Analysis of Cross-Linked Products
After cross-linking has been accomplished, the next step is usually puriŠcation and characterization of the product(s) of the reaction. The target product must Šrst be isolated from excess or unreacted reagent. In many cases, simple dialysis may sufŠce to remove unreacted reagent from the reaction solution. Typically, the solution is placed in a dialysis bag made of a semipermeable material usually based on cellulose. The dialysis tubing has pores that will allow smaller molecules to pass through while retaining larger species, that is, macromolecules. Some type of chromatography, for example, size-exclusion chromatography (SEC) (discussed below), may also be used to either remove excess reagent or isolate and characterize the cross-linked product. The isolated cross-linked protein may then be further characterized by biochemical or biophysical techniques. In the following sections, various analytical methodologies and examples of their application to protein cross-linking will be described. Once the product has been puriŠed, it may be subjected to many different types of studies including spectroscopic (e.g., ¦uorescence, NMR, EPR, and Raman), immunochemical, biochemical, and enzymatic, and numerous examples of these type of studies have been given throughout this book. In this chapter, however, we shall focus on methods to purify and characterize the cross-linked product.