NTR366L_spring2011_week10_westernblot_instruction

NTR366L_spring2011_week10_westernblot_instruction - Lab...

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Lab Days 1 and 2, Week of April 18, 2011 Western blot analysis (immunoblotting) Goal: To introduce protein analysis by western transfer or blotting (also known as “immuno-blotting”). Brief description of the experiment: Protein samples are denatured by boiling them in a detergent (sodium dodecylsulfate, SDS) and β -mercaptoethanol. Boiling destroys the higher-order structure of the proteins, SDS forces them to unfold and gives them a net negative charge, and β -mercaptoethanol reduces and disulfide bonds that may be present; as a result, molecular weight is the sole factor that determines how the proteins migrate through the gel when subjected to electrophoresis. Polyacrylamide gels are used to provide the resolution necessary to separate proteins. After electrophoresis, the proteins are blotted to a membrane using the iBlot system for further analysis. On Lab Day 2 the membrane is rewetted and nonspecific binding sites are blocked by incubation with milk protein. A specific protein (bovine serum albumin, BSA) is detected by incubation of the membrane with an antibody that specifically recognizes and binds to BSA; this is
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