You can cross-link, or cause chemical bonds to form between, two different protein structures. Cross-linking can provide you information on the identities of two different proteins, as well as the regions at which they form bonds. The process of cross-linking is rather simple, involving treating proteins in buffer solutions with cross-linking agents that bond to functional groups of the proteins. The extent to which a protein bonds to a cross-linking agent, inducing conformational changes in the protein, provides valuable insight into the number of bonding sites that protein has and how responsive the proteins are to bonding agents.
- Buffer solution, i.e. phosphate buffer at pH 7.5
- Eppendorf tubes
- Glutaraldehyde, or another cross-linking agent of choice
- Micropipettes
- Tris-HCl
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Instructions
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1
Create a solution of total volume around 100 microliters. Pipette 50 to 100 micrograms of interacting proteins into an Eppendorf containing 20 mM phosphate buffer.
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2
Add 5 microliters of 2.3 percent glutaraldehyde, via pipetting, for two to five minutes. Incubate solution at 37 degrees Celsius.
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3
Terminate the cross-linking reaction by adding, via pipetting, 10 microliters of 1 M Tris-HCl, pH 8.0.