Crosslinking Protein Interaction Analysis Service


Introduction

Under physiological condition, most protein-protein  interactions are transient, and happen in a very short duration, which make  them difficult to be studied. Crosslinking reagents, or crosslinkers, provide  the analytical solution to capture protein-protein complexes by covalently  binding them together as they interact, to freeze even transient, weak  interactions for consequent isolation and characterization.

Crosslinking  condition

The crosslinking can be performed in vivo or in  vitro. Whether to use in vivo or in vitro crosslinking depends on the specific  needs of the project. Proteins are crosslinked in a native state during In vivo  crosslinking, whereas, for in vitro crosslinking protein may be denatured.

In vivo crosslinking

For  in vivo crosslinking, proteins are linked inside cells, hence the risk of false  positive interaction or loss of protein complex stability can be eliminated.  Hydrophobic and lipid-soluble crosslinking reagents are often used to react  with proteins located within cell membrane, while hydrophilic, water-soluble  crosslinking reagents are used to crosslink proteins  located on the cell membrane, such as plasma-anchored receptors. Though  proteins are remain in native status, due to the complexity of proteins in cells,  optimization of in vivo crosslinking can be complicated as well, and possible  nonspecific crosslinking may still occur. The nonspecific crosslinking problem  can be eliminated by applying crosslinking reagents with shorter spacer arms.

In vitro crosslinking

During  in vitro crosslinking process, cells are homogenized and lysed. Therefore, this  method is more suitable for analyzing more stable protein-protein interactions.  As cells are lysed in specific lysing buffer, the reaction conditions are  easier to control, such as temperature, pH, ion strength, etc. Without cell  boundary, many types of crosslinking reagents are suitable for this  application. Nonspecific crosslinking is easier to be controlled.

Crosslinking Protein Interaction Analysis

Type of crosslinking methods:

Chemical crosslinking

Crosslinking  reagents covalently link together interacting proteins, domains or peptides by  forming chemical bonds between specific amino acid functional groups of two or  more biomolecules that occur in close proximity because of their interaction. There  are many commercial chemical crosslinkers to choose, homobifunctional or  heterobifunctional, long and short length of arm, cleavable and non-cleavable,  water-soluble and –insoluble, based on the specific requirements of the  projects. Homobifunctional molecules target the same group on the protein, while  heterobifunctional crosslinkers targets different functional groups on separate  proteins for greater variability or specificity. Crosslinker molecule can also  be designed to include cleavable elements, such as esters or disulfide bonds  (diagrammed below), to reverse or break the linkage by the addition of  hydroxylamine or reducing agents, respectively. Crosslinkers can be hydrophobic  to allow passage into hydrophobic protein domains or through the cell membrane  or hydrophilic to limit crosslinking to aqueous compartments.

Photoreactive  crosslinking

As  addition of crosslinkers to cell suspensions or cell lysates may cause many unspecific  crosslinks to be formed, in addition to the target protein-protein interaction,  more sophisticated crosslinker designs were created that incorporates  photoreactive groups, which react at selected times and only in response to  irradiation by UV light. Heterobifunctional crosslinkers with a chemical crosslinking  group at one end and a photoreactive group on the other end can be selectively  reacted to the target proteins through a two-step process.

Features of Crosslinking Protein  Interaction Analysis

Workflow of Crosslinking Protein Interaction Analysis

Ordering Procedure:

Ordering Procedure


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