Determining protein localisation and membrane topology
Flow cytometry (FCM) is a multi-parameter cell analysis technique used for detecting and measuring the physical and chemical properties of a particular cell population, including cell count, cell size, and cell cycle. While the method has been routinely used for basic cell biological research, it also plays a vital role in the diagnosis, treatment, and management of certain bone and soft tissue tumors and blood cancers such as leukemia, lymphoma, and myeloma.
Recombinant proteins are usually expressed with an affinity tag that facilitates binding of the protein to an affinity matrix and results in high yield purification of proteins (usually above 90% yield) by exploiting avidity between the tag and affinity matrix. Tags are usually small peptides or proteins that facilitate selective binding to an affinity matrix. There are many types of tags available to facilitate protein purification, some common examples are poly-histidine tag, GST tag, FLAG tag. The bound proteins can be eluted using a chemical agent usually known as eluent that has a higher affinity for affinity matrix and results in the release of bound tagged proteins from the affinity matrix by chemically competing against the bound proteins for the binding sites on affinity matrix.