Immunoblotting(Western blottingorProtein blotting
Immunoblotting (Western blotting or Protein blotting)
ImmunoblottingProteins separated byPAGEmaybetransferred (orblotted)from the gel to a thinsupport matrix, usually a nitrocellulosemembrane, which strongly binds andimmobilizes proteins. The protein blots onthe membrane surface are more accessibleto chemical or biochemical reagents forfurtheranalysis.Whenthetransferprocessis coupled with protein identification usinghighlyspecificand sensitive immunologicaldetectiontechnigues,theprocedureiscalled Western blotting or immunoblotting
Immunoblotting Proteins separated by PAGE may be transferred (or blotted) from the gel to a thin support matrix, usually a nitrocellulose membrane, which strongly binds and immobilizes proteins. The protein blots on the membrane surface are more accessible to chemical or biochemical reagents for further analysis. When the transfer process is coupled with protein identification using highly specific and sensitive immunological detection techniques, the procedure is called Western blotting or immunoblotting
ImmunoblottingOncetransferredontonitrocellulose,theseparated proteins canbe examinedfurtherThis involves probing the blot, usually usingan antibody to detect a specific protein. Theblotisfirstlyincubatedinaproteinsolution.forexample10%(w/v)bovineserumalbumin,or5%(w/v)non-fatdriedmilk(theso-called blotto technigue), which will blockall remaining hydrophobic binding sites onthenitrocellulosesheet
Immunoblotting Once transferred on to nitrocellulose, the separated proteins can be examined further. This involves probing the blot, usually using an antibody to detect a specific protein. The blot is firstly incubated in a protein solution, for example 10% (w/v) bovine serum albumin, or 5% (w/v) non-fat dried milk (the so-called blotto technique), which will block all remaining hydrophobic binding sites on the nitrocellulose sheet
ImmunoblottingThe blot is then incubated in a dilutionofanantiserum (primary antibody)directed against the protein of interest.This IgG molecule will bind to the blot if itdetects its antigen, thus identifying theprotein of interest.In orderto visualize thisinteractiontheblotisincubatedfurtherinasolution of a secondary antibody,whichisdirectedagainstthelgGofthespeciesthat provided the primary antibody
Immunoblotting The blot is then incubated in a dilution of an antiserum (primary antibody) directed against the protein of interest. This IgG molecule will bind to the blot if it detects its antigen, thus identifying the protein of interest. In order to visualize this interaction the blot is incubated further in a solution of a secondary antibody, which is directed against the IgG of the species that provided the primary antibody
ImmunoblottingFor example, if the primary antibody wasraised in a rabbit then the secondaryantibody would be anti-rabbit IgG. Thissecondary antibodyisappropriatelylabelledsothattheinteractionofthesecondary antibody withthe primaryantibody canbe visulized on the blot
Immunoblotting For example, if the primary antibody was raised in a rabbit then the secondary antibody would be anti-rabbit IgG. This secondary antibody is appropriately labelled so that the interaction of the secondary antibody with the primary antibody can be visulized on the blot