Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies

Abstract

We have demonstrated that the gel shift assay, a powerful method to study protein [middle dot] DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA [middle dot] DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein [middle dot] DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels, which are known to form non-restrictive matrices, do not perturb anti-DNA[middle dot]DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measureed in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously 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.