*2.1.4 Specific applications*

Biochemical fractionations played a role historically in identifying some of the first extracellular signaling proteins like cytokines using activities such as macrophage migration or bacteria killing [14]. In the 21st century, this approach has identified stable soluble PPIs proteome-wide by fractionating cell lysates down to the level of co-eluting protein complexes and identifying them using MS [15]. While the specific purification steps used for soluble proteins are unlikely to be applicable to ePPIs, alternative centrifugation-based fractionation successfully recovered biochemically active membranes from crude fruit fly extracts [16]. Direct application of affinity purification from crude extracts without enriching for synaptic membranes did not recover known ePPIs [16]. However, using biochemical fractionation to enrich for synaptic components was necessary for the identification of key proteins in synapse formation using an affinity purification approach (described in the next section) [17].

#### **Figure 2.**

*Biochemical fractionation can be used to reduce the complexity of a mixture while maintaining the desired activity. While traditionally performed in series, fractionation can also be done in parallel with modern purification techniques.*

*Unbiased Identification of Extracellular Protein–Protein Interactions for Drug Target… DOI: http://dx.doi.org/10.5772/intechopen.97310*

## **2.2 Affinity purification**
