APPLICATION OF THE GEL SHIFT ASSAY TO STUDY THE AFFINITY AND SPECIFICITY OF ANTI-DNA AUTOANTIBODIES

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.