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BIOC23: Lec 9 - Immunoblotting.doc

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University of Toronto Scarborough
Biological Sciences
Rongmin Zhao

Lec 9 ImmunoblottingBlotsoSouthernDNA detectionoNorthernRNA oEasternposttranslational mod of proteinsoWesternprotein aka immunoblottingBlottingoTransferring DNA RNA or protein onto a carrier NitroceelulosePVDFNylon membraneoSubsequent detection of a specific group of the transferred moc usu by antibodyantigen detectionimmunoblotsoThe mocs to be transferred are usu separated by electrophoresis Dot blot and slot blotsamples not separated by electrophoresisAdd samples directly to carrier as dotslotoSeparate DNARNAagarose gel electrophoresisoSeparate proteinSDSPAGEseparated proteins transferred onto carrier membranes fro subsequent immunodetection to visualize location of target proteinsImmunoglobulinoVariable and constant Fc fragment regionsProtein AG bind Fc fragment and therefore proteins A and G are often used to detect antibodieso2 heavy2 light chainscrude serum sampledont see IgG see serum albuminlarge band bc very abundant proteinoafter ammonium precipitationget rid of albumin enriched IgG chainsWestern blotting aka immunoblotting StepsoSDSPAGE to separate mocsoSDSPAGE proteins transferred to special blotting membraneoWestern blotimmunodetection w antibody whichll bind specific antigenproteinoBiochemical method to visualize bandproteindetermine sizeoSEPARATETRANSFERBINDDETECTHow Antibodies are produced1 Innatebornprovided by epithelial barriers skin phagocytes2 Adaptivebody cells will recognize antigen from infectious particle then produces immunoresponse one of which is Blymphocyte recognizes and produces antibodies which will recognize antigen and cause specific rxn that lets cell eliminate foreign particleoBlymphocyteseach cell only produces one type of IgG this antibody can only bind one type of antigen
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