TY - JOUR
T1 - Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies
AU - Stevens, Shawn Y.
AU - Swanson, Patrick C.
AU - Glick, Gary D.
N1 - Funding Information:
This work was supported by NIH grant GM 46831. Additional funding was provided by the National Arthritis Foundation and The University of Michigan Multipurpose Arthritis Center (NIH grant AR 20557).
PY - 1994/12/28
Y1 - 1994/12/28
N2 - 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) 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.
AB - 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) 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.
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U2 - 10.1016/0022-1759(94)90155-4
DO - 10.1016/0022-1759(94)90155-4
M3 - Article
C2 - 7822825
AN - SCOPUS:0028564754
VL - 177
SP - 185
EP - 190
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -