MBIO 2420 Lecture Notes - Lecture 7: Western Blot, Southern Blot, Skimmed Milk
7 Dec 2016
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Immobilize protein on solid substrate then detect it with antibody. Moves protein from a page gel onto a solid substrate (membrane, usually nitrocellulose or pvdf - polyvinylidene fluoride: don"t need to bake it. They are too big - they will not diffuse out. Use blocking buffer rich in protein (bsa or skim milk powder: binds to membrane where protein is not bound to block the antibody, prevents background. Detection is generally antibody based and uses ecl or fluorescently-tagged antibodies: ecl is more sensitive - but they signal doesn"t last very long. Submersed in buffer or can be semi dry. Have sponges and pads, filter paper, membranes, the gel is in the middle: you want the membrane on the side of the positive electrode. After proteins leave to membrane (because of current) they get stuck to the membrane: smaller the pore size - the more proteins that get accumulated there.
