I am interested in perform a chemical cross-linking assay. Has anyone can give me good advice to perform it correctly.
Hi, I'm not sure what you are wanting to cross-link.
I have used glutaraldehyde to perform protein-protein cross-linking. It non-specifically binds amide groups, so you would need to have two pure samples to cross-link. You would need a way of separating the different species you get (like gel filtration) because you will get ProteinA-ProteinB complexes but also ProteinA-ProteinA and ProteinB-ProteinB too. You will also get large aggregates and will have to perform a few reactions limiting the proteins to the reaction to get the least aggregation.
Let me know if this is what you are wanting to do and I will try and help more.
Chris
Dear all.
I would like to explain my interest on chemical cross-linking assay. I am currently working with a protein A that binds to protein B. The full length of my protein A binds directly to protein B and activate the protein.
However, when I do a point mutation nearby the area of binding domain of protein A, the binding is not longer possible with protein B, therefore the activation is affected.
Interestingly, when this binding domain of protein A is synthesized (length 30-35aa) with my point mutation, the protein B still binding.
I am proposing that somehow there is an interaction between the point mutation and an unknown amino acid. This amino acid is located in another domain of the full length protein A.
Do you think that chemical cross linking can help to identify this unknown amino acid?
I will really appreciate your help! thanks!
Crosslinking is one way you could test for a binding interaction. Other ways are also possible, such as 2-D NMR, ITC, SPR, fluorescence polarization, FRET, and the activation measurement you already made. What you might get from crosslinking is a sense of which parts of the proteins are close together when they bind. This will require identifying the site of crosslinking on each protein. This is done by protease digestion and mass spectrometry. As one example, see this paper: Shapiro, A.B., Gu, R.-F., and Gao, N. Dimerization of isolated Pseudomonas aeruginosa lipopolysaccharide transporter component LptA. Biochem. Biophys. Res. Commun. 450, 1327-1332. 2014.
Dear Edgar,
Sure, a crosslink coupled with a MS analysis will help you to find the aa involved in this interaction. The best is certainly to discuss with the lab that will carry out the analysis, each lab has their preferences and it also depends of the mass spectrometer.
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