清华大学：现代生命科学实验教学中心《生物化学实验》课程教学课件（讲稿）Immunoblotting（Western blotting or Protein blotting）

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

ImmunoblottingAnti-species lgG molecules are readilyavailablecommercially,witha choice of adifferent labels attached. One of the mostcommondetection methodsisto useanenzyme-linkedsecondary antibody.Inthiscase, following treatment with enzyme-labellled secondary antibody,the blot isincubated in enzyme-substrate solution,when the enzyme converts the substrateinto an insoluble coloured product that isprecipitated ontothe nitrocellulose

Immunoblotting Anti-species IgG molecules are readily available commercially, with a choice of a different labels attached. One of the most common detection methods is to use an enzyme-linked secondary antibody. In this case, following treatment with enzyme￾labellled secondary antibody, the blot is incubated in enzyme-substrate solution, when the enzyme converts the substrate into an insoluble coloured product that is precipitated onto the nitrocellulose

ImmunoblottingThepresence of a coloured bandthereforeindicates thepositionoftheproteinof interest.Bycarefulcomparisonsoftheblotwithastained gel ofthe same sample,theprotein of interest canbe identified

Immunoblotting The presence of a coloured band therefore indicates the position of the protein of interest. By careful comparisons of the blot with a stained gel of the same sample, the protein of interest can be identified

ImmunoblottingThe enzyme used in enzyme-linkedantibodies is usuallyeitheralkalinephosphatase,whichconvertscolourless5-bromo-4-chloro-indolylphosphate(BCiP)substrate into a blueproduct,orhorseradish peroxidase, which, with H,O,as a substrate,oxidises either 3-amino-9-ethylcarbazoleintoaninsolublebrownproduct, or 4-chloro-1-naphthol into aninsolubleblueproduct

Immunoblotting The enzyme used in enzyme-linked antibodies is usually either alkaline phosphatase, which converts colourless 5-bromo-4-chloro-indolylphosphate (BCIP) substrate into a blue product, or horseradish peroxidase, which, with H2O2 as a substrate, oxidises either 3-amino-9- ethylcarbazole into an insoluble brown product, or 4-chloro-1-naphthol into an insoluble blue product

ImmunoblottingAn alternative approach to the detection ofhorseradish peroxidase is to use the methodofenhanced chemiluminescence (ECL).Inthepresenceofhydrogenperoxideandthechemiluminescent substrate luminolhorseradishperoxidaseoxidisestheluminowith concomitant production of light, theintensity of which is increased 1000-fold bythe presence of a chemical enhancer. Thelight emission can be detected by exposingthe blot to a photographic film

Immunoblotting An alternative approach to the detection of horseradish peroxidase is to use the method of enhanced chemiluminescence (ECL). In the presence of hydrogen peroxide and the chemiluminescent substrate luminol horseradish peroxidase oxidises the luminol with concomitant production of light, the intensity of which is increased 1000-fold by the presence of a chemical enhancer. The light emission can be detected by exposing the blot to a photographic film

ImmunoblottingThe methods described sofar areparticularly useful for measuring levelsof certain known antigens or antibodies.but often it is necessary to identify andcharacterize previously unknownantigens from a complex mixture, inwhich case immunoblotting is veryuseful

Immunoblotting The methods described so far are particularly useful for measuring levels of certain known antigens or antibodies, but often it is necessary to identify and characterize previously unknown antigens from a complex mixture, in which case immunoblotting is very useful