about immobilization

hi,arnoud

when immobilization antigen to CM5 chip by amine coupling , IF different labels(Fc ,his) have effect on antigen activity?

Hi Wang,

Define activity…

When a protein is immobilized by the amine coupling, primary amines (in general lysine) are used for the coupling. This is a random process and it is difficult to predict which lysine's and how many are used for the coupling. If in the coupling a lysine is used that is close or in the interaction site this interaction will be reduced or abolished. Some coupling procedures use a protection peptide bound to the interaction site during the immobilization.
If lysine's are important for the interaction other immobilization protocols are available (thiol, aldehyde) or a capture approach is used.
When you modify an antigen with a special group (label) such as an Fc or 6xHIS, this can alter the interaction properties of your antigen. In general, the more closer to the interaction site the more the chance that the interaction changes.
If you have the same antigen with different labels the immobilization will depend on the free amines in this label. For 6xHIS there are none, but an Fc-fragment has probably some. In both cases the amount of antigen immobilized can be the same with the same binding activity but it can also be a little different.

Kind regards
Arnoud

Thank you sooooo much! Arnoud