Update profile
    Change password
    Sign out

Crosslinking Protein Interaction Analysis

When two or more proteins have specific affinity for one another that causes them to come together in biological systems, bioconjugation technology can provide the means for investigating those interactions. Most in vivo protein-protein binding is transient and occurs only briefly to facilitate signaling or metabolic function. Capturing or freezing these momentary contacts to study which proteins are involved and how they interact is a significant goal of proteomics research today.

Crosslinking reagents provide the means for capturing protein-protein complexes by covalently binding them together as they interact. The rapid reactivity of the common functional groups on crosslinkers allows even transient interactions to be frozen in place or weakly interacting molecules to be seized in a complex stable enough for isolation and characterization.

Page Contents:

Protein Methods Library Home

Related literature...

Protein Interaction
Technical Handbook

Overview of Crosslinking Reagents

The following is a only a brief description of crosslinkers required for the discussion of using crosslinkers to analyze protein-protein interactions. The topic of protein crosslinking is fully explained in the Crosslinking section of the Protein Methods Library and describes the types of crosslinkers used, their functional targets and reactive groups, their associated chemistries and applications.

Chemical Crosslinkers

Crosslinking reagents covalently bind proteins, domains or peptides by carrying reactive moieties that bind to specific amino acid funtional groups on target proteins. Commercially available crosslinking reagents have a wide range of characteristics, including:

  • Functional group specificity: the crosslinker molecule carries reactive moieties that target amines, sulfhydryls, carboxyls, carbonyls or hydroxyls.
     
  • Homobifunctional or heterobifunctional: molecules are available with identical reactive moieties on both ends (termed homobifunctional molecules; the upper molecule (A/A) shown below) that crosslink identical residues, while each reactive group on heterobifunctional crosslinkers targets different functional groups on separate proteins for greater variability or specificity, as shown in the lower molecule (A/B) below.
  • homobifunctional heterobifunctional crosslinkers

  • Variable spacer arm length: the reactive groups are spatially separated by the crosslinker molecule structure, which allows the crosslinking of amino acids that are varying distances apart, as shown below. Zero-length crosslinkers are also available, which crosslink two amino acid residues without leaving any part of the crosslinker molecule remaining in the interaction after the reaction is completed.
  • Diagram variability variable crosslinker spacer arm

  • Cleavable or non-cleavage: the 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.
  • Diagram cleavable crosslinker disulfide bond reducing agent reduced

  • Water-soluble or -insoluble: crosslinkers can be hydrophobic to allow passage into hydrophobic protein domains or through the cell membrane or hydrophilic to limit crosslinking to aqueous compartments.

Learn more...

Overview of Protein-Protein Interaction Analysis

Overview of Crosslinking and Protein Modification

 

View products...

Crosslinking Reagents

 

Related literature...

Crosslinking Technical Handbook

Photoreactive Crosslinkers

The simple addition of homobifunctional or heterobifunctional crosslinkers to cell suspensions or cell lysates will cause many protein conjugates to be formed, not just those directly involved in the target protein-protein interaction. Many cell surface protein interactions have been studied using this “shotgun” approach, but the challenge in this technique is the analysis of data after the complexes have been isolated.

To help solve these problems, more sophisticated crosslinker designs were created that incorporate 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, as indicated:

  • First, the chemical crosslinking moiety reacts with the functional group of one of the targets of interest to label the protein.
  • Second, the labeled protein, which is now essentially a bait, is incubated with a heterogenous protein solution, such as a lysate, and UV-irradiated at indicated times to crosslink any interacting proteins with the bait protein.

Proteins can also be metabolically labeled with photoreactive derivatives of amino acids, specifically leucine (L-photo-Leucine) and methionine (L-photo-methionine), after which the sample is UV-irradiated at specific time points to covalently crosslink proteins within protein-protein interaction domains in their native environment in live cells. This powerful method enables both stable and transient protein interactions in cells to be studied and characterized without the use of completely foreign chemical crosslinkers and associated reagent solvents during the interaction experiment that can adversely aspects of the cell biology being studied.

View products...

Photoreactive Crosslinkers

Photoreactive Amino Acids

In vivo and in vitro Crosslinking

In vivo Crosslinking

Besides the transient and sometimes tentative nature of some protein-protein interactions, the formation of these complexes can change in response to any number of stimuli, including changes in pH, temperature and osmolarity, and either the lack of a specific protein or co-factor or the introduction of a protein with which the protein(s) do not normally interact.

The benefit of in vivo crosslinking is that the protein-protein interaction can be captured in its native environment, which limits the risk of false positive interactions or the loss of complex stability during cell lysis. For in vivo crosslinking, hydrophobic, lipid-soluble crosslinkers are expected to be used if the target protein is within or across cell membranes, while hydrophilic, water-soluble crosslinkers can be used to crosslink cell surface proteins, such as receptor-ligand complexes.

Due to the high concentration of proteins in cells, crosslinkers with shorter spacer arms are usually recommended for in vivo crosslinking approaches to increase the specificity of conjugating actual interacting proteins as opposed to proteins that just happen to be in close proximity to each other during incubation with the crosslinker.

Although in vivo crosslinking can yield physiologically relevant, stably-crosslinked complexes for analysis, optimizing this approach can be difficult, as the reaction conditions cannot be tightly controlled and crosslinkers react with a wide array of proteins that all present functional groups against which crosslinkers specifically react.

View products...

In vivo Crosslinking Products

In vitro Crosslinking

In vitro crosslinking can better target specific crosslinking events, because more reaction conditions can be tightly controlled, including the pH, temperature, concentration of reactants and purity of the target protein(s). The ability to control all aspects of a conjugation experiment results in better analysis due to greater resolution of protein-protein interactions. Additionally, in vitro methods of conjugation allow researchers to modify interacting proteins, such as adding polyethylene glycol groups (PEGylation), blocking sulfhydryls or converting amines to sulfhydryls. Also, a greater variety of crosslinking reagents, both hydrophobic and hydrophilic, are available for in vitro applications.

Obviously, the disadvantage of using in vitro methods to conjugate proteins is the lack of physiological conditions. Additionally, rupturing and solubilizing membranes can disrupt protein-protein and protein-membrane interactions.

Because a myriad of crosslinking reagents are commercially available for many different applications, the key determinant in deciding to use in vivo or in vitro crosslinking is the target protein, specifically in term of its:

  • Cellular location: in vivo crosslinking would benefit protein targets embedded in the cell membrane, while cytoplasmic proteins could be crosslinked by either methods, depending on the next determinant.
  • Interaction stability: weak protein-protein interactions may be lost during in vitro crosslinking due to cell lysis and potential competition with other proteins, while stable interactions may be strong enough to withstand these forces.

View products...

In vivo Crosslinking Products

General Reaction Conditions

The exact sample preparation protocol varies depending on whether in vitro or in vivo crosslinking is performed, the specific crosslinker used and the protein-protein interaction that is targeted. The guidelines listed are general in nature and should be used as a starting point for further optimization.

 

Choosing the Appropriate Crosslinker

Correct identification of protein-protein interactions first requires the selection of the best crosslinker to use. Because there are multiple amino acid functional groups that may react with different crosslinkers, an empirical strategy of screening multiple types of crosslinkers should first be performed to identify the target protein conjugate. The crosslinkers tested may vary in:

  • Hydrophobicity
  • Reactive groups
  • Homo- vs. heterobifunctionality
  • Spacer arm length

Once the target interaction is detected by any of the methods listed below, then the protocol can be fine-tuned to optimize detection by adjusting crosslinker concentration, pH and other reaction conditions.

Learn more...

Overview of Crosslinking and Protein Modification

View products...

Crosslinking Reagents

Sample Preparation

The starting protein concentration or number of cells should be empirically determined for in vitro and in vivo crosslinking protocols, respectively. For in vitro crosslinking, the protein solution should be prepared in a nonreactive buffer, such as phosphate buffered saline (PBS), that has the proper pH for the specific crosslinker. For in vivo crosslinking applications, cells should be in the exponential phase of growth and at a subconfluent density during the crosslinking procedure. To avoid the possibility of culture media reacting with the crosslinker, the media can be replaced with PBS through a series of cell washes.

View products...

Buffers for Protein Methods

Cell Lysis Reagents

Reaction Conditions

Crosslinkers should be prepared as per the manufacturer's instructions; hydrophobic crosslinkers are first dissolved in the appropriate solvent, such as methanol or acetone. The optimum amount of reagent to add also depends on the crosslinker, but usually a 20- to 500-fold molar excess (relative to the lysate protein concentration) is appropriate. Ensure that pH of the reaction buffer is favorable for the crosslinker. Most amine-reactive crosslinkers require alkaline pH for activity.

The crosslinking reaction time may also be important, depending upon the experiment and crosslinker being used. While 30 minutes is a good incubation time to start with, multiple experiments can be performed concurrently to test other lengths of time to determine the optimal time of incubation with the specific crosslinker. Long incubation periods should generally be avoided, not only because it may cause formation of large, crosslinked protein aggregates, but also because the crosslinker may lose stability. In cases where extended incubation periods are required, though, fresh crosslinker can be added at specific time points throughout the procedure to maintain the proper molar ratio of reagent and maximize the formation of the target product. The formation of aggregates due to extensive crosslinking, though, should also be considered in determining the optimal reaction time.

View products...

Buffers for Protein Methods

Quenching the Reaction

With most amine-reactive crosslinkers used for protein-interaction analysis, the reaction can be halted at the desired time by adding excess nucleophile, such as Tris or glycine, which outcompetes the lysate proteins for reaction with the crosslinker. The crosslinked product can then be purified through multiple approaches, including precipitation, chromatography, dialysis or ultrafiltration.

A rapid method that combines quenching the reaction and denaturing the proteins in preparation for gel electrophoresis is to add boiling sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer, which contains both Tris and β-mercaptoethanol, and then boiling the solution for 5 minutes. The sample can then be directly analyzed by gel electrophoresis.

 

Protein-Protein Interaction Analysis

Crosslinking is typically used to capture and stabilize transient or labile interactions so that they can be further isolated and analyzed by downstream methods such as electrophoresis, staining, Western blot, immunoprecipitation or co-immunoprecipitation and mass spectrometry.

 

Western Blot

When two proteins are covalently crosslinked, the gel migration patterns of both proteins shift in relation to the uncrosslinked proteins. Therefore, if antibodies that detect each target protein are available, the most straightforward method to detect the shift of the interacting proteins is by SDS-PAGE and Western blot analysis.

Learn more...

Western Blot Analysis

Immunoprecipitation and Co-immunopreciptation

Both immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are methods to detect protein expression and protein-protein interactions, respectively, via affinity purification. Crosslinking is commonly performed in both applications to, either alone or in combination with affinity binding, immobilize antibody to the beaded support and or freeze weak antibody-antigen interactions to prevent sample loss during immune complex extraction. Crosslinking is also used to stabilize transient or weak protein-protein interactions prior to co-IP protocols. Following both approaches, samples are commonly analyzed by SDS-PAGE.

Learn more...

Immunoprecipitation

Co-Immunoprecipitation

Mass Spectrometry

When analysis by mass spectrometry (MS) is available, the peptide fragments that are crosslinked between interacting proteins can be identified by the change in mass resulting from the attached crosslinker molecule. In this approach, identical samples are crosslinked with either deuterated (heavy) or nondeuterated (light) crosslinkers. The crosslinked proteins are then pooled together and analyzed by MS to identify and quantify the heavy product based on its shift from the light product. This method also commonly employs SDS-PAGE as a first-stage purification step prior to digestion in preparation for MS analysis.

Learn more...

Mass Spectrometry Workflow

View products...

Mass-Tagged Crosslinkers

References

  1. Golemis E. (2002) Protein-protein interactions : A molecular cloning manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. ix, 682.
  2. Hermanson G.T. (2008) Bioconjugate Techniques, 2nd Edition. Academic Press, Inc. 1323.
  3. Ohh M. et al. (1998) The von hippel-lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol Cell. 1, 959-68.
  4. Yakovlev A.A. (2009) Crosslinkers and their utilization for studies of intermolecular interactions. Neurochem J. 3, 139-144.
  5. Yang L et al. (2010) A photocleavable and mass spectrometry identifiable cross-linker for protein interaction studies. Anal. Chem. 82, 3556-3566.
 
Written and/or reviewed by Douglas Hayworth, Ph.D.

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

Recommend this page

Follow Us
Email Sign-up  Email Announcements

 



Antibodies  |   Molecular Biology   |   Cell Biology   |  Thermo Scientific  |   * Trademarks  |   Privacy Statement
© 2014 Thermo Fisher Scientific Inc.   |   3747 N Meridian Rd, Rockford, IL USA 61101   |   1-800-874-3723  or  815-968-0747