Ubiquitination refers to the binding of the protein ubiquitin to other proteins by three different enzymes. A ubiquitination assay is a test that determines how much of this process occurs within the cell. Tests can be used to determine how different conditions affect protein binding, steps and structures that are important in the ubiquitinnation pathway, and the amount of protein in the cell. Inadequate or overabundant amounts have been linked to the development of many different diseases.
A protein found in cells, the function of ubiquitin is to bind to proteins while functioning as a "tag" or signal to other structures. This "tag" then signals enzymes to know that the protein can be destroyed, recycled, or moved. The result is the destruction or transportation of proteins.
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These tagging proteins are important for DNA repair, viral infections and immune response. Other important functions include roles in cell death, cellular division and development of cells. Too much ubiquitin, or lack thereof, may play a role in the formation of many diseases, such as cancer, genetic disorders and diseases of the immune system.
Researchers will use a ubiquitination assay to try to determine which conditions alter the concentration of the protein in the cell and comparing diseased cells with healthy cells. Scientists also use it to investigate what conditions will cause a changes in binding. Additionally, they may try to link specific DNA code with the the amount of enzymes in the cell.
There is more than one type of ubiquitination assay that research scientists specifically use. One will determine the percentage of proteins that are bound with ubiquintin. Another examines the activity of enzymes or the presence of any other intermediates found in the ubiquination pathway. Different assays are developed based on the target protein that ubiquitin will bind to.
The basis of a ubiquitination assay is to incorporate a compound that is labeled using another easily measured chemical. In oncology assays, ubiquitin can be labeled with cryptate, a compound which contains two nitrogen atoms. This can be used to determine which enzyme is most active in the cell.
A ubiquitination assay will have two steps. The first is an enzymatic step where enzymes will bind the labeled ubiquitin to the target protein. The second is a detection step in which binding of the target protein is examined using a conjugate such as an antibody. The antibody will fluoresce, or change color, which then allows the researcher to determine the activity of the enzymes.