Determination of protein oligomerization state: Two approaches based on glutaraldehyde crosslinking

Department of Biology, University of Crete, PO Box 2208, GR-71409 Heraklion, Crete, Greece.
Analytical Biochemistry (Impact Factor: 2.22). 03/2008; 373(2):404-6. DOI: 10.1016/j.ab.2007.10.027
Source: PubMed


Many biochemical and biophysical methods can be used to characterize the oligomerization state of proteins. One of the most widely used is glutaraldehyde crosslinking, mainly because of the minimum equipment and reagents required. However, the crosslinking procedures currently in use are impaired by the low specificity of the reagent, which can chemically bond any two amino groups that are close in space. Thus, extensive and time-consuming investigation of the reaction conditions is usually required. Here we describe two approaches based on glutaraldehyde that readily give reliable results.

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    • "The crosslinking reaction was then quenched with 8 mL of 0.5 M Tris/HCl, pH 8.0. Buffer C (8 mL) (300 mM imidazole, 50 mM NaCl, 50 mM phosphate buffer, pH 8.0) was then used to elute the bound crosslinked protein [18]. The covalent oligomers were then resolved on an 8% SDS–PAGE. "
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    • "The oligomerization state of FosR protein was determined by glutaraldehyde cross-linking (Fadouloglou et al., 2008). "
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    • "To investigate this possibility, two different approaches were used. The first approach used a modified glutaraldehyde cross-linking method (Fadouloglou et al., 2008). In this method, glutaraldehyde treatment stabilized the oligomerization state of AAP-1 immobilized to the beads, and then the treated AAP-1 was eluted, and analyzed by immunoblotting with the anti-Histag antibody. "
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