Blocking solution a prevents non specific binding of antibodies to the

Blocking solution a prevents non specific binding of

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5.Blocking solutiona.prevents non-specific binding of antibodies to the nitrocellulose membrane (Correct)b.prevents binding of primary and secondary antibodies while they are incubating together.c.inhibits the activity of cellular enzymes, especially proteases, during the incubation.d.is used to wash out unbound primary antibodies before the secondary antibody solution is added to nitrocellulose.e.improves binding of immunoglobulins to the nitrocellulose membrane.Pre Lab 1.Testing Liver and Tripe Protein Extracts for the presence of Albumin Proteina.Western Blottingor Immunoblottinga.i.Identifying a specific protein with a labeled antibodyb.Blocking the Electroblotted Membraneb.i.Before antibodies can be applied, the membrane must first be treated with Blocking Solutionb.i.1.Composed of proteins, which come from a variety of sourcesb.ii.What is the purpose of blocking solution?b.ii.1.is to cover sites on the membrane to which no protein had been previously been bound during electroblotting
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b.iii.If this step is not performed?b.iii.1.When the primary and secondary antibodies solutions are added, the antibodies would be trapped and bound to the membrane directly, since antibodies are themselves proteinsb.iv.Why is milk used?b.iv.1.Rich sources of proteins that are not likely to be recognizedand bound by primary antibodies that have been raised against a specific antigen that is not a component of milkc.Primary antibodiesc.i.One that binds directly to the target proteinc.ii.Named after the target molecule against which it was raisedc.ii.1.Primary antibody that recognizes Albumin isc.ii.1.a.Anti-Albumin Antibodyc.ii.2.Full named antibodiesc.ii.2.a.If raised in rabbitsc.ii.2.a.i.Rabbit Anti-Albumin Antibodyc.ii.2.b.If raised in Mousec.ii.2.b.i.Mouse Anti-Albumin Antibodyd.Secondary Antibodiesd.i.Usually have been raised in goats by injecting a bulk mixture of antibodies from either rabbits or miced.ii.Secondary antibody named for the target protein against which it has been raised, and for the organism in which it was raisedd.ii.1.Eitherd.ii.1.a.Goat Anti Rabbit Immunoglobulin Antibodyd.ii.1.b.Goat Anti Mouse Immunoglobulin Antibody e.Detection Of Bound Antibody With A Detectable Labele.i.Detectable labelse.i.1.Most common label for antibodies is to covalently link an enzymeto the antibodye.i.2.Alkaline Phosphatasee.i.2.a.chemically linked (covalently) to one end of antibody molecule. Catalyzes a reaction when the substrate is added. A Blue color precipitates on the bands that contain the Antibody/Enzyme complex after the substrate is added.e.i.2.b.Catalyzes reaction that converts colorlesssubstrate Nitroblue Tetrazolium Phosphateintocoloredproduct Nitro Blue Tetrazolium Lecture slides: -Western Blot Immunoassays: for proteins -Indirect Immunoblot: uses secondary antibody which is conjugated with an enzyme, detects the products of the reaction catalyzed by the bound enzyme when its substrate is added, secondary antibody does not bind to the protein of interest, but to primary antibody
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