Immunoblotting

Immunoblotting

1. Dr. J. P. Saranraj
IMMUNOBLOTTING –
WESTERN BLOTTING

2. WESTERN BLOTTING
Western blotting,also known as
ImmunoblottingorProteinblotting,isacore
techniqueincellandmolecularbiology.
Theterm“blotting”referstothetransferof
biologicalsamplesfromaGeltoaMembrane
andtheirsubsequentdetectiononthesurfaceof
themembrane.
ItwasfirstdescribedbyTowbinetal.(1979).
Itisusedtodetectthepresenceofaspecific
proteininacomplexmixtureextractedfrom
cellsortissue.

4. Steps in Western Blotting
Step–1:ExtractionofProtein
Proteinisextractedfromcellbymechanicalor
chemicallysisofcell.Thisstepisalsoknownas
Tissuepreparation.
TopreventdenaturingofproteinProtease
inhibitorisused.
Theconcentrationofproteinisdeterminedby
Spectroscopy.
Whensufficientamountofproteinsampleis
obtained,itisdilutedinloadingbuffercontaining
glycerolwhichhelpstosinkthesampleinwell.
Trackingdye(Bromothymolblue)isalsoadded
insampletomonitorthemovementofproteins.

5. Step–2:GelElectrophoresis
ThesampleisloadedinwellofSDS-PAGE.
Theproteinsareseparatedonthebasisof
electriccharge,isoelectricpoint,molecular
weight,orcombinationoftheseall.
Thesmallsizeproteinmovesfasterthanlarge
sizeprotein.
Protein are negatively charged, so they move
toward positive (Anode) pole as electric current
is applied.

6. Step–3:Blotting
TheNitrocellulosemembraneisplacedonthe
gel.Theseparatedproteinfromgelget
transferredtonitrocellulosepaperbycapillary
action.Thistypeofblottingistimeconsuming
andmaytake1-2days.
Forfastandmoreefficienttransferofdesired
proteinfromthegeltonitrocellulosepaper
electro-blottingcanbeused.
Inelectro-blottingnitrocellulosemembraneis
sandwichbetweengelandcassetteofFilter
paperandthenelectriccurrentispassedthrough
thegelcausingtransferofproteintothe
membrane.

7. Step–4:Blocking
BlockingisveryimportantstepinWestern
blotting.
Antibodiesarealsoproteinsotheyarelikelyto
bindthenitrocellulosepaper.Sobeforeadding
thePrimaryantibodythemembraneisnon-
specificallysaturatedormaskedbyusingcasein
orBovineserumalbumin(BSA).

8. Step–5:TreatmentwithPrimaryAntibody
ThePrimaryantibody(1°Ab)isspecificto
desiredproteinsoitformAg-Abcomplex.
Step–6:TreatmentwithSecondaryAntibody
TheSecondaryantibodyisenzymelabelled.For
eg.AlkalinephosphataseorHorseradish
peroxidaseislabelledwithSecondaryantibody.
Secondaryantibody(2°Ab)isantibodyagainst
primaryantibody(anti-antibody)soitcanbind
withAg-Abcomplex.

9. Step–7:TreatmentwithsuitableSubstrate
Tovisualizetheenzymeaction,thereaction
mixtureisincubatedwithspecificsubstrate.
Theenzymeconvertthesubstratetogivevisible
coloredproduct,sobandofcolorcanbe
visualizedinthemembrane.

10. Applications of Western Blotting
Todeterminethesizeandamountofproteinin
givensample.
Diseasediagnosis-Detectsantibodyagainst
virusorbacteriainserum.
WesternblottingtechniqueistheConfirmatory
testforHIV.ItdetectsAnti-HIVantibodyin
patient’sserum.
UsefultodetectDefectiveproteins.Foreg
Prionsdisease.
DefinitivetestforCreutzfeldt-Jacobdisease
(Braindisorder),Lymedisease(Borreliosis
causedbyBorreliasp.),HepatitisBvirusand
Herpesvirus.