Sy. Stevens et al., APPLICATION OF THE GEL SHIFT ASSAY TO STUDY THE AFFINITY AND SPECIFICITY OF ANTI-DNA AUTOANTIBODIES, Journal of immunological methods, 177(1-2), 1994, pp. 185-190
We have demonstrated that the gel shift assay, a powerful method to st
udy protein DNA interactions under equilibrium conditions, is both an
accurate and precise method to measure the affinity of anti-DNA DNA im
mune complexes. One difficulty in performing gel shift assays is disru
ption of protein DNA equilibria during the time needed for complexes t
o enter the gel matrix. However, we have found that highly cross-linke
d polyacrylamide gels, which are known to form non-restrictive matrice
s, do not perturb anti-DNA DNA complexes. Using anti-ssDNA BV04-01 as
a model antibody, we find good agreement between the dissociation cons
tants (K-d) measured in the gel shift assay using a 5.4% polyacrylamid
e gel cross-linked with 0.6% (bis)acrylamide, and those obtained previ
ously by fluorescence quenching. Because gel shift assays require only
nanogram quantities of analyte and can be performed in several hours,
it is well suited for a range of anti-DNA binding studies.