Immunoprecipitation Technique - Coursework Example

The importance and reliability of this technique lie in the fact that it is the most common method used for the purpose of separation of proteins. The basic idea behind this technique is the separation of one single protein from a mixture of proteins. This could give us the idea of several characters of a protein such as its relative occurrence in a solution, its up and downregulation as well as its affinity for a specific antibody.

Technique/Methodology

Usually, the process of immunoprecipitation can be completed in two ways: in sequential or in one step. Commonly the antibody on which the required protein is supposed to be attached is immobilized on a solid support such as beads. This solution containing the immobilized antibody and the beaded support is then incubated with the solution that contains the protein mixture containing the required protein. Incubation allows the specific protein to bind to the antigen and form a complex. This complex can then be separated from the solution and studied with different techniques such as ELISA or Western Blot according to the requirements.
The diagrams courtesy of ‘Pierce Biotechnology’ show the process of immunoprecipitation starting from preparation of solid support along with the antigen to the incubation and the formation of the antigen-protein complexes till the precipitation of the required protein.
Types
Their main types of immunoprecipitation are:
1. Co-immunoprecipitation: the technique in which associated proteins are also precipitated out and studied by SDS-PAGE or Western Blot analysis.
2. Chromatin Immunoprecipitation: this type of technique is used for the identification of genomes containing Transcription factors and other DNA binding proteins.
3. RNA immunoprecipitation: Same as Chromatin only difference lies in the detection of RNA binding proteins

Uses
Immunoprecipitation has been useful in many aspects such as It has enabled scientists to know the activation of the proteins, their molecular weight and also separate some protein binding molecules too. This technique has also been helpful in detecting the abundance and activity of a protein.
Protocols
After collecting the required number of cells and washing them in ice-cold PBS, the solution was spun at 1000g at a temperature of 4 degrees. This would help in separating the supernatant fluid. The next step involves the resuspension of the pellet that contains the cells in an ice-cold buffer after which the cells are lysed by centrifuge. The supernatant fluid is removed after spinning it again at 13000g at the same temperature for fifteen minutes. Bradford assay is then used to measure the quantity of the protein after the supernatant fluid is removed by spinning briefly. This supernatant-free solution is then incubated in a cold room with the required amount of specific antibody solution. After the addition of Protein A or G beads to the tube, it is again incubated for an hour and then spun briefly again so that further supernatant is removed. This beaded fraction is then washed with ice-cold buffer and spun to remove the supernatant and the resultant solution is kept for further analysis.

BCL-2 Proteins
Bcl 2 family proteins have been identified to play a major role in the process of cell death i.e. apoptosis. These proteins play both anti and pro-apoptotic roles. Some members of the family are supposed to increase while some are supposed to decrease this process of apoptosis. In this project, the interaction of Bcl 2 family proteins with PUMA has been identified and studied.