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Lecture 11

BIOL 3096 Lecture 11: 11


Department
Biology
Course Code
BIOL 3096
Professor
Joel Sheffield
Lecture
11

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AffinityChromatography
Anbodies
Iftheanbodiesonly
interactwith1ofthe
proteinsthenwash
thispetridish
ChangepHsoanbodies
aresllstuckonthe
boom
pHdisruptsthestructure
oftheproteinsothatit
cannolongerbindtothe
anbodies
Bindtheanbodiestothegelandrunthemixture
through
Proteinsthat
didn'tsck
ChangepH Proteinthatwas
boundandpurified
Troublewiththistechnique:
Anbodiesneedtobeveryspecificandvery
pure
Affinityligand‐whatsckstheproteininqueson
I.e..Anbodies,insulin,nickel‐whichbindshisdine
AddhisdinetoaproteininquesontogetaHIStag
Electrophoresis
Insteadofliquidasolidsupportisused
I.e..Paper
Starch
PolyAcrylamide‐plascpolymer(mainoneused)
Polyacrylamide
Force‐electriceld
Propertyofmolecules‐charge
Whatholds/pullthem‐amtofelectriccharge&thematrixthatthesampleis
in
Direcon‐duetochargeofthemolecule
SodiumDodecylsulfate‐detergent
(S‐laurelSulfate)(SDS)
WhenaddedtoproteinitbreaksalltheHbondsunfoldingtheproteinandapplyachargethatis
proporonaltothemolecularweightoftheprotein(bigger=moreSDSbinds)
CanalsoaddBMEtobreaksulfidebonds
‐biggerproteinstravelfurtherbecausethereismoreSDSboundtoitsothereismorenegavecharge
andthusitismorearactedtotheposivecharge
+
Lecture11
Sunday,September20,2015 2:29PM
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