Lab#7

WesternWrapUp
And
PlateOverlayAssay

 Whatisanantibody?

Animmunoglobulinthat
iscapableofcombining
withspecificitytothe
antigenthatelicitedits
production.
Huh?And,whydowe
care?

 Whatisanantigen?

Anantigenisanysubstancethatelicitsan
immuneresponseandisthencapableofbinding
tothesubsequentlyproducedantibodies.
Antigensaregenerallyproteinsor
polysaccharides,butothersubstancessuchas
nucleicacidscanalsobeantigens.


 How,WhyandWhereareAntibodies
Produced?
(Massivelycondensedversion)

ProducedinVertebrateCellsinresponsetoanonself
substancetermedanantigen.
Theprocessbeginswithmacrophagesroamingthe
bloodstream.Macrophagesengulfnonselfmolecules
andthendisplayportionsoftheengulfedmoleculeson
theirouterplasmamembranes.
AftermanyrecognitionstepsBcellsarestimulatedto
producespecificantibodies.
Theantibodiesthengooffandbindtotheforeign
substancetherebymarkingthesubstancefordestruction.

 WhatisanEpitope
Anepitopeisthesmallsiteontheantigen
whichisrecognizedbytheantibody.
Usuallybetweenoneandsixsugarsor
aminoacidsonthesurfaceoftheantigen.
AntibodyUniqueness:
Bcellsproducesomewherebetween1x108
and1x1010IgGantibodieswithdifferent
bindingsites.

 AntibodyAntigenInteractions
Bindingofantibodytoantigenisdependentonhydrogen
bonds,electrostaticattractionsandVanderWaals
attractions.
Thesebondsareweakcomparedtocovalentbondsbut
thelargenumberofweakbondsresultinastable
complex.
Antibodyantigenbindingisreversible.
Bindingsitedifferencesareduetoaminoacidsequence
differences.
Thevariableregionismadeupof110130aminoacids.




 Howdoesantibodystaining
work?
Antibodyantigenreactionishighlyspecific
foragivenantibodyantigenpair.
Immunostainingtechniquesrelyonthe
extremespecificityofanantibodyforits
antigen.

 IndirectvsDirectAntibody
Staining
Whatisthedifferencebetweendirectandindirect
immunostaining?
Asinglesecondarydetectsmanyprimariessoyou
onlyneedonelabeledantibodytobeabletovisualize
thelocationoflargenumbersofprimaryantibodies.

WhydoindirectImmunostaining?
Also,thesignalisstronger.
Cheaper

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 PolyclonalAntibodies
Producedbyimmunizingananimalwiththe
appropriateantigen.
Theimmunizedanimalsserumiscollected.
Antibodiescanthenbepurifiedfromtheserum.
Sinceoneantigeninducestheproductionofmany
antibodiestheresultisapolyclonalmixtureof
antibodies.
Polyclonalantibodiesaremuchlessexpensive
thanmonoclonalantibodies.

 MonoclonalAntibodies
Muchmorecomplicatedtoproducethanpolyclonal
antibodies.
Processbeginsbyimmunizingananimal(mostcommonly
amouse)withanantigen.
Theanimalsspleenisremoved.
Bcellsarefusedwithmyelomacellsresultingin
hybridomas.
Hybridomasarescreenedtofindthoseproducing
antibodiestotheantigenwithwhichtheywereimmunized.
EachhybridomacellisderivedfromoneBcellsothe
antibodiesthataclonalpopulationofhybridomacells
producearemonoclonalantibodies.
Monoclonalantibodiesrecognizeoneepitopeonly.

 PlateOverlayAssay
YoubeginprocedureatDay2.
Whatareyoulookingfor?
CPYinvertaseactivity
Wheredoestheglucosecomefrom?

 Whathappenswhenyoupourthetop
agarsolutionontoyouryeastplates?
Step1:Invertasehydrolyzessucroseintoglucoseand
fructose:Sucrose+H2O>glucose+fructose
Step2:CatalyzedbyGlucoseOxidase(GO):
Glucose+O2+GOFADH2>Dglucono1,4lactone+
GOFAD+H2O2
Step3:CatalyzedbyPeroxidase:
H2O2+odianisidine(H2)(colorless)>odianisidine
(colored)+H2O
Oxidationofodianisidine(colorless)resultsinacolored
(visible)product.

 FM464Staining
Fluorescentvitalstain
Whythepulseandthechase?

 Whatisafluorophore?
Fluorophoreistheregionofthemolecule
capableofexhibitingfluorescence.
Fluorochromeisadyeormoleculecapable
ofexhibitingfluorescence.

 HowDoesFluorescenceMicroscopy
Work?
Excitiation:Energyintheformofaphotonoflight
isabsorbedbyafluorophore.
Whileintheexcitedstate,thefluorophore
undergoesconformationalchange.
Emission:aphotonofenergyisemittedandthe
fluorophorereturnstoitsoriginalstate.
Theprocessrepeatsinacyclicalmannerunlessthe
fluorophoreisdestroyedbyphotobleaching.

 Theenergyoftheemittedphotonislowerthanthe
energyoftheexcitationphotonduetoenergy
dissipationintheexcitedstate.
Lowerenergy=Longerwavelength
Thedifferenceinenergyorwavelength(hEX
hEM)istheStokesshift.
TheStokesshiftisthekeytothefluorescence
microscopytechniquebecauseitallowsthe
emissionphotonstobedetectedandisolatedaway
fromtheexcitationphotons.
Asinglefluorophorecanberepeatedlyexcitedand
detected.

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