Protein blot (Western) hub

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Revision as of 09:21, 15 October 2008

The Western blot is one of the most tried and true experiments for antibody-dependent determination of protein expression/presence in a sample.

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-==Overview==
+The Western blot is one of the most tried and true experiments for [http://openwetware.org/wiki/Griffin:Antibody_Basics antibody]-dependent determination of protein expression/presence in a sample.
-A Western Blot allows for the semiquantitative determination of protein expression.
-Crude cell lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current can then be passed through the gel and the proteins will migrate through the gel, with the largest proteins traveling the slowest, resulting in a lane where proteins become separated on the basis of their weight. The proteins can then be transferred from the gel onto a membrane (often nitrocellulose or PVDF), which can then by incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can now detect specifically the protein of interest.
 ==General Western blot protocol==