凝胶层析.docx
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凝胶层析.docx
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凝胶层析
GelFiltrationChromatography
Bio96zoumi
2009030069
1.Objectives
1)Tounderstandtheprincipleofgelchromatographyanditsapplication.
2)Bymeasuringtheproteinmolecularweighttraining,initiallymastergelchromatography.
2.principles
Gelchromatography,alsoknownasexclusionchromatography,gelfiltration,molecularsievechromatography,filtrationorinfiltration.Itiswidelyusedinseparation,Purification,concentrationanddesaltingofbiologicalmacromolecules,andsoon,whilethedeterminationofproteinmolecularweightisalsooneofitsimportantapplications.
Gelisakindofthree-dimensionalnetworkstructurewasporousandthebead-shapedinsolubleparticulatematerial.Whenusingittoseparatesubstances,itismainlyaccordingtothedifferentradiiofproteinmolecules(near-spherical)withdifferentexclusioneffects.Thatis,itisbasedonthemolecularsizeofthephysicalpropertiesoftheseparationandpurification.Forcertaintypesofgel,somemoleculescannotentertheparticles,andcanonlyflowalongtheoutercolumn;whileanumberofsmallmoleculesarenotexcluded.Theyarefreetospread,infiltratingintothegelinsidethesieve,andlateristakenout.Thesmallermolecules,thedeeperinsideintothegel,themoredistancetheygo,sothesmallmoleculesarethelasttoflowout.Somemedium-sizedmoleculesbetweenmacromoleculesandsmallmoleculescanonlyenterthepartofthelargergelpores,whichispartlyexcluded,sothesemoleculescomeoutbetweenlargeandsmallmolecules.Thissampleaftergelchromatography,themoleculeswillbeinaccordancewithbothlargeandsmallintheflowingordertoachievethepurposeofseparation.
Foranykindofcompoundsseparatedbygel,theexclusionchromatographycolumnsareintherangeof0~100%,anditsrankeddegreeofbeingexcludedcanbedescribedbyKav(isolatedcompounds,includingouterwaterandexternalwatervolumeintheproportionalrelationship).AsforKavandthetotalvolumeofgelcolumnbed(Vt),outerwatervolume(V0),andtheelutionvolume(Ve)thereisaformulalikethis:
Kav=(Ve-V0)/(Vt-V0).
Chromatographyunderthefixedconditions,VtandV0isaconstantvalue,Vechangeswiththechangesofthemassofthemoleculestobeseparated.Thelargerthemolecularweightis,thesmallerthesmallerVevalueandKavvaluewillbe.Onthecontrary,thesmallerthemolecularis,thebiggertheVevalueandKavvaluewillbe.
Kavisimportanttoestimatetheseparationeffectandthemassofproteinmolecules.Inthesamechromatographicconditions,themoredifferenttheKavvaluesofseparatedmaterialis,thebetterseparationeffectwillbe.Onthecontrary,separationefficiency,otherwisethemoleculescan’tbeseparatedortheeffectisverybad.Intheactualexperiment,wemeasuredaVt,V0andVevalues,andthencalculatetheKav.Foraparticulartypeofgel,inthecontextofacertainmolecularweight,Kav,andlgMr(Mrindicatedthatsubstancemolecularweight)intoalinearrelationship:
Kav=-blgMr+C
Thesamecanbe:
Ve=-b'lgMr+C'
Whereb',C'areconstant.Namely,VeandlgMrhasalinearrelationship.WecanisolateKnown-weightmolecularproteinsinavarietyofgelcolumn,andinaccordancewiththeabove-mentionedlinearrelationshipbetweentheplottedstandardcurves,andthenmeasuretheotherunknownproteinmolecularweightusingthesamegelcolumn.
3.Equipmentandreagents
1)Equipment
(1)Glasschromatography
(2)DHL-BPCconstantpump(ShanghaiJingBranchedIndustrialCo.,Ltd.)
(3)BSZ-100automaticpartofthecollector(ShanghaiQingpuInstrumentFactoryHuxi)
(4)8823BUVdetector
(5)UNICCOUV-2102Cuv-visspectrophotometer
(6)TYPE3057PortableRecorder(SichuanNo.4InstrumentFactory)
(7)TB-600gradientmixer(ShanghaiUnityBiochemicalInstrumentFactory)
(8)100mlreagentbottle
(9)1000mlgraduatedcylinder
(10)250mlbeaker
(11)50ml,100mlbeaker
(12)10mltest-tubescale
2)Reagents
Standardprotein
Bovineserumalbumin:
Mr=67000(ShanghaiBiochemistry).
Eggalbumenprotein:
Mr=45000(U.S.SIGMAcompany).
Lysozyme:
Mr=14300.
Unknownproteinsamples
Elute0.025mol/LKCL~0.1mol/LHAC
Bluedextran2000
4.Experimentprocedures
1)Gelswelling
Weigh7gSephadexG-75inthe250mlbeaker,add100mlelate,swellatroomtemperaturefor2~3d,repeatedlyPourremovethefineparticles,andthenplacegelinvacuumtocasttheairbetweentheparticles,thenintoboilingwaterbathtoboil2~3h(torunawayInternalairoftheparticles,andtosterilized).
2)Loadingthecolumn
(1)Takeacleanglasschromatographycolumnandverticallyfixeittothemetalframeworkstage.
(2)Determiningthetotalvolumeofgelcolumn(Vt)
Makeamark5cmawayuptothecolumnend,closethecolumnoutlet,adddeionizedwater,opentheoutlet.Whentheliquidleveldroptothemark,closetheoutlet.Thenusethemeasuringcylindertoacceptthede-ionizedwater(surfacedowntotheglasssieve),readthecolumn.OutofthetotalvolumeofthecolumnbedvolumeshallbeVt.WecanalsomeasuringVtafterhavingdonetheunknown-protein.
(3)addingelatetothecolumn(about1/3columnbedheight),slowlypourtthickslurrytothecolumnuntilthegelbecomes1cm~2cmhighandthenopentheoutlet,theflowratetendstobeabout3~6ml/10min.whenthegelgoestothemark,andtheloadingiscompleted,becarefulthattheloadinggelcannotbelayered.Andthenclosetheoutlet,putitasideforamoment,untilthegeldeposits,thenconnectittotheconstant-current.Use1~2timesofthebedvolumeofelatetobalancecolumn,sothatmakesureofthestability.
3)DeterminingV0andtheVeoftheunknownprotein
Drawawaytheelateonthesurface(mustnotupsetthesurface,filterpaperornylonnetcanbecovered).Opentheoutlet,sothatresidualliquidisreducedtogluefacialcut(butnotletitdry).Closetheoutlet.Suckupwithafinedropper0.5mlmixtureof(4.5mg/ml)blue-color-glucan-2000andunknownprotein(5mg/ml)mixture,carefullyadditcirclingaroundthecolumnwall(2mmabovethegelsurface),andthenopentheoutlet(Startcollecting!
),Afterthesolutionintothegelbed,Closetheoutletwithalittleeluentaddingtothebed,fillthetopwitheluent.Startelutingatthespeedof5~6ml/10min.automaticcollectorforeachtubeissetat5mincollection.Finallymakeelutioncurve
Finallymadeelutioncurve.CollectionandplanningbasedonexpenditurefromtheCanadian
KindofbeginningtotheelutionconcentrationofbluedextranelutionvolumeshallbethehighestpointoftheV0.Note:
BlueDextranwashdown
Afterwards,usetheelate(1~2timesthebedvolume)tobalance,preparingforthenextstep
4)Thestandardcurve
(1)Thestandardproteinsolutionwiththeeluentispreparedforclassuse,thesolutionofvariousconcentrationsofthethreeproteinsare:
bovineserumAlbumin(5mg/ml),chickeneggalbumin(8mg/ml),lysozyme(4.5mg/ml).
(2)Accordingtotheoperationmethod(3)addthestandardprotein0.5mlofthesolution5~6ml/10minandcollecteluent.
(3)ByusingultravioletspectrophotometerdetermineA280tubebytubeanddecidethepeakpointofproteinelution,andthenmeasuredproteinelutionvolumeVe.
Notethataftertheexperimentiscompleted,getallthegelIrecycledforthenexttesttouse.It’sstrictlyprohibitedtodiscard,orpourthegelintothePool.
5.Records
1)rawdata
EluteSpeed:
0.6ml/min
Automatedportionofthecollectorspeed:
(forthefirsttime)5min/tube,(second)3min/tube
RecordingPaper’sspeed:
2cm/h
UVDetector:
(forthefirsttime)0.5A,(second)0.2A
Thetotalvolumeofgelcolumns:
Vt=86.0ml
Thebluedextranelutionvolume(externalwatervolume):
V0=31.3ml
Unknownproteinelutionvolume:
Vg=51.4ml
Standardproteins:
Ve1=32.2mlVe2=41.2mlVe3=65.6ml
2)AbsorbanceA280Data
number
A280
V
Number
A280
V
1
0
16
0.093
2
0.002
17
0.080
3
0.002
18
0.124
4
0.008
19
0.274
5
0.003
20
0.454
6
0.003
21
0.531
51.4ml
7
-0.003
22
0.436
8
-0.003
23
0.266
9
0.001
24
0.125
10
0.011
25
0.026
11
0.001
26
0.000
12
0.397
27
-0.017
13
1.170
31.3ml
14
0.590
15
0.188
Table2-1280nmUVabsorbanceofsample1
number
A280
V
number
A280
V
1
0.000
26
0.059
2
0.001
27
0.065
3
-0.001
28
-0.022
4
-0.003
29
-0.048
5
-0.002
30
-0.049
6
0.003
31
0.047
7
0.001
32
0.025
8
0.002
33
0.102
9
-0.001
34
0.184
10
-0.001
35
0.251
11
-0.001
36
0.272
12
0.001
37
0.276
65.6ml
13
0.002
38
0.2
14
0.011
39
0.131
15
0.004
40
0.059
16
0.011
41
0.101
17
0.142
42
0.07
18
0.206
32.2ml
43
0.123
19
0.173
44
0.031
20
0.141
45
0.019
21
0.124
46
0.004
22
0.082
47
0.083
23
0.165
41.2ml
48
0.019
24
0.129
49
0.005
25
0.116
50
0.003
Table2-2280nmUVabsorbanceofsample2
标准蛋白洗脱曲线Figure2-1Standardproteinelutioncurve
6.DataProcessing
1)Thetotalvolumeofgelcolumn
Vt=86.0ml
2)ExternalwatervolumeV0andtheunknownproteinelutionvolumeVe
Figure2-2AbsorbanceGraphofsample1
ThefirstelutionpeakisfortheexternalwatervolumeV0.
ThesecondpeakisforunknownproteinVe.
Betweenthecolumnandtheautomaticalcollector,thereisabout5mlliquid.Whencounttheelutionvolume,itshouldbesubtracted.
V0=
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