Immunoblotting vs. Immunoprecipitation/immunoblotting - (Dec/27/2007 )

hallo,

maybe this is a stupid question, but i want to know the advantage of immunoprecipitation/immunoblotting compared to immunoblotting only?

when one make a immunoprecipitation/immunoblotting rather than immunoblotting?

isn´t it enough to make only immunoblotting to get information of protein levels and regulations on protein level?

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The primary use of IP-IB is to demonstrate a physical associaton between two molecules(eg. ubiquitlylation of targeted proteins). This requires gentle, non-denaturing lysis conditions, and an 1' antibody that recognizes the native form of the first antigen. Using the previous example, you can use an anti-Ub antibody in the IP, to enrich ubiquitylated proteins from the lysate, then use the IP'd sample for an IB with an antibody for a second protein. For control purposes, you could run a pre-IP and non-IP (with an irrelevant 1'Ab) sample in parallel lanes to deomionstrate that the enrichment worked and that the 2' antigen was present in the initial lysate.

Another case to use IP-IB is to harvest/enrich an engineered protein (His/GST tags etc) from a total cell lysate, though this mechanism can be used as above to demonstrate intermolecular association when a suitable commercial antibody is not available.



If you're not interested in a specific intermolecular interaction, and just want to know the relative abundance of a particular antigen/protein, a standard Western blot would suffice. Good Luck-JAH

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i understand. it is only necessary for demonstration of protein interactions?!

but i often read in publications that they perform IP-IB when it is not really necessary. is there any additional advantage?

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