I have a question wrt to amine coupling. In the SPR pages it is mentioned that amine coupling should not be used for acidic ligands. Can someone please explain me the reason for the same. Has it anything to do with the preconcentration ie since the dextran surface will have a negative charge at physlogical pH and our peptide will have a-ve charge alos, so there will be no preconcentration.
However if we want to immobilize the acidic peptides on a carboxyl functiolized silica particle, will we face the same problem? Secondly can an acidic ligand be immobilized on an amine functionlized silica particle employing glutaraldehyde chemistry?
I am sorry that it is a bit out of SPR topic.
But if someone can help me with this, I would be really grateful.
Yes, the dextran matrix will lose its pre-concentration ability below pH 3.5. At a pH below the pI of a protein, it will have an overall positive charge. At a pH above approximately pH 3.0 the carboxyl groups (pKa COOH = 2.19) will have an overall negative charge. The matrix is fully negatively charged at pH values above 7(1).
I am not familiar with silica particles, so I do not know if it will work.
Iam not sure what you mean with gluteraldehyde chemistry, but the aldehyde protocol used with Biacore depends on ligands having aldehyde groups. By activating the dextran surface to a hydrazide the aldehyde will react forming a covalent bond. The reaction is done a pH 4.0.
One reference about peptide immobilization: Houseman, B. T., Huh, J. H., Kron, S. J., and Mrksich, M. Peptide chips for the quantitative evaluation of protein kinase activity Nat.Biotechnol. 20: 270-274 (2002).
1. BIACORE AB BIACORE Technology Handbook (1998) .