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4 Cards in this Set
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-Specific & sensitive technique -anti-his tag antibody that has a peroxidase enzyme conjugated to it. -Direct: the primary antibody that is used to detect an antigen on the blot is labelled with an enzyme or fluorescent dye. -Indirect: a primary antibody is added first to bind to the antigen. This is followed by a labeled secondary antibody that is directed against the primary antibody. |
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-reaction of peroxide radicals with 4-chloronaphthol produces a dark purple stain that can be photographed without using specialized photographic film. -TBS washes away unbound molecules -TBS helps simulate physiological conditions so that the antibodies bind (thus the pH 7.4 is the same as in the body) -Milk Protein; attaches to all the place where target proteins have not been attached so that when the antibody is added it HAS to bind to the binding sites of the specific target protein -leads to clearer results -Tween mostly used as a wash buffer as it prevents non-specific antibody binding |
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-Better to use an enzyme than a dye as there are more substrates available for the enzyme conjugates, and it helps with a direct visualisation of the bands. -Antibody binds to histidine residues. |
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-two bands on the gel show the two sub units of the protein that were coated in SDS to separate them, they gained an overall -ve net charge. -one band on the blot shows to which subunit the antibody has bound -as the line hasn't travelled far it suggests it's a large molecule, thus the beta subunit. |
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