what is the different between immunoprecipitation-western blot and western blot? For measurements of proteins, that contributed in necroptosis, such as RIP-1/3, immunoprecipitation- western blot was used. Why western blot no used in measurements of them?
I don't know about the particular system with RIP-1/3, but there could be several reasons for immunoprecipitation before a Western blot. One is that immunoprecipitation would concentrate a protein of interest. This would be useful when a crude lysate could have a lot of other proteins, but only a small amount of the protein of interest. Also the crude lysate could have so much protein that it overloads the gel and creates artifacts and poor bands. Another use for immunoprecipitation before Western blot could be that different antibodies are used for each procedure. For example there could be variants of a protein, and the antibody used for immunoprecipitation could recognize the common region of all the variants, and the antibody used for Western blot could recognize a different and specific epitope of the variants.
I don't know about the particular system with RIP-1/3, but there could be several reasons for immunoprecipitation before a Western blot. One is that immunoprecipitation would concentrate a protein of interest. This would be useful when a crude lysate could have a lot of other proteins, but only a small amount of the protein of interest. Also the crude lysate could have so much protein that it overloads the gel and creates artifacts and poor bands. Another use for immunoprecipitation before Western blot could be that different antibodies are used for each procedure. For example there could be variants of a protein, and the antibody used for immunoprecipitation could recognize the common region of all the variants, and the antibody used for Western blot could recognize a different and specific epitope of the variants.
Hi Elizabeth
Thank you for your answers to my question. It seem, which probably the expression of this proteins was low in normal conditions. Thus, immunoprecipitation was used befor a Western blot .
Hello Alireza
I hope you are doing well.
As dear Elizabeth said before all antigens that you want to detect carefully, the best method is immune reaction (antigen-antibody) as direct and highly specific method.
in general, simple western blot used for new & unknown proteins that after detection you can analyze its amino-acid chain sequel and determine its tri-dimensional and functional activities.
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