Western Blot

Comprehensive insight into western Blot

1. Western Blot
Sunil Bhandari

3. •Western blots are labor intensive and expensive but provide a
method of confirming the identity of a reactive antigen
•Westernblotsaredonebyelectrophoresisofantigen(sample
protein)bysodiumdodecylsulfate(polyacrylamidegel
electrophoresis(PAGE)
•Theproteinsinamixtureareseparatedbysizeinthegeland
aretransferredfromthegeltoasolidpaperlikesubstrate
oftencalledamembrane.
•Themembraneisincubatedwithpatientseraandthepresence
ofpatientantibodiesisdetectedbysubsequentlyincubating
themembranewithlabeledsecondaryantibodies.
•The specificity of the western blot lies in its ability to demonstrate
the molecular weights ( of the proteins recognized by patient sera

4. Principle:
•Westernblottingisamethodwherealabelledantibodyagainst
particularproteinisusedtoidentifythedesiredprotein,soitisa
specifictest.Westernblottingisalsoknownasimmunoblottingbecause
ituseantibodiestodetecttheprotein.
•Westernblotisperformedbyusingpolyacrylamidegel
electrophoresis(SDS-PAGE)whichallowsproteinsamplestobe
separatedandtransferredtoasolidsupportsuchasnitrocellulose
membrane.
•This solid support can absorb the protein and keep its biological
activity unchanged.
•Theproteintransferredsolidsupportmembraneiscalledablotand
istreatedwithaproteinsolutiontoblockthehydrophobicbinding
siteonthemembrane.

5. •Themembraneistreatedwiththeantibody(primaryantibody)
ofthetargetproteins.Onlytheproteinstobestudiedcan
specificallybindtotheprimaryantibodytoformanantigen
antibodycomplex.
•Theprimaryantibodytreatedmembranesaretreatedwitha
labeledsecondaryantibodyafterwashing.Aftertreatment,the
labeledsecondaryantibodythatbindstotheprimaryantibody
formsanantibodycomplexthatcanindicatethelocationofthe
primaryantibody,iethelocationoftheproteinbeingstudied.

6. Process of western blot