Hello Leslie Traqueña,

As you know that the immunoprecipitation is a method, which enables the purification of a protein from cell/tissue lysate. In this method, an antibody for the protein of interest is incubated with a cell extract enabling the antibody to bind to the protein in solution. The antibody/antigen complex is then pulled out of the sample using protein A/G-coupled agarose beads. This isolates the protein of interest from the rest of the sample. The sample can then be separated by SDS-PAGE for western blot analysis.

The non-denaturing lysis buffer is used if the antigens are detergent soluble and recognized in native form by the antibody. For the purpose, Triton X-100 is substituted for NP-40.

However, some soluble proteins do not require use of detergents. Hence, Detergent-free soluble protein lysis buffer is used if the lysate is prepared by mechanical cell lysis such as homogenization with a Dounce homogenizer.

On the other hand, epitopes of native proteins are not accessible to antibodies that only recognize denatured proteins. Hence, denaturing lysis buffer is used for non-detergent soluble antigens. Therefore, while harvesting and lysing the cells, heat the cells in denaturing lysis buffer. This method can also be used for antigens that cannot be extracted from the cell with non-ionic detergents. Use of DNase1 will aid extraction of proteins from chromatin.

Hope this helps

Best

Porque se debe asegurar la separación del analito de interés directamente o indirectamente. Te ayuda a eliminar o desnaturalizar los componentes acompañantes en una etapa de la técnica.

Both can work. Depends on the question you try to answer. Clearly for co-immunoprecipitation, one cannot use denaturing conditions in order to preserve native-like complexes. IP under denaturing conditions are arguably more challenging (Ab affinity might be impacted) but may be needed to expose buried epitopes. To neutralize possible interference with SDS, one can use excess Triton to form mixed micelles with SDS. See Article Effect of detergents on antibody-antigen interaction