What is Protein Cross-Linking and Which Reagents are Used in it?
Protein cross-linking is the process of binding two or more protein molecules together to facilitate scientific probes on protein-protein interactions. To achieve this effect, specific crosslinking reagents (crosslinkers) are used to chemically join the protein molecules.
Understanding the Functions and Behaviors of Crosslinking Reagents
Protein cross-linking reagents usually contain two or more chemically reactive ends that attach themselves to the functional groups found in proteins and other molecules. Such reactions make the molecules stable enough to allow for intensive scientific analysis.
Additionally, intermolecular crosslinks can also be used to catalyze the production of specific conjugates to identify an unknown protein interactor, conjugate an enzyme, immobilize proteins for assays and affinity-purification and facilitate peptide handling and storage by attaching them to larger carrier proteins.
There are three types of crosslinking reagents:
1. Homobifunctional crosslinking reagent
2. Heterobifunctional crosslinking reagent
3. Photoreactive crosslinking reagent
Homobifunctional crosslinking reagents have identical reactive groups and are therefore used to bind like functional groups while heterobifunctional crosslinking reagents have dissimilar reactive groups and can be used to link unlike functional groups. The first type is mainly used to form intramolecular crosslinks and in the preparation of polymers from monomers while the latter is used to produce multiple intermolecular crosslinks and conjugates using two different biomolecules.
Photoreactive crosslinking reagents, on the other hand, are a special type of heterobifunctional crosslinkers that only becomes reactive when exposed to UV or visible light.
Given the number of protein cross-linking reagents available, how do you choose which one to use?
To determine which protein crosslinker to use in any particular application, you should take the chemical reactivities and other chemical properties of a specific reagent into consideration. Your choice of reagent will ultimately depend on its chemical specificity (whether it binds with similar or dissimilar reactive groups), spacer arm length, is the crosslinker reversible or cleavable, cell membrane permeability and water solubility. You should also consider whether you want the reagent to immediately react with the sample or whether you prefer to activate it at a specific time.
In summary. These features are taken into consideration when making selection of a cross-linker:
1. Reagent solubility
2. The nature of reactive groups
3. Homobifunctional or heterobifunctional
4. Photoreactive or thermoreactive groups
5. The length of the spacer arm
6. Conjugated product cleavable or not
7. Potential for further labeling
8. Reaction condition needed for conjugation