholderadaptorontothestripholderwhilekeeptheextrudingstreaksincontactwiththeholderandjustbelowthecurveinIPGphorlid.ClosetheIPGphorlid.Thenopenthepower.Edittheprogram.Pressstarttorun.Note:

Thelimittemperatureandcurrentcanneverbechanged!

Becauseofthehighvoltage,turnonthepoweronlyafterclosethelid.Andwhenyouwanttotouchtheholderorelectrode,shutthepowerdown.MakesurethatIPGphorishorizontalplaced.

HerebelowaretheexperimentaldataforthestripsItriedbefore.Whenincreasingloadingamount,changingstrippHrange,lengthorsomerecombinantproteinincludedinone’sloadingsample,oneshouldtrydifferentfocusingvolt-hourstogetoptimizedresult.

pH3-1024cmstrip,100ngproteindeterminedbythekit

Step

Volt/v

Volt-hour/vh

Time/hour

1

30

6

2

60

6

3

200

1

4

500

1

5

1000

1

6

8000

0.5

7

8000

56,000

dependsoncurrent

8-end

0

0

Temperature

20℃

Maxielectriccurrent

50uA

pH3-10NL24cmstrip,100ngproteindeterminedbythekit

Step/Grad

Volt/v

Volt-hour/vh

Time/hr

Step

30

12

Step

100

1

Step

250

1

Grad

500

1

Grad

1000

1

Grad

3000

2

Grad

6000

1

Grad

8000

1

Grad

8000

45,000

dependsoncurrent

Temperature

20℃

Maxielectriccurrent

50uA

pH4-718cmstrip,100ngproteindeterminedbythekit

Step/Grad

Volt/v

Volt-hour/vh

Time/hr

Step

30

12

Step

100

1

Step

250

1

Grad

500

1

Grad

1000

1

Grad

3000

2

Grad

6000

1

Grad

8000

1

Grad

8000

99,000

dependsoncurrent

Temperature

20℃

Maxielectriccurrent

50uA

AfterIEF,ifonedonotwanttorunthe2nddimensionimmediately,keepthestripsintotheplastictubesandstoreat-80℃

WashthestripholderafterusewithcleaningsolutionprovidedbyAmersham,andsoftbrush.FinallywashwithMilli-Qwater.

4.GelCasting

GelCastingCassette

Assemblethegelcastingcassettesandputthemintogelcasterwithplasticfillersheetbetweeneachtwocastingcassette.Allthe13fillersheetsshouldbeputbetweenthecastingcassettes.Addfaceplatecontainingsealinggasket.Closetheclampscrewstightlyandaddspringclampsintocorrectposition.

AssembledGelCaster

Pourthepreparedgelfromfillingchannelifyouwanttomakehomogeneousgel.Makesurethatgelcasterishorizontalplaced.

Forgradientgel:

ReservoirChamber

MixingChamber

Pourlightgelmixtureintothemixingchamber,whilepourtheheavygelmixtureintothereservoirchamber.Beforecastingtheslidevalveshouldbeclosed.Whencasting,openthepinneartheoutletconnectorandtheslidevalveatthesametimewhilekeepthepinnearthefillerportopen.Makesurethatgelcasterishorizontalplaced.

SlideValve

Pin

MagneticStir

Closethepinnearthefillerport,untilthegelsurfaceupto0.5~1cmtotheglassedge.

Pin

Aftercastingcoverthegelsurfacewith70%ethanol.Keeptheminroomtemperatureforatleastthreehoursforpolymerization.

Note:

Onecanatmostcast6piecegelseachtime

5.Equilibration

Firstequilibration:

Add100mgDTTinto10mlequilibrationbuffer;rotatetoresolve.Putthestripintothetubecontaining1stequilibrationbuffer.Shakeslightlyforexactly15min.

Secondequilibration:

Add400mgiodoacetamideinto10mlequilibrationbuffer;rotatetoresolve.Pulloutthestripsfromthe1stequilibrationbuffer.Removetheextraliquidwithpaper.Putthestripintothetubecontaining2ndequilibrationbuffer.Shakeslightlyforexactly15min.Pulloutthestripsfromthe2ndequilibrationbuffer.Removetheextraliquidwithpaper.QuicklydipthestripintoMilli-Qwaterandremovetheextrawaterwithpaper.

6.SDS-PAGE:

Addstriptothe2nddimensiongel:

Attachtheequilibratedstripontothesurfaceof2nddimensiongel.Makesurethattheplasticsideofthestripincontactwiththehigherglasspiece.

Sealthesurfacewithpreheated0.5%agarosesealingsolution.Nobubbleshouldexistbetweenthestripand2nddimensiongel,aswellasagarose.Aftertheagarosesealingsolutionbecomesolid,insertthecassettesintothecassettecarrierandputthecarrierintoelectrophoresisunit.Addabout5~6Lrunningbuffer.

cassette

electrophoresisunit

cassettecarrier

RunningCondition:

15℃,100v,overnightisrecommended.Therunningtimedependsontheconcentrationofthe2nddimensiongel.

Note:

MultiTempIIIshouldbeopenpreviouslybeforerunningandsetthetemperatureto15℃

7.Staining

SilverStaining:

Method1:

Step

Solution（1Leverytwopieces）

Time（1mmunbackedgel）

Fix

100mlaeticacid,400mlmethanol

20minX2

and500mlmilli-Qwater

Sensitize

300mlmethanol,2gNa2S2O3,68gNaAc

30min

Addmilli-Qwaterto1000ml

Wash

1000mlmilli-Qwater

5minX3

Silverstain

2.5gAgNO3,milli-Qwaterto1000ml

20min

Wash

1000mlmilli-Qwater

1minX2

Develop

25gNa2CO3,400ulformaldehyde

1~4minapprox.

addmilli-Qwaterto1000ml

Stop

14.6gEDTA,addmilli-Qwaterto1000ml

10min

Wash

1000mlmilli-Qwater

5minX3

Note:

“Method1”willprovideyoumorenice-lookinggel.Butsometimes,whenyourinterestedspotsareinlowamountandyouwanttoidentifyitwithMALDI,youarerecommendedtouse“Method2”asthisonestainlighterandonewillgetmorepeptidepeaksafterdigestion.

Method2:

Step

Solution（1Leveryt