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

Shawn Y. Stevens, Patrick C. Swanson, Gary D. Glick

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)185-190
Number of pages6
JournalJournal of Immunological Methods
Volume177
Issue number1-2
DOIs
StatePublished - Dec 28 1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

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