It can be carried out by direct quantification of the immobilized antibodies. Please refer to Protocol 107 on our website.
The best beads are the NHS beads with activated COOH radicals on the COOH bead surface. The ε-NH2 radicals in the antibody lysine residue bond with the COOH radicals on the bead surface. It is also possible to bind antibodies to streptavidin and NeutrAvidinTM beads with biotin-labeled antibodies.
If binding is performed when 50 μg of antibodies is supplied to 1 mg of NHS beads, approximately 20 – 50 μg will be bound, although the result varies depending on the antibody animal, subclass, and clone. It is possible to increase the amount that is bound by increasing the amount on feed. When streptavidin beads are used, up to approximately 10 μg of biotin-modified antibodies will bond to 1 mg of streptavidin beads or NeutrAvidinTM beads, although this also varies depending on the antibody.
Please perform the dispersion of the beads by the manual agitation. When you cannot disperse the beads easily, disperse them in a short time by using an ice-cold ultrasonic homogenizer or ultrasonic washer. Please refer to the video on website for details.
It may be improved by stirring with a microtube mixer instead of inverting mixing. And please use a protein low bind tube.
Antibodies or proteins immobilized beads may easily precipitate. Please disperse the beads well before use. In addition, dispersibility improves if salt is removed from the buffer.