Epitope Tagging

 

                    

                      Epitope Tagging

1.      Epitope tagging is used for localization and purification of protein.

2.      Using standard engineering techniques, a short peptide tag can be added to protein interest.

3.      It is used to study protein-protein or DNA-protein interaction.

4.      If a tag itself is an antigen determinant or epitope it can be targeted by an appropriate commercially available antibody.

5.      The antibody, suitably labelled can be used to determine the location of protein in cell to purify it by immunoprecipitation of affinity chromatography.

6.      In an immunoprecipitation antibody directed against the epitope tag re added to a solution as antibody protein complex.

7.      Richard young and his co-workers originally identified 10 polypeptide that are authentic polymerase 2 sub at least tightly bond contained.

8.      The method they used is called epitope tagging, in which they attach a small foreign epitope to one of yeast poly II subunit (RPBS)

9.      Then they introduce this gene into yeast cell, lacking a functional RPB₃ gene. labelled the protein with either S³⁵ or P³² and use as Ab directed against the foreign epitope to precipitate whole enzyme.

10.                              After immunoprecipitation, they separated the labelled polypeptide of precipitated protein by SDS page and detected them by autoradiography.