BE115+-+Lab07 - Lab 7: Western blotting 1 Introduction...

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Page 1 of 5 Bioengineering 115: Cell Biology for Engineers Laboratory 7: Western Blotting Last updated: January 21, 2011 Lab 7: Western blotting 1 Introduction ‘Western’ is the common term for electrophoretic or capillary transfer of proteins to a solid support, followed by immunostaining. Proteins that have been resolved in SDS-PAGE are transferred through the ~1 mm thickness of the gel to a membrane made of nitrocellulose or nylon. Once the proteins have been moved over to the membrane, the rest of the membrane surface is blocked with a protein solution (the protein in the block is chosen such that it will not interfere with the rest of the experiment). After the block, the membrane is washed and then incubated with an antibody recognizing the specific protein of interest. This is followed by steps to detect where that antibody (and thus the specific protein) is present. The end result: a blot which, when developed, identifies the presence of only a single protein (of the many proteins on the blot). In some instances, image analysis of the final product can be used to quantify the amount of the protein of interest present on the membrane. During the last lab, you used electrophoresis to resolve a known amount of total protein from each of your culture extracts. After SDS-PAGE you began the Western blotting. The instructor completed the protocol by taking apart the blotting apparatus, storing the membranes, and blocking them. 2
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BE115+-+Lab07 - Lab 7: Western blotting 1 Introduction...

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