How can tagging a protein help you purify it How can tagging

How can tagging a protein help you purify it? How can tagging a protein help you determine when and where it is expressed in a cell or organism?

Solution

A tagged protein is used to simplify the purification protocol. It also improves yield and solubility of protein of interest and proper folding. Most commonly affinity tags are used for the purpose. During purification, the tagged protein is reversibly bound with a complementary ligand that recognizes the tag. Now the bound tagged protein is eluted or recovered using a competitive ligand. Cellular proteins are washed through the column and those proteins that interact with the target bind to the affinity matrix. The unbound material is washed out of the column. The identity of eluted proteins is determined by mass spectrometry or another suitable method.

Understanding how cells work requires understanding how proteins function. Tagging a protein can help understand where and when it is expressed in a cell. For example: GFP is the green fluorescent protein isolated from jelly fish. The GFP gene can be used as a visual tag for the expression of other genes.To identify the expression of - galactosidase gene which produces -galactosidase enzyme that digests lactose, the -gal gene is tagged with GFP gene. The gene expressed can be analyzed visually by fluorescence microscopy.

A fluorescently-labelled antibody generated against a protein of choice allows direct examination of endogenous unmodified proteins. This makes it possible to analyze the localization of endogenous expression.

Serial analysis of gene expression (SAGE) is the techniques used to identify which genes are turned on and which are turned off within cells. SAGE identifies and counts the mRNA transcripts in a cell with the help of tags.