Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports.
Very weak protein-protein interactions are very difficult to detect because these complexes could be under the detection limit or they tend to dissociate. Here, using as a model the antibody-antigen interaction weaken by the presence of dioxane, we have shown a strategy for the protein complexes purification by selective adsorption of the associated proteins. This strategy is based on the use of poorly activated anionic exchanger supports to selectively adsorb large complexes. This selective adsorption of the associated proteins shifted the association equilibrium of the soluble proteins toward the associated form. Thus, in the presence of 15% v/v dioxane, a concentration that is able to almost fully break the immunocomplex (less that 3% of the immunocomplex appeared associated when soluble antigen-antibody mixture was cross-linked with aldehyde-dextran), we can obtain more than 90% of the fully pure immunocomplex from the non-associated protein, adsorbed on anionic exchanger supports having a very low activation. This simple strategy may be a very useful tool to solve one of the most relevant challenges in the modern proteomics, the detection of very weak protein-protein interactions.
Fuentes
Manuel
M
Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Madrid, Spain.
Mateo
Cesar
C
Pessela
Benevides C C
BC
Guisán
Jose M
JM
Fernandez-Lafuente
Roberto
R
D000327:Adsorption; D000818:Animals; D000936:Antigen-Antibody Complex; D003432:Cross-Linking Reagents / Q000737:chemistry; D004146:Dioxanes / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D009195:Peroxidase / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D012685:Sepharose / Q000737:chemistry; D013499:Surface Properties
D000936:Antigen-Antibody Complex; D003432:Cross-Linking Reagents; D004146:Dioxanes; D007074:Immunoglobulin G; D012685:Sepharose; D006735:Horseradish Peroxidase; D009195:Peroxidase; C025223:1,4-dioxane