We are using the protocol that the manufacturer suggests for the covalent
binding of the antibody to the beads, with a polyclonal rabbit antibody to
MAPK kinase from Santa Cruz (+ gelatin) and we have also tried a monoclonal
from Transduction (+ BSA), both have yielded less than favorable results.
Both antibodies have carrier proteins in them that we believe are
interferring with the binding of antibody to the beads. But even increasing
the amount of beads did not produce an improvement. A major problem is
that there is no depletion of antibody when comparing the load and
breakthrough of antibody. Also, when eluting all proteins off the bead we
do not see any antibody heavy chain coming off with the carrier proteins.
We have also tested the blocking and found that
it is not sufficient. We also get very poor binding of purified MAPK kinase
to the antibody-dynabeads, and what is binding is not eluting very well
(probably because the blocking isn't adequate).
We have used a variety of buffers to wash the beads and for antibody
binding: Phosphate buffer pH 7.4
Acetate buffer pH 4.0
Borate buffer pH 9.0
We have also used two different bead volumes
first with 20ul of dynabead solution
and later with 100ul
We have varied coating solution volumes from 50ul-300ul, and decided larger
volumes work better ie) 200-300ul
We have blocked the beads with BSA and more recently with ethanolamine.
We have also varied the amount of antibody added to the coating solution.
Dynal recommends 1-3ug/1X10 7 beads. We have tried several amounts within
this ratio as well as above and below.
Katheryn Resing
Dept Chem and Biochem
Univ Colorado
Boulder CO 80303
303-492-6273
fax 492-2439