兔磷酸激酶实验报告英文Word文件下载.docx

兔磷酸激酶实验报告英文Word文件下载.docx

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Therearemanywaysofpurifyingcreatinekinase,andthepurificationmethodusedinthisstudyisimprovedKubymethod.

3.Measurementofcreatinekinaseactivities

Therearemanywaysofmeasuringtheactivitiesofcreatinekinase,andweusepH-colorimetryinthisstudy.

pH-colorimetry

Whencreatinekinaseiscatalyzeforwardreaction,withthetransferofPhosphorylfromATPtocreatine,theequimolarH+isproduced,whoseoptimumpHis7.5~9.0.Withinthiscontext,thegenerationrateofH+measuredcanbeusedasindicatorsofactivity.Inthisstudy,thymolblueisusedasapHindicator.TheactivitiesofcreatinekinaseismeasuredbypH-colorimetryonspectrophotometer,whichismonitoringchangesinabsorbanceunderthewavelengthof597nm.Inthecuvette,H+producedbytheenzyme-catalyzedreactionlowerthepHvalueofsolution.ThecolorofsubstratesolutiongraduallychangefromdeeppurpleredyellowgreenandlowertheAbsorbancevalueofA597.InthepH-colorimetry,thesubstratesolutionrequirestobenew.

4.Measurementofcreatinekinasesolution

280nmlightabsorption

Methodforproteinmeasurement

Sensitivity

Time

Principle

Interference

Comments

Spectrophotometric

（A280）

Moderate

50-1000g

Rapid

5-10min

Absorptionof280nmlightbyaromaticresidues

Purines,pyrimidines,nucleicacids

Usefulformonitoringcolumneluents.Nucleicacidabsorptioncanbecorrected.Nondestructivetoproteinsamples.Varieswithproteins.

II.Experimental

1.ReagentsandEquipments

1）Reagents

1.Preparationofcrudeextract:

0.01MKCl,NH4Cl,5MNH4OH,anhydrous 

ethanol,pH8.5-9.02MMgSO4

a.KCl：

0.01mol/L,200ml.（0.15gKCladdwaterto200ml）

b.NH4OH：

1.7mmol/L,1000ml.　（take0.12mlNH3H2O,addH2Oto1000ml）

c.NH4Cl

d.Anhydrousethanol

e.MgSO4：

2.0mol/L，pH8.5，20ml

f.MgAC2：

0.07mol/L，pH9.0，100ml

g.Tris-HCl：

0.1mol/L，pH8.0，1000ml（Tris12.1g,HCl23ml,pH为8.0，addwaterto1000ml）

h.Salt

2.Chromatography

a.48mmol/Lcreatinekinasesolution

b.0.1mol/LMgAC2

c.0.1％Thymolblue（100mgThymolblue，dissolvewith20mlethanoland60mlddwater）.

d.0.1mol/LpH9.0Gly-NaOHbuffer

e.ATP

f.0.1mol/LNaOH

g.0.07M.MgAC2pH9.0

h.0.01MTris-HClpH8.0

2）Equipments

Knife,meatchopper,plasticbasin,1/100balance,High-Speeddiffuser,refrigerator;

100mlbeaker,25mlbeaker,100mlmeasuringcylinder,250mlmeasuringcylinder;

Pippet,burette,glassstick;

0.5mlEptube,1.5mlEptube;

centrifugalmachine,50mlcentrifugaltube,stainlesssteelscoop,mortar;

drinkingpaper,accuratepHtestpaper

2.Measurementofenzymesactivities:

SPECORD200Spectrophotometer,Pippete,50mlbeaker,burette.

3.Measurementofproteinsolutions:

SPECORD200Spectrophotometer.

2.Procedure

1）Preparationofcrudeextract

1.Withasharpknife,cutthemuscleintocubes4to5cmwidefromtheice-coldrabbitmuscle.

2.Quicklyweighabout50gofthemusclecubesandsuspendthecubesin140mLof0.01mol/LKClat0C.

3.Homogenize140mLofthesuspensioninaglassbeakerwithablender（turnontheblenderathighspeedfor20seconds）andstirthemwithaglassstirringrodat0Cfor15minutes.

4.Centrifugethehomogenatefor10minutesat10000r/minat0C.

5.Carefullydecantthesupernatantandmeasurethevolumeofthesupernatant（V1）（keep0.6mLforproteinassayat–20C）.Discardthepellet.

6.Addgroundammoniumchloride（NH4Cl）tomake0.1mol/LsolutionandthenadjustpHto9.0with5mol/Lammoniumhydroxide（NH4OH）,keepstirringthesolutionat0Cfor30minutes.

7.Add1-foldV1ofcold95%ethanolandstirthesolutionat20Cfor30minutes.

8.Centrifugethesolutionfor10minutesat10000r/minat-8C.Discardthepelletandmeasurethevolumeofthesupernatant（V2）（keep0.6mLforproteinassayat–20C）.

9.Takethesupernatant（V2）andaddVA（mL）of2mol/LMgSO4（pH8.5）toafinalconcentrationof0.03mol/L.

10.Add1-foldVAofcold95%ethanolandstirthesolutionat0Cfor30minutes.

11.Centrifugethesolutionfor10minutesat10000r/minat-8C.Pouroffanddiscardthesupernatant.

12.Suspendthepelletwith1/10V10.07mol/LMgAC2（pH9.0）.

13.Stirthesuspensionat0Cfor30minutes.Centrifugethesuspensionfor10minutesat12000r/minat0C.Pouroffanddiscardthepellet.

14.Measurethevolumeofthesupernatant（V3）（keep0.6mLforproteinassayat–20C）.

15.AdjustthepHofthesupernatantto8.0with1mol/LNaOHandaddVBofcold95%ethanoltoafinalconcentrationof36%inanice-saltbath.CalculatetheVBusing:

[（V3-thevolumeofMgAC2）x0.5+VB]/（V3+VB）=0.36.

16.Stirthesolutionat0Cfor30minutes.Centrifugefor10minutesat12000r/minat-8C.Pouroffanddiscardthepellet.Measurethevolumeofthesupernatant（V4）（keep0.6mLforproteinassayat–20C）.

17.AddVCcold95%ethanoltoafinalconcentrationof50%inanice-saltbath.CalculatetheVCusing:

（V4x0.36+VC）/（V4+VC）=0.6.

18.Stirthesolutionat0Cfor30minutes.Centrifugefor10minutesat12000r/minat-8C.Pouroffanddiscardthesupernatant.Suspendthepelletinabout5mLof0.01mol/LTris-HCl（pH7.5）.Centrifugefor10minutesat12000r/minat0C.Measurethevolumeofthesupernatant（V5）（keep3-4mLforproteinassayat–20C）.

19.StoretheV5at–20Covernight.

2）Chromatography

1.QSepharoseHighPerformancecolumnhasbeenpreparedforyouruse.Beforestartingarun,thecolumnhastobeequilibrated.Thisisdonebypumping5columnvolumesofstartbuffer（bufferA:

0.01mol/LTris-HCl,pH7.5）throughthecolumn.

2.Measureout4~5mLofV5forapplicationtothecolumn,thenwashthecolumnwith2columnvolumesofbufferAandelutethesamplewithbufferB（0.01mol/LTris-HCl,0.3mol/LNaCl,pH7.5）inalineargradient,flowrateofthecolumnisabout1mL/min.

3.Collect1mLfractionsintesttubesinafractioncollector.Transferthecontentsofeveryotherfractiontoaquartzcuvetteandmeasuretheabsorbanceat280nmoruseaUVmonitor.RecordtheA280valuesinyournotebook.Whenyoubegintodetectproteinelutingfromthecolumn（A280>

0）,measuretheA280ofeveryfraction.

3）MeasurementofEnzymeActivities

1.ThedetectionofthespecificactivityofthecreatinekinaseisbasedonthepH-colorimetry.ItisrathersensitivefortheadvancedspectroscopytomonitorthealterationofthecolorofThymolblue.

2.Thewaytocalculatethespecificactivityisaccordingtothefollowingequation:

1.3isaconvertingcoefficient.Ustandsforspecificactivity.VAstandsforthevolumeofsubstrate（VA=1.0mL）.VBstandsforthevolumeofenzymesolution（VB=0.01mL）.Cistheconcentrationofenzymesolution,anditisdeterminedbytheabsorbanceofthesolutionat280nm.

3.Fillthespecificactivityofthecreatinekinaseineachfractionintheformbellow.

Fraction

ProteinConcentration（mg/mL）

SpecificActivity

FoldPurification

Recovery（%）

4）SDS-PAGE

Thesamplesobtainedineachstepofcrudeextractionandthefinalproductsfromion-exchangechromatographyareloadedontosodiumdodecylsulfate-poly-acrylamidegelelectrophoresis（SDS-PAGE）.Thestackinggelwasmadeof3%acrylamide,andtheseparatinggelwasmadeof12.5%acrylamide.

III.Experimentalrecord

1.Thevolumeineachpurificationstage

Chart1.Volumeoftheprotein.

V1

V2

V3

V4

V5

46.8ml

83.1ml

4.98ml

5