The Protein Man's Blog | A Discussion of Protein Research

What is Protein Cross-Linking and Which Reagents are Used in it?

Posted by The Protein Man on Oct 12, 2012 6:00:00 AM
The Protein Man

Question:

What is Protein Cross-Linking and Which Reagents are Used in it?

The Protein Man Says:

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

Download Free Protein  Crosslinker Selection Guide

                              

Topics: Cross-Linkers

Want more Protein Man blogs?

Purification_resins
Ellyn Daugherty's Biotechnology: Science for the New Millennium

CB™ PROTEIN ASSAY: A Bradford Protein Assay

CB Protein Assay Graph

An improved Coomassie Dye based protein assay based on the Bradford Protein Assay. This assay is suitable for the simple and rapid estimation of protein concentration. This assay is based on a single Coomassie dye based reagent. The binding of protein to the dye results in a change of color from brown to blue. The change in color density is proportional to protein concentration. Protein estimation can be performed using as little as 0.5µg protein.

Features

  • Sensitivity: Linear responses over the range of 0.5µg-50µg protein
  • Flexible Protocols: Suitable for tube or Titer plate assays
  • Ready to use assay reagents and no preparation required
  • Long shelf life, stable for 12 months
Click for CB Protein Assay