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 study protein.DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA.DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein.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.DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measured 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.
Stevens
S Y
SY
University of Michigan, Department of Chemistry, Ann Arbor 48109-1055.
Swanson
P C
PC
Glick
G D
GD
D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000915:Antibody Affinity; D000918:Antibody Specificity; D004277:DNA, Single-Stranded / Q000276:immunology* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques
D000974:Antibodies, Antinuclear; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins