Proteingelelectrophoresis

usedtoseparateproteinsbasedonanumberof
characteristics
Denaturinggelelectrophoresis
separatebysize
Nondenaturing(native)gelelectrophoresis
separatebysize,shape,charge

Proteingelelectrophoresis
Polyacrylamide gel electrophoresis

TEMEDcatalyzesfreeradicalformation
APSfreeradicaldonor
Bisacrylamidecrosslinkingagent(19:1ratioofacrylamideto
bismaximizescrosslinking)

Higher%ofgelsmallerpores(holes)sosmallerfragments
canberesolved

Proteingelelectrophoresis

GradientgelsAsproteinsmigratethroughincreasing
acrylamideconcentration,smallerpores,mobilitydecreases
Onceproteinsreachtheirporelimitlittlemovementandcan
calcMW

Proteingelelectrophoresis
Agarose gel electrophoresis
Agarose is a long sugar molecule

Heated,>70C

Roomtemp

Agaroseseparationoflargemolecules
8kDto800,000kD
Polyacrylamideseparationofsmallermolecules
0.2kDto500kD

Gelelectrophoresis

Roomtemp

Proteingelelectrophoresis

Proteingelelectrophoresis
SDSGelElectrophoresis(agaroseorpolyacrylamide)
Denaturingcondition
DenatureproteinbyaddingSDS(thenseparatebysizeonly)

SDSformsmicellesand
bindstoproteins

Proteingelelectrophoresis
SDSGelElectrophoresis(agaroseorpolyacrylamide)
Usedtoestimatepurityandmolecularweight,separateproteinsbysize
ElectrophoresisofSDSsolvatedproteinonpolyacrylamidegel
StaingelwithCoomassieBlue(bindstoproteins)

Proteingelelectrophoresis
Nativegelelectrophoresis
polypeptidesretaintheirhigherorderstructureandoftenretain
enzymaticactivityandinteractionwithotherpolypeptides
migrationofproteinsdependsonmanyfactors,includingsize,
shape,andnativecharge.
nativegelsomittheSDSandreducingagent(DTT)
donotputSDSorDTTinthesamplebuffer
donotheatthesamples
preparethegelandtankbuffersolutionswithoutSDS.

Proteingelelectrophoresis
Separationofhemoglobinproteins
Hemoglobininvolvedinoxygentransportin
body

NormaladultHb(HbA)twosubunitsand2
subunits
Sickletraithemoglobin(HbAS)onlyone
inheritedmutation,50%HbSand50%HbA
Sicklehemoglobin(HbS)Glu>Val
mutation,HbSinheritedfrombothparents
WhenHbSisdeoxygenateditcrystallizesin
RBCswhichleadstodistortionofredcellsand
reductioninnumberofRBCs

Hemoglobin
SickleCellAnemia
GeneticdiseaseinwhichpersoninheritsgeneforsicklecellHbfromboth
parents
Hb
Hbsickle

VHLTPEEK
VHLTPVEK

Hbsickleisdeoxygenated,insolubleandformspolymersthataggregate
Valinehashydrophobicsidechain,glutamatehasnegativecharge
ValinecreatesstickyhydrophobiccontactpointwheredeoxyHbsickle
moleculesassociateforminglong,fibrousaggregates

Symptoms:

weak,dizzy,shortofbreath,heartmurmurs
sicklecellsfragileanemia
capillariesblockedabnormalorganfunction

Patientswithsicklecellanemiahavetohaveinherited2copiesofmutantgene
Inheritonly1copyresistancetomalaria
Sicklecelltrait~10%ofAfricanAmericanpopulation

Hemoglobin
AbnormalHbsdetectedinlabbyelectrophoresis
HbatpH9.2hasanet()chargesomovesinelectricfield
toward(+)electrode
pIofnormalHbis6.9butchangeswithmutations
InHbSGlu>Val(chain),so2fewer()charges
ExampleofvariationsinmigrationonagelwhenHb
mutationspresent

SickleCellAnemia
ExamineelectrophoreticbehaviorofHbA,HbS,HbAS
1.Setupproteingel(gelsarepremade)
2.LoadHbsamples
3.SeparateHbproteinsbyapplyingelectricfield
4.Takegelapparatusapart
5.StaingelinCoomassieBlue(entiregelisblueatfirst)
6.Destaingel(removesnonspecificstainingtoreveal
proteinbandsongel)